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Sample records for high-affinity protein-complex purification

  1. Affinity Purification of Protein Complexes Using TAP Tags

    PubMed Central

    Gerace, Erica; Moazed, Danesh

    2016-01-01

    This protocol is used for the isolation and analysis of protein complexes using the tandem affinity purification (TAP) tag system. The protocol describes the purification of a protein fused to a TAP tag comprised of two protein A domains and the calmodulin binding peptide separated by a TEV cleavage site. This is a powerful technique for rapid purification of protein complexes and the analysis of their stoichiometric composition, posttranslational modifications, structure, and functional activities. PMID:26096502

  2. Purification of high affinity benzodiazepine receptor binding site fragments from rat brain

    SciTech Connect

    Klotz, K.L.

    1984-01-01

    In central nervous system benzodiazepine recognition sites occur on neuronal cell surfaces as one member of a multireceptor complex, including recognition sites for benzodiazepines, gamma aminobutyric acid (GABA), barbiturates and a chloride ionophore. During photoaffinity labelling, the benzodiazepine agonist, /sup 3/H-flunitrazepam, is irreversibly bound to central benzodiazepine high affinity recognition sites in the presence of ultraviolet light. In these studies a /sup 3/H-flunitrazepam radiolabel was used to track the isolation and purification of high affinity agonist binding site fragments from membrane-bound benzodiazepine receptor in rat brain. The authors present a method for limited proteolysis of /sup 3/H-flunitrazepam photoaffinity labeled rat brain membranes, generating photolabeled benzodiazepine receptor fragments containing the agonist binding site. Using trypsin chymotrypsin A/sub 4/, or a combination of these two proteases, they have demonstrated the extent and time course for partial digestion of benzodiazepine receptor, yielding photolabeled receptor binding site fragments. These photolabeled receptor fragments have been further purified on the basis of size, using ultrafiltration, gel permeation chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as well as on the basis of hydrophobicity, using a high performance liquid chromatography (HPLC) precolumn, several HPLC elution schemes, and two different HPLC column types. Using these procedures, they have purified three photolabeled benzodiazepine receptor fragments containing the agonist binding site which appear to have a molecular weight of less than 2000 daltons each.

  3. Structures of the Ultra-High-Affinity Protein-Protein Complexes of Pyocins S2 and AP41 and Their Cognate Immunity Proteins from Pseudomonas aeruginosa.

    PubMed

    Joshi, Amar; Grinter, Rhys; Josts, Inokentijs; Chen, Sabrina; Wojdyla, Justyna A; Lowe, Edward D; Kaminska, Renata; Sharp, Connor; McCaughey, Laura; Roszak, Aleksander W; Cogdell, Richard J; Byron, Olwyn; Walker, Daniel; Kleanthous, Colin

    2015-08-28

    How ultra-high-affinity protein-protein interactions retain high specificity is still poorly understood. The interaction between colicin DNase domains and their inhibitory immunity (Im) proteins is an ultra-high-affinity interaction that is essential for the neutralisation of endogenous DNase catalytic activity and for protection against exogenous DNase bacteriocins. The colicin DNase-Im interaction is a model system for the study of high-affinity protein-protein interactions. However, despite the fact that closely related colicin-like bacteriocins are widely produced by Gram-negative bacteria, this interaction has only been studied using colicins from Escherichia coli. In this work, we present the first crystal structures of two pyocin DNase-Im complexes from Pseudomonas aeruginosa, pyocin S2 DNase-ImS2 and pyocin AP41 DNase-ImAP41. These structures represent divergent DNase-Im subfamilies and are important in extending our understanding of protein-protein interactions for this important class of high-affinity protein complex. A key finding of this work is that mutations within the immunity protein binding energy hotspot, helix III, are tolerated by complementary substitutions at the DNase-Immunity protein binding interface. Im helix III is strictly conserved in colicins where an Asp forms polar interactions with the DNase backbone. ImAP41 contains an Asp-to-Gly substitution in helix III and our structures show the role of a co-evolved substitution where Pro in DNase loop 4 occupies the volume vacated and removes the unfulfilled hydrogen bond. We observe the co-evolved mutations in other DNase-Immunity pairs that appear to underpin the split of this family into two distinct groups.

  4. Structures of the Ultra-High-Affinity Protein–Protein Complexes of Pyocins S2 and AP41 and Their Cognate Immunity Proteins from Pseudomonas aeruginosa

    PubMed Central

    Joshi, Amar; Grinter, Rhys; Josts, Inokentijs; Chen, Sabrina; Wojdyla, Justyna A.; Lowe, Edward D.; Kaminska, Renata; Sharp, Connor; McCaughey, Laura; Roszak, Aleksander W.; Cogdell, Richard J.; Byron, Olwyn; Walker, Daniel; Kleanthous, Colin

    2015-01-01

    How ultra-high-affinity protein–protein interactions retain high specificity is still poorly understood. The interaction between colicin DNase domains and their inhibitory immunity (Im) proteins is an ultra-high-affinity interaction that is essential for the neutralisation of endogenous DNase catalytic activity and for protection against exogenous DNase bacteriocins. The colicin DNase–Im interaction is a model system for the study of high-affinity protein–protein interactions. However, despite the fact that closely related colicin-like bacteriocins are widely produced by Gram-negative bacteria, this interaction has only been studied using colicins from Escherichia coli. In this work, we present the first crystal structures of two pyocin DNase–Im complexes from Pseudomonas aeruginosa, pyocin S2 DNase–ImS2 and pyocin AP41 DNase–ImAP41. These structures represent divergent DNase–Im subfamilies and are important in extending our understanding of protein–protein interactions for this important class of high-affinity protein complex. A key finding of this work is that mutations within the immunity protein binding energy hotspot, helix III, are tolerated by complementary substitutions at the DNase–Immunity protein binding interface. Im helix III is strictly conserved in colicins where an Asp forms polar interactions with the DNase backbone. ImAP41 contains an Asp-to-Gly substitution in helix III and our structures show the role of a co-evolved substitution where Pro in DNase loop 4 occupies the volume vacated and removes the unfulfilled hydrogen bond. We observe the co-evolved mutations in other DNase–Immunity pairs that appear to underpin the split of this family into two distinct groups. PMID:26215615

  5. The tandem affinity purification method: an efficient system for protein complex purification and protein interaction identification.

    PubMed

    Xu, Xiaoli; Song, Yuan; Li, Yuhua; Chang, Jianfeng; Zhang, Hua; An, Lizhe

    2010-08-01

    Isolation and identification of protein partners in multi-protein complexes are important in gaining further insights into the cellular roles of proteins and determining the possible mechanisms by which proteins have an effect in the molecular environment. The tandem affinity purification (TAP) method was originally developed in yeast for the purification of protein complexes and identification of protein-protein interactions. With modifications to this method and many variations in the original tag made over the past few years, the TAP system could be applied in mammalian, plant, bacteria and other systems for protein complex analysis. In this review, we describe the application of the TAP method in various organisms, the modification in the tag, the disadvantages, the developments and the future prospects of the TAP method. PMID:20399864

  6. Tandem Affinity Purification of Protein Complexes in Mouse Embryonic Stem Cells Using In Vivo Biotinylation

    PubMed Central

    Wang, Jianlong; Cantor, Alan B.; Orkin, Stuart H.

    2009-01-01

    In dissecting the pluripotent state in mouse embryonic stem (ES) cells, we have employed in vivo biotinylation of critical transcription factors for streptavidin affinity purification of protein complexes and constructed a protein-protein interaction network. This has facilitated discovery of novel pluripotency factors and a better understanding of stem cell pluripotency. Here we describe detailed procedures for in vivo biotinylation system setup in mouse ES cells and affinity purification of multi-protein complexes using in vivo biotinylation. In addition, we present a protocol employing SDS-PAGE fractionation to reduce sample complexity prior to submission for mass spectrometry (MS) protein identification. PMID:19306258

  7. Affinity purification of protein complexes for analysis by multidimensional protein identification technology.

    PubMed

    Banks, Charles A S; Kong, Stephanie E; Washburn, Michael P

    2012-12-01

    Characterizing protein complexes and identifying their subunits promote our understanding of the machinery involved in many in vivo processes. Proteomic studies can identify a protein's binding partners, and this can provide insight into how protein complexes function and how they are regulated. In addition, the composition of a protein complex within an organism can be investigated as a function of time, as a function of location, or during the response of an organism to a change in environment. There are many ways to isolate a complex and identify its constituents. This review will focus on complex isolation using affinity purification and will address issues that biochemists should bear in mind as they isolate protein complexes for mass spectrometric analysis by multidimensional protein identification technology (MudPIT)(1). Protein complex analysis by mass spectrometry frequently involves the collaborative efforts of biochemists or biologists who purify protein complexes and proteomic specialists who analyze the samples - for fruitful collaborations it can be helpful for these specialized groups to be acquainted with basic principles of their collaborator's discipline. With this in mind, we first review the variety of affinity purification methods which might be considered for preparing complexes for analysis, and then provide brief primers on the principles of MudPIT mass spectrometry and data analysis. From this foundation, we then discuss how these techniques are integrated and optimized and suggest salient points to consider when preparing purified samples for protein identification, performing mass spectrometry runs, and analyzing the resulting data.

  8. Engineering a reversible, high-affinity system for efficient protein purification based on the cohesin-dockerin interaction.

    PubMed

    Karpol, Alon; Kantorovich, Lia; Demishtein, Alik; Barak, Yoav; Morag, Ely; Lamed, Raphael; Bayer, Edward A

    2009-01-01

    Efficient degradation of cellulose by the anaerobic thermophilic bacterium, Clostridium thermocellum, is carried out by the multi-enzyme cellulosome complex. The enzymes on the complex are attached in a calcium-dependent manner via their dockerin (Doc) module to a cohesin (Coh) module of the cellulosomal scaffoldin subunit. In this study, we have optimized the Coh-Doc interaction for the purpose of protein affinity purification. A C. thermocellum Coh module was thus fused to a carbohydrate-binding module, and the resultant fusion protein was applied directly onto beaded cellulose, thereby serving as a non-covalent "activation" procedure. A complementary Doc module was then fused to a model protein target: xylanase T-6 from Geobacillus stearothermophilus. However, the binding to the immobilized Coh was only partially reversible upon treatment with EDTA, and only negligible amounts of the target protein were eluted from the affinity column. In order to improve protein elution, a series of truncated Docs were designed in which the calcium-coordinating function was impaired without appreciably affecting high-affinity binding to Coh. A shortened Doc of only 48 residues was sufficient to function as an effective affinity tag, and highly purified target protein was achieved directly from crude cell extracts in a single step with near-quantitative recovery of the target protein. Effective EDTA-mediated elution of the sequestered protein from the column was the key step of the procedure. The affinity column was reusable and maintained very high levels of capacity upon repeated rounds of loading and elution. Reusable Coh-Doc affinity columns thus provide an efficient and attractive approach for purifying proteins in high yield by modifying the calcium-binding loop of the Doc module. PMID:18979459

  9. Modular Broad-Host-Range Expression Vectors for Single-Protein and Protein Complex Purification

    PubMed Central

    Fodor, Barna D.; Kovács, Ákos T.; Csáki, Róbert; Hunyadi-Gulyás, Éva; Klement, Éva; Maróti, Gergely; Mészáros, Lívia S.; Medzihradszky, Katalin F.; Rákhely, Gábor; Kovács, Kornél L.

    2004-01-01

    A set of modular broad-host-range expression vectors with various affinity tags (six-His-tag, FLAG-tag, Strep-tag II, T7-tag) was created. The complete nucleotide sequences of the vectors are known, and these small vectors can be mobilized by conjugation. They are useful in the purification of proteins and protein complexes from gram-negative bacterial species. The plasmids were easily customized for Thiocapsa roseopersicina, Rhodobacter capsulatus, and Methylococcus capsulatus by inserting an appropriate promoter. These examples demonstrate the versatility and flexibility of the vectors. The constructs harbor the T7 promoter for easy overproduction of the desired protein in an appropriate Escherichia coli host. The vectors were useful in purifying different proteins from T. roseopersicina. The FLAG-tag-Strep-tag II combination was utilized for isolation of the HynL-HypC2 protein complex involved in hydrogenase maturation. These tools should be useful for protein purification and for studying protein-protein interactions in a range of bacterial species. PMID:14766546

  10. Surfactant-free purification of membrane protein complexes from bacteria: application to the staphylococcal penicillin-binding protein complex PBP2/PBP2a

    NASA Astrophysics Data System (ADS)

    Paulin, Sarah; Jamshad, Mohammed; Dafforn, Timothy R.; Garcia-Lara, Jorge; Foster, Simon J.; Galley, Nicola F.; Roper, David I.; Rosado, Helena; Taylor, Peter W.

    2014-07-01

    Surfactant-mediated removal of proteins from biomembranes invariably results in partial or complete loss of function and disassembly of multi-protein complexes. We determined the capacity of styrene-co-maleic acid (SMA) co-polymer to remove components of the cell division machinery from the membrane of drug-resistant staphylococcal cells. SMA-lipid nanoparticles solubilized FtsZ-PBP2-PBP2a complexes from intact cells, demonstrating the close physical proximity of these proteins within the lipid bilayer. Exposure of bacteria to (-)-epicatechin gallate, a polyphenolic agent that abolishes β-lactam resistance in staphylococci, disrupted the association between PBP2 and PBP2a. Thus, SMA purification provides a means to remove native integral membrane protein assemblages with minimal physical disruption and shows promise as a tool for the interrogation of molecular aspects of bacterial membrane protein structure and function.

  11. An improved toolbox to unravel the plant cellular machinery by tandem affinity purification of Arabidopsis protein complexes.

    PubMed

    Van Leene, Jelle; Eeckhout, Dominique; Cannoot, Bernard; De Winne, Nancy; Persiau, Geert; Van De Slijke, Eveline; Vercruysse, Leen; Dedecker, Maarten; Verkest, Aurine; Vandepoele, Klaas; Martens, Lennart; Witters, Erwin; Gevaert, Kris; De Jaeger, Geert

    2015-01-01

    Tandem affinity purification coupled to mass spectrometry (TAP-MS) is one of the most advanced methods to characterize protein complexes in plants, giving a comprehensive view on the protein-protein interactions (PPIs) of a certain protein of interest (bait). The bait protein is fused to a double affinity tag, which consists of a protein G tag and a streptavidin-binding peptide separated by a very specific protease cleavage site, allowing highly specific protein complex isolation under near-physiological conditions. Implementation of this optimized TAP tag, combined with ultrasensitive MS, means that these experiments can be performed on small amounts (25 mg of total protein) of protein extracts from Arabidopsis cell suspension cultures. It is also possible to use this approach to isolate low abundant protein complexes from Arabidopsis seedlings, thus opening perspectives for the exploration of protein complexes in a plant developmental context. Next to protocols for efficient biomass generation of seedlings (∼7.5 months), we provide detailed protocols for TAP (1 d), and for sample preparation and liquid chromatography-tandem MS (LC-MS/MS; ∼5 d), either from Arabidopsis seedlings or from cell cultures. For the identification of specific co-purifying proteins, we use an extended protein database and filter against a list of nonspecific proteins on the basis of the occurrence of a co-purified protein among 543 TAP experiments. The value of the provided protocols is illustrated through numerous applications described in recent literature.

  12. Identification of protein complexes in Escherichia coli using sequential peptide affinity purification in combination with tandem mass spectrometry.

    PubMed

    Babu, Mohan; Kagan, Olga; Guo, Hongbo; Greenblatt, Jack; Emili, Andrew

    2012-01-01

    Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems(1, 2). Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria(1-6). In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes(1, 2, 7). Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast(8, 9), and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system(10). Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large

  13. Dual-tagging system for the affinity purification of mammalian protein complexes

    SciTech Connect

    Giannone, Richard J; McDonald, W Hayes; Hurst, Gregory {Greg} B; Huang, Ying; Wu, Jun; Liu, Yie; Wang, Yisong

    2007-01-01

    Although affinity purification coupled with mass spectrometry (MS) provides a powerful tool to study protein-protein interactions, this strategy has encountered numerous difficulties when adapted to mammalian cells. Here we describe a Gateway{reg_sign}-compatible dual-tag affinity purification system that integrates regulatable expression, tetracysteine motifs, and various combinations of affinity tags to facilitate the cloning, detection, and purification of bait proteins and their interacting partners. Utilizing the human telomere binding protein TRF2 as a benchmark, we demonstrate bait protein recoveries upwards of approximately 16% from as little as 1-7 x 10{sup 7} cells and successfully identify known TRF2 interacting proteins, suggesting that our dual-tag affinity purification approach is a capable new tool for expanding the capacity to explore mammalian proteomic networks.

  14. Purification of effector-target protein complexes via transient expression in Nicotiana benthamiana.

    PubMed

    Win, Joe; Kamoun, Sophien; Jones, Alexandra M E

    2011-01-01

    Effectors of plant pathogens play important roles in not only pathogenesis but also plant immunity. Plant pathogens use these effectors to manipulate host cells for colonization, and their activities likely influence the evolution of plant immune responses. Analyses of genome sequences revealed that oomycete pathogens, such as Phytophthora spp., possess hundreds of RXLR effectors that are thought to be delivered into the host cells and hence function inside the cells by interacting with the host protein complexes. This article describes a co-immunoprecipitation protocol aimed at identifying putative target complexes of the effectors by transiently overexpressing the tagged effectors in planta. The identification of the eluted protein complexes was achieved by LC-MS/MS mass spectrometry and peptide spectrum matching. PMID:21359809

  15. The 5-S RNA . protein complex from an extreme halophile, Halobacterium cutirubrum. Purification and characterization.

    PubMed

    Smith, N; Matheson, A T; Yaguchi, M; Willick, G E; Nazar, R N

    1978-09-01

    A 5-S RNA . protein complex has been isolated from the 50-S ribosomal subunit of an extreme halophile, Halobacterium cutirubrum. The 50-S ribosomal subunit from the extreme halophile requires 3.4 M K+ and 100 mM Mg2+ for stability. However, if the high K+ concentration is maintained but the Mg2+ concentration lowered to 0.3 mM, the 5-S RNA . protein complex is selectively extracted from the subunit. After being purified on an Agarose 0.5-m column the complex had a molecular weight of about 80000 and contained 5-S RNA and two proteins, HL13 and HL19, with molecular weights (by sedimentation equilibrium) of 18700 and 18000, respectively. No ATPase or GTPase activity could be detected in the 5-S RNA . protein complex. The amino acid composition and electrophoretic mobility on polyacrylamide gels indicated both proteins were much more acidic than the equivalent from Escherichia coli or Bacillus stearothermophilus. Partial amino acid sequence data suggest HL13 is homologous to EL18 and HL19 to EL5.

  16. High Throughput Identification, Purification and Structural Characterization of Water Soluble Protein Complexes in Desulfovibrio vulgaris

    SciTech Connect

    Dong,, Ming; Han, Bong-Gyoon; Liu, Hui-Hai; Malik, J.; Geller, Jil; Yang, Li; Choi, M.; Chandonia, John-Marc; Arbelaez, Pablo; Sterling, H. J.; Typke, Dieter; Shatsky, Max; Brenner, Steve; Fisher, Susan; Williams, Evan; Szakal, Evelin; Allen, S.; Hall, S. C.; Hazen, Terry; Witkowska, H. E.; Jin, Jiming; Glaeser, Robert; Biggin, Mark

    2010-05-17

    Our scheme for the tagless purification of water soluble complexes. 10 g of protein from a crude bacterial extract is first fractionated by ammonium sulfate precipitation and then by a series of chromatographic steps: anion exchange (IEX), hydrophobic interaction (HIC), and finally size exclusion (Gel Filtration). Fractions from the last chromatography step are trypsin digested and peptides labeled with iTRAQ reagents to allow multiplexing and quantitation during mass spectrometric analysis. Elution profiles of identified proteins are then subjected to clustering analysis.

  17. Isolation of Endogenously Assembled RNA-Protein Complexes Using Affinity Purification Based on Streptavidin Aptamer S1

    PubMed Central

    Dong, Yangchao; Yang, Jing; Ye, Wei; Wang, Yuan; Ye, Chuantao; Weng, Daihui; Gao, Huan; Zhang, Fanglin; Xu, Zhikai; Lei, Yingfeng

    2015-01-01

    Efficient isolation of endogenously assembled viral RNA-protein complexes is essential for understanding virus replication mechanisms. We have developed an affinity purification strategy based on an RNA affinity tag that allows large-scale preparation of native viral RNA-binding proteins (RBPs). The streptavidin-binding aptamer S1 sequence was inserted into the 3′ end of dengue virus (DENV) 5′–3′ UTR RNA, and the DENV RNA UTR fused to the S1 RNA aptamer was expressed in living mammalian cells. This allowed endogenous viral ribonucleoprotein (RNP) assembly and isolation of RNPs from whole cell extract, through binding the S1 aptamer to streptavidin magnetic beads. Several novel host DENV RBPs were subsequently identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS), including RPS8, which we further implicate in DENV replication. We proposed efficient S1 aptamer-based isolation of viral assembled RNPs from living mammalian cells will be generally applicable to the purification of high- and low-affinity RBPs and RNPs under endogenous conditions. PMID:26389898

  18. Isolation of Endogenously Assembled RNA-Protein Complexes Using Affinity Purification Based on Streptavidin Aptamer S1.

    PubMed

    Dong, Yangchao; Yang, Jing; Ye, Wei; Wang, Yuan; Ye, Chuantao; Weng, Daihui; Gao, Huan; Zhang, Fanglin; Xu, Zhikai; Lei, Yingfeng

    2015-09-16

    Efficient isolation of endogenously assembled viral RNA-protein complexes is essential for understanding virus replication mechanisms. We have developed an affinity purification strategy based on an RNA affinity tag that allows large-scale preparation of native viral RNA-binding proteins (RBPs). The streptavidin-binding aptamer S1 sequence was inserted into the 3' end of dengue virus (DENV) 5'-3' UTR RNA, and the DENV RNA UTR fused to the S1 RNA aptamer was expressed in living mammalian cells. This allowed endogenous viral ribonucleoprotein (RNP) assembly and isolation of RNPs from whole cell extract, through binding the S1 aptamer to streptavidin magnetic beads. Several novel host DENV RBPs were subsequently identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS), including RPS8, which we further implicate in DENV replication. We proposed efficient S1 aptamer-based isolation of viral assembled RNPs from living mammalian cells will be generally applicable to the purification of high- and low-affinity RBPs and RNPs under endogenous conditions.

  19. Optimisation of Downscaled Tandem Affinity Purifications to Identify Core Protein Complexes

    PubMed Central

    Haura, Eric B.; Sacco, Roberto; Li, Jiannong; Müller, André C.; Grebien, Florian; Superti-Furga, Giulio; Bennett, Keiryn L.

    2013-01-01

    In this study we show that via stable, retroviral-expression of tagged EGFR del (L747-S752 deletion mutant) in the PC9 lung cancer cell line and stable doxycycline-inducible expression of tagged Grb2 using a Flp-mediated recombination HEK293 cell system, the SH-TAP can be downscaled to 5 to 12.5 mg total protein input (equivalent to 0.5 - 1 × 15 cm culture plate or 4 - 8 × 106 cells). The major constituents of the EGFR del complex (USB3B, GRB2, ERRFI, HSP7C, GRP78, HSP71) and the Grb2 complex (ARHG5, SOS1, ARG35, CBL, CBLB, PTPRA, SOS2, DYN2, WIPF2, IRS4) were identified. Adjustment of the quantity of digested protein injected into the mass spectrometer reveals that optimisation is required as high quantities of material led to a decrease in protein sequence coverage and the loss of some interacting proteins. This investigation should aid other researchers in performing tandem affinity purifications in general, and in particular, from low quantities of input material. PMID:24077984

  20. Isolation of a High-Affinity Functional Protein Complex between OmcA and MtrC: Two Outer Membrane Decaheme c-type Cytochromes of Shewanella oneidensis MR-1

    SciTech Connect

    Shi, Liang; Chen, Baowei; Wang, Zheming; Elias, Dwayne A.; Mayer, M. Uljana; Gorby, Yuri A.; Ni, Shuisong; Lower, Brian H.; Kennedy, David W.; Wunschel, David S.; Mottaz, Heather M.; Marshall, Matthew J.; Hill, Eric A.; Beliaev, Alex S.; Zachara, John M.; Fredrickson, Jim K.; Squier, Thomas C.

    2006-07-01

    SUMMARY Shewanella oneidensis MR-1 is a facultatively anaerobic bacterium that is capable of using insoluble oxidized metals, such as manganese [Mn(III, IV)] and iron [Fe(III)] oxides and oxyhydroxides, as terminal electron acceptors during anaerobic respiration. The ability of S. oneidensis MR-1 to reduce oxidized Mn and/or Fe has previously been linked to OmcA and MtrC: two decaheme c-type cytochromes that are localized to the outer membrane. To investigate how the electron transport proteins OmcA and MtrC are organized, we expressed and purified recombinant OmcA and MtrC from wild type S. oneidensis MR-1 as well as a mutant that lacked OmcA and MtrC (ΔomcA/mtrC). After purification to the nearly electrophoretic homogeneity from the ΔomcA/mtrC mutant, the recombinant OmcA and MtrC exhibited the characteristics of c-type cytochromes, and each of their polypeptides was confirmed to contain 10 hemes. When purified from wild type cells, endogenous MtrC or OmcA was always co-purified with recombinant OmcA or MtrC, respectively. Fluorescence polarization experiment showed that recombinant OmcA bound to the FlAsH-labeled MtrC with a dissociation constant of 7 ×10-7 M. The purified recombinant OmcA or MtrC alone displayed intrinsic ferric reductase activity with NADH used as an electron donor. Ferric reductase specific activity increased by 35 to 41% when nearly equimolar concentrations of OmcA and MtrC were assayed relative to the two proteins assayed independently. These results demonstrate that OmcA and MtrC directly interact with each other to form a stable complex with high ferric reductase activity.

  1. Pigment-protein complexes

    SciTech Connect

    Siegelman, H W

    1980-01-01

    The photosynthetically-active pigment protein complexes of procaryotes and eucaryotes include chlorophyll proteins, carotenochlorophyll proteins, and biliproteins. They are either integral components or attached to photosynthetic membranes. Detergents are frequently required to solubilize the pigment-protein complexes. The membrane localization and detergent solubilization strongly suggest that the pigment-protein complexes are bound to the membranes by hydrophobic interactions. Hydrophobic interactions of proteins are characterized by an increase in entropy. Their bonding energy is directly related to temperature and ionic strength. Hydrophobic-interaction chromatography, a relatively new separation procedure, can furnish an important method for the purification of pigment-protein complexes. Phycobilisome purification and properties provide an example of the need to maintain hydrophobic interactions to preserve structure and function.

  2. Purification and partial characterization of a lectin protein complex, the clathrilectin, from the calcareous sponge Clathrina clathrus.

    PubMed

    Gardères, Johan; Domart-Coulon, Isabelle; Marie, Arul; Hamer, Bojan; Batel, Renato; Müller, Werner E G; Bourguet-Kondracki, Marie-Lise

    2016-10-01

    Carbohydrate-binding proteins were purified from the marine calcareous sponge Clathrina clathrus via affinity chromatography on lactose and N-acetyl glucosamine-agarose resins. Proteomic analysis of acrylamide gel separated protein subunits obtained in reducing conditions pointed out several candidates for lectins. Based on amino-acid sequence similarity, two peptides displayed homology with the jack bean lectin Concanavalin A, including a conserved domain shared by proteins in the L-type lectin superfamily. An N-acetyl glucosamine - binding protein complex, named clathrilectin, was further purified via gel filtration chromatography, bioguided with a diagnostic rabbit erythrocyte haemagglutination assay, and its activity was found to be calcium dependent. Clathrilectin, a protein complex of 3200kDa estimated by gel filtration, is composed of monomers with apparent molecular masses of 208 and 180kDa estimated on 10% SDS-PAGE. Nine internal peptides were identified using proteomic analyses, and compared to protein libraries from the demosponge Amphimedon queenslandica and a calcareous sponge Sycon sp. from the Adriatic Sea. The clathrilectin is the first lectin isolated from a calcareous sponge and displays homologies with predicted sponge proteins potentially involved in cell aggregation and interaction with bacteria. PMID:27113336

  3. Biochemical isolation of Argonaute protein complexes by Ago-APP

    PubMed Central

    Hauptmann, Judith; Schraivogel, Daniel; Bruckmann, Astrid; Manickavel, Sudhir; Jakob, Leonhard; Eichner, Norbert; Pfaff, Janina; Urban, Marc; Sprunck, Stefanie; Hafner, Markus; Tuschl, Thomas; Deutzmann, Rainer; Meister, Gunter

    2015-01-01

    During microRNA (miRNA)-guided gene silencing, Argonaute (Ago) proteins interact with a member of the TNRC6/GW protein family. Here we used a short GW protein-derived peptide fused to GST and demonstrate that it binds to Ago proteins with high affinity. This allows for the simultaneous isolation of all Ago protein complexes expressed in diverse species to identify associated proteins, small RNAs, or target mRNAs. We refer to our method as “Ago protein Affinity Purification by Peptides“ (Ago-APP). Furthermore, expression of this peptide competes for endogenous TNRC6 proteins, leading to global inhibition of miRNA function in mammalian cells. PMID:26351695

  4. Extraction of functionally active photosystem 2 pigment-protein complexes from pea thylakoids and their purification on Sepharose DEAE 6B.

    PubMed

    Khristin, M S; Nikitishena, O V; Smolova, T N; Zastrizhnaya, O M

    1997-01-01

    A method for selective extraction of photosystem 2 (PS 2) pigment-protein complexes (PPC) from pea thylakoids and their purification from Sepharose DEAE 6B was developed. It was shown that extraction of thylakoids (3 mg chlorophyll/ml) with Triton X-100 (Triton X-100:chlorophyll = 15-20:1 w/w) in the presence of 1 M sucrose, 1 M NaCl, 50 mM MES in the solubilization medium (pH 5.0), tenfold dilution with 50 mM MES buffer (pH 5.0), and centrifugation at 16,000 g for 10 min provided selective extraction of PS 2 PPC. Spectral, electrophoretic and functional properties of the isolated PPC were determined. This method allowed us to isolate native pigment-protein oxygen-evolving complexes (core complexes) and D1-D2-cytochrome b559 complexes of the reaction centre (RC). The core complex immobilized on a Sepharose DEAE 6B column was treated with 2 mM hydroxylamine or 1 M CaCl2. In the former case, modification by hydroxylamine led to the removal of 4 atoms of Mn, but not of the 33-kDa protein; in the latter case, modification by CaCl2 led to the removal of the 33 kDa protein. The modified core complexes were unstable upon native electrophoresis in the presence of n-dodecyl-beta-d-maltoside and deriphat-160. We suggest that the structural organization of the Mn cluster and 33 kDa protein stabilized the core complex and increased its stability upon the action of the detergents. PMID:9354396

  5. The Center for Optimized Structural Studies (COSS) platform for automation in cloning, expression, and purification of single proteins and protein-protein complexes.

    PubMed

    Mlynek, Georg; Lehner, Anita; Neuhold, Jana; Leeb, Sarah; Kostan, Julius; Charnagalov, Alexej; Stolt-Bergner, Peggy; Djinović-Carugo, Kristina; Pinotsis, Nikos

    2014-06-01

    Expression in Escherichia coli represents the simplest and most cost effective means for the production of recombinant proteins. This is a routine task in structural biology and biochemistry where milligrams of the target protein are required in high purity and monodispersity. To achieve these criteria, the user often needs to screen several constructs in different expression and purification conditions in parallel. We describe a pipeline, implemented in the Center for Optimized Structural Studies, that enables the systematic screening of expression and purification conditions for recombinant proteins and relies on a series of logical decisions. We first use bioinformatics tools to design a series of protein fragments, which we clone in parallel, and subsequently screen in small scale for optimal expression and purification conditions. Based on a scoring system that assesses soluble expression, we then select the top ranking targets for large-scale purification. In the establishment of our pipeline, emphasis was put on streamlining the processes such that it can be easily but not necessarily automatized. In a typical run of about 2 weeks, we are able to prepare and perform small-scale expression screens for 20-100 different constructs followed by large-scale purification of at least 4-6 proteins. The major advantage of our approach is its flexibility, which allows for easy adoption, either partially or entirely, by any average hypothesis driven laboratory in a manual or robot-assisted manner.

  6. Mass Spectrometry of Intact Membrane Protein Complexes

    PubMed Central

    Laganowsky, Arthur; Reading, Eamonn; Hopper, Jonathan T.S.; Robinson, Carol V.

    2014-01-01

    Mass spectrometry of intact soluble protein complexes has emerged as a powerful technique to study the stoichiometry, structure-function and dynamics of protein assemblies. Recent developments have extended this technique to the study of membrane protein complexes where it has already revealed subunit stoichiometries and specific phospholipid interactions. Here, we describe a protocol for mass spectrometry of membrane protein complexes. The protocol begins with preparation of the membrane protein complex enabling not only the direct assessment of stoichiometry, delipidation, and quality of the target complex, but also evaluation of the purification strategy. A detailed list of compatible non-ionic detergents is included, along with a protocol for screening detergents to find an optimal one for mass spectrometry, biochemical and structural studies. This protocol also covers the preparation of lipids for protein-lipid binding studies and includes detailed settings for a Q-ToF mass spectrometer after introduction of complexes from gold-coated nanoflow capillaries. PMID:23471109

  7. Protein Complexes in Bacteria

    PubMed Central

    Caufield, J. Harry; Abreu, Marco; Wimble, Christopher; Uetz, Peter

    2015-01-01

    Large-scale analyses of protein complexes have recently become available for Escherichia coli and Mycoplasma pneumoniae, yielding 443 and 116 heteromultimeric soluble protein complexes, respectively. We have coupled the results of these mass spectrometry-characterized protein complexes with the 285 “gold standard” protein complexes identified by EcoCyc. A comparison with databases of gene orthology, conservation, and essentiality identified proteins conserved or lost in complexes of other species. For instance, of 285 “gold standard” protein complexes in E. coli, less than 10% are fully conserved among a set of 7 distantly-related bacterial “model” species. Complex conservation follows one of three models: well-conserved complexes, complexes with a conserved core, and complexes with partial conservation but no conserved core. Expanding the comparison to 894 distinct bacterial genomes illustrates fractional conservation and the limits of co-conservation among components of protein complexes: just 14 out of 285 model protein complexes are perfectly conserved across 95% of the genomes used, yet we predict more than 180 may be partially conserved across at least half of the genomes. No clear relationship between gene essentiality and protein complex conservation is observed, as even poorly conserved complexes contain a significant number of essential proteins. Finally, we identify 183 complexes containing well-conserved components and uncharacterized proteins which will be interesting targets for future experimental studies. PMID:25723151

  8. Development of an efficient E. coli expression and purification system for a catalytically active, human Cullin3-RINGBox1 protein complex and elucidation of its quaternary structure with Keap1

    SciTech Connect

    Small, Evan; Eggler, Aimee; Mesecar, Andrew D.

    2010-10-01

    Research highlights: {yields} A novel expression strategy was used to purify Cul3-Rbx1 from E. coli. {yields} The Cul3-Rbx1 complex is fully active and catalyzes ubiquitination of Nrf2 in vitro. {yields} Cul3, Rbx1, and Keap1 form a complex with unique stoichiometry of 1:1:2. -- Abstract: The Cullin3-based E3 ubiquitin ligase complex is thought to play an important role in the cellular response to oxidative stress and xenobiotic assault. While limited biochemical studies of the ligase's role in these complex signaling pathways are beginning to emerge, structural studies are lagging far behind due to the inability to acquire sufficient quantities of full-length, highly pure and active Cullin3. Here we describe the design and construction of an optimized expression and purification system for the full-length, human Cullin3-RINGBox 1 (Rbx1) protein complex from Escherichia coli. The dual-expression system is comprised of codon-optimized Cullin3 and Rbx1 genes co-expressed from a single pET-Duet-1 plasmid. Rapid purification of the Cullin3-Rbx1 complex is achieved in two steps via an affinity column followed by size-exclusion chromatography. Approximately 15 mg of highly pure and active Cullin3-Rbx1 protein from 1 L of E. coli culture can be achieved. Analysis of the quaternary structure of the Cullin3-Rbx1 and Cullin3-Rbx1-Keap1 complexes by size-exclusion chromatography and analytical ultracentrifugation indicates a 1:1 stoichiometry for the Cullin3-Rbx1 complex (MW = 111 kDa), and a 1:1:2 stoichiometry for the Cullin3-Rbx1-Keap1 complex (MW = 280 kDa). This latter complex has a novel quaternary structural organization for cullin E3 ligases, and it is fully active based on an in vitro Cullin3-Rbx1-Keap1-Nrf2 ubiquitination activity assay that was developed and optimized in this study.

  9. High-affinity neuropeptide Y receptor antagonists.

    PubMed Central

    Daniels, A J; Matthews, J E; Slepetis, R J; Jansen, M; Viveros, O H; Tadepalli, A; Harrington, W; Heyer, D; Landavazo, A; Leban, J J

    1995-01-01

    Neuropeptide Y (NPY) is one of the most abundant peptide transmitters in the mammalian brain. In the periphery it is costored and coreleased with norepinephrine from sympathetic nerve terminals. However, the physiological functions of this peptide remain unclear because of the absence of specific high-affinity receptor antagonists. Three potent NPY receptor antagonists were synthesized and tested for their biological activity in in vitro, ex vivo, and in vivo functional assays. We describe here the effects of these antagonists inhibiting specific radiolabeled NPY binding at Y1 and Y2 receptors and antagonizing the effects of NPY in human erythroleukemia cell intracellular calcium mobilization perfusion pressure in the isolated rat kidney, and mean arterial blood pressure in anesthetized rats. PMID:7568074

  10. Solubilization and partial characterization of a microsomal high affinity GTPase

    SciTech Connect

    Nicchitta, C.; Williamson, J.R.

    1987-05-01

    Isolated rat liver microsomes release sequestered Ca/sup 2 +/ following addition of GTP. In contrast to permeabilized cells, GTP dependent microsomal Ca/sup 2 +/ release requires low concentrations of polyethylene glycol (PEG). They have identified a microsomal, PEG-sensitive high affinity GTPase which shares a number of characteristics with the GTP-dependent Ca/sup 2 +/ release system. To aid in further characterization of this activity they have initiated studies on the solubilization and purification of the microsomal GTPases. When microsomes are solubilized under the following conditions (150 mM NaCl, 5 mg protein/ml, 1% Triton X-114) PEG sensitive GTPase activity selectively partitions into the detergent rich phase of the Triton X-114 extract. As observed in intact microsomal membranes the Triton X-114 soluble GTPase is maximally stimulated by 3% PEG. Half maximal stimulation is observed at 1% PEG. PEG increases the Vmax of this activity; no effects on Km were observed. The Km for GTP of the detergent soluble GTPase is 5 ..mu..M. This GTPase is sensitive to inhibition by sulfhydryl reagents. PEG-sensitive GTPase activity was completely inhibited in the presence of 25 ..mu..M p-hydroxymercuribenzoate (PHMB); half maximal inhibition was observed at 5 ..mu..M. Labeling of the Triton X-114 extract with the photosensitive compound (/sup 32/P) 8-azido GTP indicated the presence of two prominent GTP binding proteins of approximate molecular weights 17 and 54 kD.

  11. RNA mango aptamer-fluorophore: a bright, high-affinity complex for RNA labeling and tracking.

    PubMed

    Dolgosheina, Elena V; Jeng, Sunny C Y; Panchapakesan, Shanker Shyam S; Cojocaru, Razvan; Chen, Patrick S K; Wilson, Peter D; Hawkins, Nancy; Wiggins, Paul A; Unrau, Peter J

    2014-10-17

    Because RNA lacks strong intrinsic fluorescence, it has proven challenging to track RNA molecules in real time. To address this problem and to allow the purification of fluorescently tagged RNA complexes, we have selected a high affinity RNA aptamer called RNA Mango. This aptamer binds a series of thiazole orange (fluorophore) derivatives with nanomolar affinity, while increasing fluorophore fluorescence by up to 1,100-fold. Visualization of RNA Mango by single-molecule fluorescence microscopy, together with injection and imaging of RNA Mango/fluorophore complex in C. elegans gonads demonstrates the potential for live-cell RNA imaging with this system. By inserting RNA Mango into a stem loop of the bacterial 6S RNA and biotinylating the fluorophore, we demonstrate that the aptamer can be used to simultaneously fluorescently label and purify biologically important RNAs. The high affinity and fluorescent properties of RNA Mango are therefore expected to simplify the study of RNA complexes. PMID:25101481

  12. Reconstitution of high-affinity opioid agonist binding in brain membranes

    SciTech Connect

    Remmers, A.E.; Medzihradsky, F. )

    1991-03-15

    In synaptosomal membranes from rat brain cortex, the {mu} selective agonist ({sup 3}H)dihydromorphine in the absence of sodium, and the nonselective antagonist ({sup 3}H)naltrexone in the presence of sodium, bound to two populations of opioid receptor sites with K{sub d} values of 0.69 and 8.7 nM for dihydromorphine, and 0.34 and 5.5 nM for naltrexone. The addition of 5 {mu}M guanosine 5{prime}-({gamma}-thio)triphosphate (GTP({gamma}S)) strongly reduced high-affinity agonist but not antagonist binding. Exposure of the membranes to high pH reduced the number of GTP({gamma}-{sup 35}S) binding sites by 90% and low K{sub m}, opioid-sensitive GTPase activity by 95%. In these membranes, high-affinity agonist binding was abolished and modulation of residual binding by GTP({gamma}S) was diminished. Alkali treatment of the glioma cell membranes prior to fusion inhibited most of the low K{sub m} GTPase activity and prevented the reconstitution of agonist binding. The results show that high-affinity opioid agonist binding reflects the ligand-occupied receptor - guanine nucleotide binding protein complex.

  13. Monochromatic multicomponent fluorescence sedimentation velocity for the study of high-affinity protein interactions

    PubMed Central

    Zhao, Huaying; Fu, Yan; Glasser, Carla; Andrade Alba, Eric J; Mayer, Mark L; Patterson, George; Schuck, Peter

    2016-01-01

    The dynamic assembly of multi-protein complexes underlies fundamental processes in cell biology. A mechanistic understanding of assemblies requires accurate measurement of their stoichiometry, affinity and cooperativity, and frequently consideration of multiple co-existing complexes. Sedimentation velocity analytical ultracentrifugation equipped with fluorescence detection (FDS-SV) allows the characterization of protein complexes free in solution with high size resolution, at concentrations in the nanomolar and picomolar range. Here, we extend the capabilities of FDS-SV with a single excitation wavelength from single-component to multi-component detection using photoswitchable fluorescent proteins (psFPs). We exploit their characteristic quantum yield of photo-switching to imprint spatio-temporal modulations onto the sedimentation signal that reveal different psFP-tagged protein components in the mixture. This novel approach facilitates studies of heterogeneous multi-protein complexes at orders of magnitude lower concentrations and for higher-affinity systems than previously possible. Using this technique we studied high-affinity interactions between the amino-terminal domains of GluA2 and GluA3 AMPA receptors. DOI: http://dx.doi.org/10.7554/eLife.17812.001 PMID:27436096

  14. Monochromatic multicomponent fluorescence sedimentation velocity for the study of high-affinity protein interactions.

    PubMed

    Zhao, Huaying; Fu, Yan; Glasser, Carla; Andrade Alba, Eric J; Mayer, Mark L; Patterson, George; Schuck, Peter

    2016-01-01

    The dynamic assembly of multi-protein complexes underlies fundamental processes in cell biology. A mechanistic understanding of assemblies requires accurate measurement of their stoichiometry, affinity and cooperativity, and frequently consideration of multiple co-existing complexes. Sedimentation velocity analytical ultracentrifugation equipped with fluorescence detection (FDS-SV) allows the characterization of protein complexes free in solution with high size resolution, at concentrations in the nanomolar and picomolar range. Here, we extend the capabilities of FDS-SV with a single excitation wavelength from single-component to multi-component detection using photoswitchable fluorescent proteins (psFPs). We exploit their characteristic quantum yield of photo-switching to imprint spatio-temporal modulations onto the sedimentation signal that reveal different psFP-tagged protein components in the mixture. This novel approach facilitates studies of heterogeneous multi-protein complexes at orders of magnitude lower concentrations and for higher-affinity systems than previously possible. Using this technique we studied high-affinity interactions between the amino-terminal domains of GluA2 and GluA3 AMPA receptors. PMID:27436096

  15. Proteomics-Based Analysis of Protein Complexes in Pluripotent Stem Cells and Cancer Biology

    PubMed Central

    Sudhir, Putty-Reddy; Chen, Chung-Hsuan

    2016-01-01

    A protein complex consists of two or more proteins that are linked together through protein–protein interactions. The proteins show stable/transient and direct/indirect interactions within the protein complex or between the protein complexes. Protein complexes are involved in regulation of most of the cellular processes and molecular functions. The delineation of protein complexes is important to expand our knowledge on proteins functional roles in physiological and pathological conditions. The genetic yeast-2-hybrid method has been extensively used to characterize protein-protein interactions. Alternatively, a biochemical-based affinity purification coupled with mass spectrometry (AP-MS) approach has been widely used to characterize the protein complexes. In the AP-MS method, a protein complex of a target protein of interest is purified using a specific antibody or an affinity tag (e.g., DYKDDDDK peptide (FLAG) and polyhistidine (His)) and is subsequently analyzed by means of MS. Tandem affinity purification, a two-step purification system, coupled with MS has been widely used mainly to reduce the contaminants. We review here a general principle for AP-MS-based characterization of protein complexes and we explore several protein complexes identified in pluripotent stem cell biology and cancer biology as examples. PMID:27011181

  16. Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice

    NASA Astrophysics Data System (ADS)

    de Boer, Ernie; Rodriguez, Patrick; Bonte, Edgar; Krijgsveld, Jeroen; Katsantoni, Eleni; Heck, Albert; Grosveld, Frank; Strouboulis, John

    2003-06-01

    Proteomic approaches require simple and efficient protein purification methodologies that are amenable to high throughput. Biotinylation is an attractive approach for protein complex purification due to the very high affinity of avidin/streptavidin for biotinylated templates. Here, we describe an approach for the single-step purification of transcription factor complex(es) based on specific in vivo biotinylation. We expressed the bacterial BirA biotin ligase in mammalian cells and demonstrated very efficient biotinylation of a hematopoietic transcription factor bearing a small (23-aa) artificial peptide tag. Biotinylation of the tagged transcription factor altered neither the factor's protein interactions or DNA binding properties in vivo nor its subnuclear distribution. Using this approach, we isolated the biotin-tagged transcription factor and at least one other known interacting protein from crude nuclear extracts by direct binding to streptavidin beads. Finally, this method works efficiently in transgenic mice, thus raising the prospect of using biotinylation tagging in protein complex purification directly from animal tissues. Therefore, BirA-mediated biotinylation of tagged proteins provides the basis for the single-step purification of proteins from mammalian cells.

  17. Blotting protein complexes from native gels to electron microscopy grids.

    PubMed

    Knispel, Roland Wilhelm; Kofler, Christine; Boicu, Marius; Baumeister, Wolfgang; Nickell, Stephan

    2012-01-08

    We report a simple and generic method for the direct transfer of protein complexes separated by native gel electrophoresis to electron microscopy grids. After transfer, sufficient material remains in the gel for identification and characterization by mass spectrometry. The method should facilitate higher-throughput single-particle analysis by substantially reducing the time needed for protein purification, as demonstrated for three complexes from Thermoplasma acidophilum.

  18. High affinity retinoic acid receptor antagonists: analogs of AGN 193109.

    PubMed

    Johnson, A T; Wang, L; Gillett, S J; Chandraratna, R A

    1999-02-22

    A series of high affinity retinoic acid receptor (RAR) antagonists were prepared based upon the known antagonist AGN 193109 (2). Introduction of various phenyl groups revealed a preference for substitution at the para-position relative to the meta-site. Antagonists with the highest affinities for the RARs possessed hydrophobic groups, however, the presence of polar functionality was also well tolerated.

  19. Affinity proteomics to study endogenous protein complexes: Pointers, pitfalls, preferences and perspectives

    PubMed Central

    LaCava, John; Molloy, Kelly R.; Taylor, Martin S.; Domanski, Michal; Chait, Brian T.; Rout, Michael P.

    2015-01-01

    Dissecting and studying cellular systems requires the ability to specifically isolate distinct proteins along with the co-assembled constituents of their associated complexes. Affinity capture techniques leverage high affinity, high specificity reagents to target and capture proteins of interest along with specifically associated proteins from cell extracts. Affinity capture coupled to mass spectrometry (MS)-based proteomic analyses has enabled the isolation and characterization of a wide range of endogenous protein complexes. Here, we outline effective procedures for the affinity capture of protein complexes, highlighting best practices and common pitfalls. PMID:25757543

  20. High affinity ligands from in vitro selection: Complex targets

    PubMed Central

    Morris, Kevin N.; Jensen, Kirk B.; Julin, Carol M.; Weil, Michael; Gold, Larry

    1998-01-01

    Human red blood cell membranes were used as a model system to determine if the systematic evolution of ligands by exponential enrichment (SELEX) methodology, an in vitro protocol for isolating high-affinity oligonucleotides that bind specifically to virtually any single protein, could be used with a complex mixture of potential targets. Ligands to multiple targets were generated simultaneously during the selection process, and the binding affinities of these ligands for their targets are comparable to those found in similar experiments against pure targets. A secondary selection scheme, deconvolution-SELEX, facilitates rapid isolation of the ligands to targets of special interest within the mixture. SELEX provides high-affinity compounds for multiple targets in a mixture and might allow a means for dissecting complex biological systems. PMID:9501188

  1. Selective high affinity polydentate ligands and methods of making such

    DOEpatents

    DeNardo, Sally; DeNardo, Gerald; Balhorn, Rodney

    2010-02-16

    This invention provides novel polydentate selective high affinity ligands (SHALs) that can be used in a variety of applications in a manner analogous to the use of antibodies. SHALs typically comprise a multiplicity of ligands that each bind different region son the target molecule. The ligands are joined directly or through a linker thereby forming a polydentate moiety that typically binds the target molecule with high selectivity and avidity.

  2. Integrating Mass Spectrometry of Intact Protein Complexes into Structural Proteomics

    PubMed Central

    Hyung, Suk-Joon; Ruotolo, Brandon T.

    2013-01-01

    Summary Mass spectrometry analysis of intact protein complexes has emerged as an established technology for assessing the composition and connectivity within dynamic, heterogeneous multiprotein complexes at low concentrations and in the context of mixtures. As this technology continues to move forward, one of the main challenges is to integrate the information content of such intact protein complex measurements with other mass spectrometry approaches in structural biology. Methods such as H/D exchange, oxidative foot-printing, chemical cross-linking, affinity purification, and ion mobility separation add complementary information that allows access to every level of protein structure and organization. Here, we survey the structural information that can be retrieved by such experiments, demonstrate the applicability of integrative mass spectrometry approaches in structural proteomics, and look to the future to explore upcoming innovations in this rapidly-advancing area. PMID:22611037

  3. High affinity of water-soluble cryptophanes for cesium cations.

    PubMed

    Brotin, Thierry; Montserret, Roland; Bouchet, Aude; Cavagnat, Dominique; Linares, Mathieu; Buffeteau, Thierry

    2012-01-20

    Exceptionally high affinity for cesium cations was achieved in aqueous solution using two enantiopure cryptophanes. Complexation of cesium was evidenced by (133)Cs NMR spectroscopy and by electronic circular dichroism (ECD). Binding constants as high as 6 × 10(9) M(-1) have been measured by isothermal titration calorimetry (ITC). Very strong complexation of rubidium cations (K ~10(6) M(-1)) has also been measured. Chiral hosts allowed the detection of the two cations at low concentrations (μM) using ECD.

  4. Cationic polymer brush-modified cellulose nanocrystals for high-affinity virus binding

    NASA Astrophysics Data System (ADS)

    Rosilo, Henna; McKee, Jason R.; Kontturi, Eero; Koho, Tiia; Hytönen, Vesa P.; Ikkala, Olli; Kostiainen, Mauri A.

    2014-09-01

    Surfaces capable of high-affinity binding of biomolecules are required in several biotechnological applications, such as purification, transfection, and sensing. Therein, the rod-shaped, colloidal cellulose nanocrystals (CNCs) are appealing due to their large surface area available for functionalization. In order to exploit electrostatic binding, their intrinsically anionic surfaces have to be cationized as biological supramolecules are predominantly anionic. Here we present a facile way to prepare cationic CNCs by surface-initiated atom-transfer radical polymerization of poly(N,N-dimethylaminoethyl methacrylate) and subsequent quaternization of the polymer pendant amino groups. The cationic polymer brush-modified CNCs maintained excellent dispersibility and colloidal stability in water and showed a ζ-potential of +38 mV. Dynamic light scattering and electron microscopy showed that the modified CNCs electrostatically bind cowpea chlorotic mottle virus and norovirus-like particles with high affinity. Addition of only a few weight percent of the modified CNCs in water dispersions sufficed to fully bind the virus capsids to form micrometer-sized assemblies. This enabled the concentration and extraction of the virus particles from solution by low-speed centrifugation. These results show the feasibility of the modified CNCs in virus binding and concentrating, and pave the way for their use as transduction enhancers for viral delivery applications.Surfaces capable of high-affinity binding of biomolecules are required in several biotechnological applications, such as purification, transfection, and sensing. Therein, the rod-shaped, colloidal cellulose nanocrystals (CNCs) are appealing due to their large surface area available for functionalization. In order to exploit electrostatic binding, their intrinsically anionic surfaces have to be cationized as biological supramolecules are predominantly anionic. Here we present a facile way to prepare cationic CNCs by surface

  5. 01-ERD-111 - The Development of Synthetic High Affinity Ligands

    SciTech Connect

    Perkins, J; Balhorn, R; Cosman, M; Lightstone, F; Zeller, L

    2004-02-05

    The aim of this project was to develop Synthetic High-Affinity Ligands (SHALs), which bind with high affinity and specificity to proteins of interest for national security and cancer therapy applications. The aim of producing synthetic ligands for sensory devices as an alternative to antibody-based detection assays and therapeutic agents is to overcome the drawbacks associated with antibody-based in next-generation sensors and systems. The focus area of the project was the chemical synthesis of the SHALs. The project concentrated on two different protein targets. (a) The C fragment of tetanus and botulinum toxin, potential biowarfare agents. A SHAL for tetanus or botulinum toxin would be incorporated into a sensory device for the toxins. (b) HLA-DR10, a protein found in high abundance on the surface of Non-Hodgkins Lymphoma. A SHAL specific to a tumor marker, labeled with a radionuclide, would enable the targeted delivery of radiation therapy to metastatic disease. The technical approach used to develop a SHAL for each protein target will be described in more detail below. However, in general, the development of a SHAL requires a combination of computational modeling techniques, modern nuclear magnetic resonance spectroscopy (NMR) and synthetic chemistry.

  6. Paracetamol and cytarabine binding competition in high affinity binding sites of transporting protein

    NASA Astrophysics Data System (ADS)

    Sułkowska, A.; Bojko, B.; Równicka, J.; Sułkowski, W. W.

    2006-07-01

    Paracetamol (acetaminophen, AA) the most popular analgesic drug is commonly used in the treatment of pain in patients suffering from cancer. In our studies, we evaluated the competition in binding with serum albumin between paracetamol (AA) and cytarabine, antyleukemic drug (araC). The presence of one drug can alter the binding affinity of albumin towards the second one. Such interaction can result in changing of the free fraction of the one of these drugs in blood. Two spectroscopic methods were used to determine high affinity binding sites and the competition of the drugs. Basing on the change of the serum albumin fluorescence in the presence of either of the drugs the quenching ( KQ) constants for the araC-BSA and AA-BSA systems were calculated. Analysis of UV difference spectra allowed us to describe the changes in drug-protein complexes (araC-albumin and AA-albumin) induced by the presence of the second drug (AA and araC, respectively). The mechanism of competition between araC and AA has been proposed.

  7. Complex high affinity interactions occur between MHCI and superantigens

    NASA Technical Reports Server (NTRS)

    Chapes, S. K.; Herpich, A. R.; Spooner, B. S. (Principal Investigator)

    1998-01-01

    Staphylococcal enterotoxins A and C1 (SEA or SEC1) bound to major histocompatibility-I (MHCI) molecules with high affinity (binding constants ranging from 1.1 microM to 79 nM). SEA and SEC1 directly bound MHCI molecules that had been captured by monoclonal antibodies specific for H-2Kk, H-2Dk, or both. In addition, MHCI-specific antibodies inhibited the binding of SEC1 to LM929 cells and SEA competitively inhibited SEC1 binding; indicating that the superantigens bound to MHCI on the cell surface. The affinity and number of superantigen binding sites differed depending on whether MHCI was expressed in the membrane of LM929 cells or whether it was captured. These data support the hypothesis that MHCI molecules can serve as superantigen receptors.

  8. Interactions by 2D Gel Electrophoresis Overlap (iGEO): a novel high fidelity approach to identify constituents of protein complexes

    PubMed Central

    2013-01-01

    Background Here we describe a novel approach used to identify the constituents of protein complexes with high fidelity, using the integrin-associated scaffolding protein PINCH as a test case. PINCH is comprised of five LIM domains, zinc-finger protein interaction modules. In Drosophila melanogaster, PINCH has two known high-affinity binding partners—Integrin-linked kinase (ILK) that binds to LIM1 and Ras Suppressor 1 (RSU1) that binds to LIM5—but has been postulated to bind additional proteins as well. Results To purify PINCH complexes, in parallel we fused different affinity tags (Protein A and Flag) to different locations within the PINCH sequence (N- and C-terminus). We expressed these tagged versions of PINCH both in cell culture (overexpressed in Drosophila S2 cell culture in the presence of endogenous PINCH) and in vivo (at native levels in Drosophila lacking endogenous PINCH). After affinity purification, we analyzed PINCH complexes by a novel 2D-gel electrophoresis analysis, iGEO (interactions by 2D Gel Electrophoresis Overlap), with mass spectrometric identification of individual spots of interest. iGEO allowed the identification of protein partners that associate with PINCH under two independent purification strategies, providing confidence in the significance of the interaction. Proteins identified by iGEO were validated against a highly inclusive list of candidate PINCH interacting proteins identified in previous analyses by MuDPIT mass spectrometry. Conclusions The iGEO strategy confirmed a core complex comprised of PINCH, RSU1, ILK, and ILK binding partner Parvin. Our iGEO method also identified five novel protein partners that specifically interacted with PINCH in Drosophila S2 cell culture. Because of the improved reproducibility of 2D-GE methodology and the increasing affordability of the required labeling reagents, iGEO is a method that is accessible to most moderately well-equipped biological laboratories. The biochemical co-purifications

  9. Affinity Crystallography: A New Approach to Extracting High-Affinity Enzyme Inhibitors from Natural Extracts.

    PubMed

    Aguda, Adeleke H; Lavallee, Vincent; Cheng, Ping; Bott, Tina M; Meimetis, Labros G; Law, Simon; Nguyen, Nham T; Williams, David E; Kaleta, Jadwiga; Villanueva, Ivan; Davies, Julian; Andersen, Raymond J; Brayer, Gary D; Brömme, Dieter

    2016-08-26

    Natural products are an important source of novel drug scaffolds. The highly variable and unpredictable timelines associated with isolating novel compounds and elucidating their structures have led to the demise of exploring natural product extract libraries in drug discovery programs. Here we introduce affinity crystallography as a new methodology that significantly shortens the time of the hit to active structure cycle in bioactive natural product discovery research. This affinity crystallography approach is illustrated by using semipure fractions of an actinomycetes culture extract to isolate and identify a cathepsin K inhibitor and to compare the outcome with the traditional assay-guided purification/structural analysis approach. The traditional approach resulted in the identification of the known inhibitor antipain (1) and its new but lower potency dehydration product 2, while the affinity crystallography approach led to the identification of a new high-affinity inhibitor named lichostatinal (3). The structure and potency of lichostatinal (3) was verified by total synthesis and kinetic characterization. To the best of our knowledge, this is the first example of isolating and characterizing a potent enzyme inhibitor from a partially purified crude natural product extract using a protein crystallographic approach. PMID:27498895

  10. Jen1p: A High Affinity Selenite Transporter in Yeast

    PubMed Central

    McDermott, Joseph R.; Rosen, Barry P.

    2010-01-01

    Selenium is a micronutrient in most eukaryotes, including humans, which is well known for having an extremely thin border between beneficial and toxic concentrations. Soluble tetravalent selenite is the predominant environmental form and also the form that is applied in the treatment of human diseases. To acquire this nutrient from low environmental concentrations as well as to avoid toxicity, a well-controlled transport system is required. Here we report that Jen1p, a proton-coupled monocarboxylate transporter in S. cerevisiae, catalyzes high-affinity uptake of selenite. Disruption of JEN1 resulted in selenite resistance, and overexpression resulted in selenite hypersensitivity. Transport assay showed that overexpression of Jen1p enables selenite accumulation in yeast compared with a JEN1 knock out strain, indicating the Jen1p transporter facilitates selenite accumulation inside cells. Selenite uptake by Jen1p had a Km of 0.91 mM, which is comparable to the Km for lactate. Jen1p transported selenite in a proton-dependent manner which resembles the transport mechanism for lactate. In addition, selenite and lactate can inhibit the transport of each other competitively. Therefore, we postulate selenite is a molecular mimic of monocarboxylates which allows selenite to be transported by Jen1p. PMID:20861301

  11. Phosphatidylserine Reversibly Binds Cu2+ with Extremely High Affinity

    PubMed Central

    Monson, Christopher F.; Cong, Xiao; Robison, Aaron; Pace, Hudson P.; Liu, Chunming; Poyton, Matthew F.; Cremer, Paul S.

    2012-01-01

    Phosphatidylserine (PS) embedded within supported lipid bilayers (SLBs) was found to bind Cu2+ from solution with extraordinarily high affinity. In fact, the equilibrium dissociation constant was in the femtomolar range. The resulting complex formed in a 1:2 Cu2+ to PS ratio and quenches a broad spectrum of lipid-bound fluorophores in a reversible and pH-dependent fashion. At acidic pH values, the fluorophores were almost completely unquenched, while at basic pH values significant quenching (85–90%) was observed. The pH at which the transition occurred was dependent on the PS concentration and ranged from approximately pH 5 to 8. The quenching kinetics was slow at low Cu2+ concentrations and basic values pH (up to several hours), while the unquenching reaction was orders of magnitude more rapid upon lowering the pH. This was consistent with diffusion limited complex formation at basic pH, but rapid dissociation under acidic conditions. The tight binding of Cu2+ to PS may have physiological consequences under certain circumstances. PMID:22548290

  12. Deciphering the specific high-affinity binding of cucurbit[7]uril to amino acids in water.

    PubMed

    Lee, Jong Wha; Lee, Hyun Hee L; Ko, Young Ho; Kim, Kimoon; Kim, Hugh I

    2015-04-01

    This work presents a systematic study on the host-guest interactions between the macrocyclic host molecule cucurbit[7]uril (CB[7]) and amino acids (AAs) including three basic AAs (Lys, Arg, and His) and three aromatic AAs (Phe, Tyr, and Trp) to elucidate the origin of the high selectivity of CB[7] toward AA residues in proteins. Complex formation between CB[7] and each AA was examined in solution (by isothermal titration calorimetry and NMR) as well as in the gas phase (by ion mobility mass spectrometry and collision-induced dissociation), and the results were further combined with computational investigations. Generally, the aromatic AAs show higher binding affinities than the basic AAs in buffer solutions with various pH values. On the contrary, the gas-phase stabilities of the basic AA complex ions are higher than those of the aromatic AA complex ions, suggesting that the direct ion-dipole interactions between the charged side chains of the basic AAs and the polar carbonyl groups of CB[7] predominate in the absence of water. The ion-dipole interactions are less significant in water, since the original interactions of the guests with water are lost upon complex formation. In contrast, the transfer of the hydrophobic groups from the bulk into the hydrophobic CB[7] cavity suffers less from the desolvation penalty, resulting in higher binding affinities in water. Therefore, initial guest solvation is another key factor which should be considered when designing high-affinity host-guest systems, in addition to the contribution from the release of high-energy water molecules from the CB[7] cavity (J. Am. Chem. Soc. 2012, 134, 15318-15323).

  13. Deciphering the specific high-affinity binding of cucurbit[7]uril to amino acids in water.

    PubMed

    Lee, Jong Wha; Lee, Hyun Hee L; Ko, Young Ho; Kim, Kimoon; Kim, Hugh I

    2015-04-01

    This work presents a systematic study on the host-guest interactions between the macrocyclic host molecule cucurbit[7]uril (CB[7]) and amino acids (AAs) including three basic AAs (Lys, Arg, and His) and three aromatic AAs (Phe, Tyr, and Trp) to elucidate the origin of the high selectivity of CB[7] toward AA residues in proteins. Complex formation between CB[7] and each AA was examined in solution (by isothermal titration calorimetry and NMR) as well as in the gas phase (by ion mobility mass spectrometry and collision-induced dissociation), and the results were further combined with computational investigations. Generally, the aromatic AAs show higher binding affinities than the basic AAs in buffer solutions with various pH values. On the contrary, the gas-phase stabilities of the basic AA complex ions are higher than those of the aromatic AA complex ions, suggesting that the direct ion-dipole interactions between the charged side chains of the basic AAs and the polar carbonyl groups of CB[7] predominate in the absence of water. The ion-dipole interactions are less significant in water, since the original interactions of the guests with water are lost upon complex formation. In contrast, the transfer of the hydrophobic groups from the bulk into the hydrophobic CB[7] cavity suffers less from the desolvation penalty, resulting in higher binding affinities in water. Therefore, initial guest solvation is another key factor which should be considered when designing high-affinity host-guest systems, in addition to the contribution from the release of high-energy water molecules from the CB[7] cavity (J. Am. Chem. Soc. 2012, 134, 15318-15323). PMID:25757499

  14. High-affinity binding of fibronectin to cultured Kupffer cells

    SciTech Connect

    Cardarelli, P.M.; Blumenstock, F.A.; McKeown-Longo, P.J.; Saba, T.M.; Mazurkiewicz, J.E.; Dias, J.A. )

    1990-11-01

    Hepatic Kupffer cells are a major component of the reticuloendothelial or macrophage system. They were the first phagocytic cell type whose phagocytosis was shown to be influenced by plasma fibronectin, a dimeric opsonic glycoprotein. In the current study, the binding of soluble radioiodinated fibronectin purified from rat serum to isolated rat hepatic Kupffer cells was investigated using a cultured Kupffer cell monolayer technique. Binding was specific, since unlabeled purified fibronectin competed in a dose-dependent manner with the 125I-fibronectin for binding to the Kupffer cells. Addition of gelatin enhanced the binding of 125I-fibronectin to Kupffer cells. The phagocytosis of gelatinized-coated red cells by Kupffer cells was increased either by preopsonizing the target particles with purified fibronectin or by the addition of purified fibronectin to the culture medium. In contrast, exposure of the Kupffer cells to medium containing purified fibronectin followed by wash-removal of the fibronectin did not increase the uptake of gelatin-coated red blood cells, even though fibronectin was detected on the surface of the Kupffer cells by immunofluorescence. Trypsinized monolayers expressed decreased capacity to bind 125I-fibronectin as well as fibronectin-coated sheep erythrocytes. The binding of 125I-fibronectin-gelatin complexes was inhibited by excess unlabeled fibronectin. We calculated that specific high-affinity (Kd = 7.46 x 10(-9) M) binding sites for fibronectin exist on Kupffer cells. There are approximately 2,800-3,500 binding sites or putative fibronectin receptors per Kupffer cell. These sites appear to mediate the enhanced phagocytosis of gelatin-coated particles opsonized by fibronectin.

  15. Development and Characterization of High Affinity Leptins and Leptin Antagonists*

    PubMed Central

    Shpilman, Michal; Niv-Spector, Leonora; Katz, Meirav; Varol, Chen; Solomon, Gili; Ayalon-Soffer, Michal; Boder, Eric; Halpern, Zamir; Elinav, Eran; Gertler, Arieh

    2011-01-01

    Leptin is a pleiotropic hormone acting both centrally and peripherally. It participates in a variety of biological processes, including energy metabolism, reproduction, and modulation of the immune response. So far, structural elements affecting leptin binding to its receptor remain unknown. We employed random mutagenesis of leptin, followed by selection of high affinity mutants by yeast surface display and discovered that replacing residue Asp-23 with a non-negatively charged amino acid leads to dramatically enhanced affinity of leptin for its soluble receptor. Rational mutagenesis of Asp-23 revealed the D23L substitution to be most effective. Coupling the Asp-23 mutation with alanine mutagenesis of three amino acids (L39A/D40A/F41A) previously reported to convert leptin into antagonist resulted in potent antagonistic activity. These novel superactive mouse and human leptin antagonists (D23L/L39A/D40A/F41A), termed SMLA and SHLA, respectively, exhibited over 60-fold increased binding to leptin receptor and 14-fold higher antagonistic activity in vitro relative to the L39A/D40A/F41A mutants. To prolong and enhance in vivo activity, SMLA and SHLA were monopegylated mainly at the N terminus. Administration of the pegylated SMLA to mice resulted in a remarkably rapid, significant, and reversible 27-fold more potent increase in body weight (as compared with pegylated mouse leptin antagonist), because of increased food consumption. Thus, recognition and mutagenesis of Asp-23 enabled construction of novel compounds that induce potent and reversible central and peripheral leptin deficiency. In addition to enhancing our understanding of leptin interactions with its receptor, these antagonists enable in vivo study of the role of leptin in metabolic and immune processes and hold potential for future therapeutic use in disease pathologies involving leptin. PMID:21119198

  16. Development and characterization of high affinity leptins and leptin antagonists.

    PubMed

    Shpilman, Michal; Niv-Spector, Leonora; Katz, Meirav; Varol, Chen; Solomon, Gili; Ayalon-Soffer, Michal; Boder, Eric; Halpern, Zamir; Elinav, Eran; Gertler, Arieh

    2011-02-11

    Leptin is a pleiotropic hormone acting both centrally and peripherally. It participates in a variety of biological processes, including energy metabolism, reproduction, and modulation of the immune response. So far, structural elements affecting leptin binding to its receptor remain unknown. We employed random mutagenesis of leptin, followed by selection of high affinity mutants by yeast surface display and discovered that replacing residue Asp-23 with a non-negatively charged amino acid leads to dramatically enhanced affinity of leptin for its soluble receptor. Rational mutagenesis of Asp-23 revealed the D23L substitution to be most effective. Coupling the Asp-23 mutation with alanine mutagenesis of three amino acids (L39A/D40A/F41A) previously reported to convert leptin into antagonist resulted in potent antagonistic activity. These novel superactive mouse and human leptin antagonists (D23L/L39A/D40A/F41A), termed SMLA and SHLA, respectively, exhibited over 60-fold increased binding to leptin receptor and 14-fold higher antagonistic activity in vitro relative to the L39A/D40A/F41A mutants. To prolong and enhance in vivo activity, SMLA and SHLA were monopegylated mainly at the N terminus. Administration of the pegylated SMLA to mice resulted in a remarkably rapid, significant, and reversible 27-fold more potent increase in body weight (as compared with pegylated mouse leptin antagonist), because of increased food consumption. Thus, recognition and mutagenesis of Asp-23 enabled construction of novel compounds that induce potent and reversible central and peripheral leptin deficiency. In addition to enhancing our understanding of leptin interactions with its receptor, these antagonists enable in vivo study of the role of leptin in metabolic and immune processes and hold potential for future therapeutic use in disease pathologies involving leptin.

  17. High Affinity Binding of Indium and Ruthenium Ions by Gastrins.

    PubMed

    Baldwin, Graham S; George, Graham N; Pushie, M Jake

    2015-01-01

    The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated forms of the hormone. Since gastrins act as growth factors in gastrointestinal cancers, and as peptides labelled with Ga and In isotopes are increasingly used for cancer diagnosis, the ability of gastrins to bind other metal ions was investigated systematically by absorption spectroscopy. The coordination structures of the complexes were characterized by extended X-ray absorption fine structure (EXAFS) spectroscopy. Changes in the absorption of gastrin in the presence of increasing concentrations of Ga3+ were fitted by a 2 site model with dissociation constants (Kd) of 3.3 x 10-7 and 1.1 x 10-6 M. Although the absorption of gastrin did not change upon the addition of In3+ ions, the changes in absorbance on Fe3+ ion binding in the presence of indium ions were fitted by a 2 site model with Kd values for In3+ of 6.5 x 10-15 and 1.7 x 10-7 M. Similar results were obtained with Ru3+ ions, although the Kd values for Ru3+ of 2.6 x 10-13 and 1.2 x 10-5 M were slightly larger than observed for In3+. The structures determined by EXAFS all had metal:gastrin stoichiometries of 2:1 but, while the metal ions in the Fe, Ga and In complexes were bridged by a carboxylate and an oxygen with a metal-metal separation of 3.0-3.3 Å, the Ru complex clearly demonstrated a short range Ru-Ru separation, which was significantly shorter, at 2.4 Å, indicative of a metal-metal bond. We conclude that gastrin selectively binds two In3+ or Ru3+ ions, and that the affinity of the first site for In3+ or Ru3+ ions is higher than for ferric ions. Some of the metal ion-gastrin complexes may be useful for cancer diagnosis and therapy.

  18. High Affinity Binding of Indium and Ruthenium Ions by Gastrins

    PubMed Central

    Baldwin, Graham S.; George, Graham N.; Pushie, M. Jake

    2015-01-01

    The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated forms of the hormone. Since gastrins act as growth factors in gastrointestinal cancers, and as peptides labelled with Ga and In isotopes are increasingly used for cancer diagnosis, the ability of gastrins to bind other metal ions was investigated systematically by absorption spectroscopy. The coordination structures of the complexes were characterized by extended X-ray absorption fine structure (EXAFS) spectroscopy. Changes in the absorption of gastrin in the presence of increasing concentrations of Ga3+ were fitted by a 2 site model with dissociation constants (Kd) of 3.3 x 10−7 and 1.1 x 10−6 M. Although the absorption of gastrin did not change upon the addition of In3+ ions, the changes in absorbance on Fe3+ ion binding in the presence of indium ions were fitted by a 2 site model with Kd values for In3+ of 6.5 x 10−15 and 1.7 x 10−7 M. Similar results were obtained with Ru3+ ions, although the Kd values for Ru3+ of 2.6 x 10−13 and 1.2 x 10−5 M were slightly larger than observed for In3+. The structures determined by EXAFS all had metal:gastrin stoichiometries of 2:1 but, while the metal ions in the Fe, Ga and In complexes were bridged by a carboxylate and an oxygen with a metal-metal separation of 3.0–3.3 Å, the Ru complex clearly demonstrated a short range Ru—Ru separation, which was significantly shorter, at 2.4 Å, indicative of a metal-metal bond. We conclude that gastrin selectively binds two In3+ or Ru3+ ions, and that the affinity of the first site for In3+ or Ru3+ ions is higher than for ferric ions. Some of the metal ion-gastrin complexes may be useful for cancer diagnosis and therapy. PMID:26457677

  19. Studying protein complexes by the yeast two-hybrid system.

    PubMed

    Rajagopala, Seesandra V; Sikorski, Patricia; Caufield, J Harry; Tovchigrechko, Andrey; Uetz, Peter

    2012-12-01

    Protein complexes are typically analyzed by affinity purification and subsequent mass spectrometric analysis. However, in most cases the structure and topology of the complexes remains elusive from such studies. Here we investigate how the yeast two-hybrid system can be used to analyze direct interactions among proteins in a complex. First we tested all pairwise interactions among the seven proteins of Escherichia coli DNA polymerase III as well as an uncharacterized complex that includes MntR and PerR. Four and seven interactions were identified in these two complexes, respectively. In addition, we review Y2H data for three other complexes of known structure which serve as "gold-standards", namely Varicella Zoster Virus (VZV) ribonucleotide reductase (RNR), the yeast proteasome, and bacteriophage lambda. Finally, we review an Y2H analysis of the human spliceosome which may serve as an example for a dynamic mega-complex.

  20. AGIA Tag System Based on a High Affinity Rabbit Monoclonal Antibody against Human Dopamine Receptor D1 for Protein Analysis

    PubMed Central

    Yano, Tomoya; Takeda, Hiroyuki; Uematsu, Atsushi; Yamanaka, Satoshi; Nomura, Shunsuke; Nemoto, Keiichirou; Iwasaki, Takahiro; Takahashi, Hirotaka; Sawasaki, Tatsuya

    2016-01-01

    Polypeptide tag technology is widely used for protein detection and affinity purification. It consists of two fundamental elements: a peptide sequence and a binder which specifically binds to the peptide tag. In many tag systems, antibodies have been used as binder due to their high affinity and specificity. Recently, we obtained clone Ra48, a high-affinity rabbit monoclonal antibody (mAb) against dopamine receptor D1 (DRD1). Here, we report a novel tag system composed of Ra48 antibody and its epitope sequence. Using a deletion assay, we identified EEAAGIARP in the C-terminal region of DRD1 as the minimal epitope of Ra48 mAb, and we named this sequence the “AGIA” tag, based on its central sequence. The tag sequence does not include the four amino acids, Ser, Thr, Tyr, or Lys, which are susceptible to post-translational modification. We demonstrated performance of this new tag system in biochemical and cell biology applications. SPR analysis demonstrated that the affinity of the Ra48 mAb to the AGIA tag was 4.90 × 10−9 M. AGIA tag showed remarkably high sensitivity and specificity in immunoblotting. A number of AGIA-fused proteins overexpressed in animal and plant cells were detected by anti-AGIA antibody in immunoblotting and immunostaining with low background, and were immunoprecipitated efficiently. Furthermore, a single amino acid substitution of the second Glu to Asp (AGIA/E2D) enabled competitive dissociation of AGIA/E2D-tagged protein by adding wild-type AGIA peptide. It enabled one-step purification of AGIA/E2D-tagged recombinant proteins by peptide competition under physiological conditions. The sensitivity and specificity of the AGIA system makes it suitable for use in multiple methods for protein analysis. PMID:27271343

  1. A Proteomic Strategy for Global Analysis of Plant Protein Complexes[W][OPEN

    PubMed Central

    Aryal, Uma K.; Xiong, Yi; McBride, Zachary; Kihara, Daisuke; Xie, Jun; Hall, Mark C.; Szymanski, Daniel B.

    2014-01-01

    Global analyses of protein complex assembly, composition, and location are needed to fully understand how cells coordinate diverse metabolic, mechanical, and developmental activities. The most common methods for proteome-wide analysis of protein complexes rely on affinity purification-mass spectrometry or yeast two-hybrid approaches. These methods are time consuming and are not suitable for many plant species that are refractory to transformation or genome-wide cloning of open reading frames. Here, we describe the proof of concept for a method allowing simultaneous global analysis of endogenous protein complexes that begins with intact leaves and combines chromatographic separation of extracts from subcellular fractions with quantitative label-free protein abundance profiling by liquid chromatography-coupled mass spectrometry. Applying this approach to the crude cytosolic fraction of Arabidopsis thaliana leaves using size exclusion chromatography, we identified hundreds of cytosolic proteins that appeared to exist as components of stable protein complexes. The reliability of the method was validated by protein immunoblot analysis and comparisons with published size exclusion chromatography data and the masses of known complexes. The method can be implemented with appropriate instrumentation, is applicable to any biological system, and has the potential to be further developed to characterize the composition of protein complexes and measure the dynamics of protein complex localization and assembly under different conditions. PMID:25293756

  2. Co-translational assembly of protein complexes.

    PubMed

    Wells, Jonathan N; Bergendahl, L Therese; Marsh, Joseph A

    2015-12-01

    The interaction of biological macromolecules is a fundamental attribute of cellular life. Proteins, in particular, often form stable complexes with one another. Although the importance of protein complexes is widely recognized, we still have only a very limited understanding of the mechanisms underlying their assembly within cells. In this article, we review the available evidence for one such mechanism, namely the coupling of protein complex assembly to translation at the polysome. We discuss research showing that co-translational assembly can occur in both prokaryotic and eukaryotic organisms and can have important implications for the correct functioning of the complexes that result. Co-translational assembly can occur for both homomeric and heteromeric protein complexes and for both proteins that are translated directly into the cytoplasm and those that are translated into or across membranes. Finally, we discuss the properties of proteins that are most likely to be associated with co-translational assembly.

  3. Identification of Post-translational Modifications of Plant Protein Complexes

    PubMed Central

    Piquerez, Sophie J. M.; Balmuth, Alexi L.; Sklenář, Jan; Jones, Alexandra M.E.; Rathjen, John P.; Ntoukakis, Vardis

    2014-01-01

    Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved. Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs. This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein. PMID:24637539

  4. Purification and subunit structure of a putative K sup + -channel protein identified by its binding properties for dendrotoxin I

    SciTech Connect

    Rehm, H.; Lazdunski, M. )

    1988-07-01

    The binding protein for the K{sup +}-channel toxin dendrotoxin I was purified from a detergent extract of rat brain membranes. The purification procedure utilized chromatography on DEAE-Trisacryl, affinity chromatography on a dendrotoxin-I-Aca 22 column, and wheat germ agglutinin-Affigel 10 with a final 3,800- to 4,600-fold enrichment and a recovery of 8-16%. The high affinity (K{sub d}, 40-100 pM) and specificity of the binding site are retained throughout the purification procedure. Analysis of the purified material on silver-stained NaDodSO{sub 4}/polyacrylamide gel revealed three bands of M{sub r} 76,000-80,000, 38,000 and 35,000. Interestingly, the binding site for {sup 125}I-labeled mast cell degranulating peptide, another putative K{sup +}-channel ligand from bee venom, which induces long-term potentiation in hippocampus, seems to reside on the same protein complex, as both binding sites copurify through the entire purification protocol.

  5. ESCRT-III-Associated Protein ALIX Mediates High-Affinity Phosphate Transporter Trafficking to Maintain Phosphate Homeostasis in Arabidopsis

    PubMed Central

    Cardona-López, Ximena; Cuyas, Laura; Marín, Elena; Irigoyen, María Luisa; Gil, Erica; Puga, María Isabel; Bligny, Richard; Nussaume, Laurent; Geldner, Niko; Paz-Ares, Javier

    2015-01-01

    Prior to the release of their cargoes into the vacuolar lumen, sorting endosomes mature into multivesicular bodies (MVBs) through the action of ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT (ESCRT) protein complexes. MVB-mediated sorting of high-affinity phosphate transporters (PHT1) to the vacuole limits their plasma membrane levels under phosphate-sufficient conditions, a process that allows plants to maintain phosphate homeostasis. Here, we describe ALIX, a cytosolic protein that associates with MVB by interacting with ESCRT-III subunit SNF7 and mediates PHT1;1 trafficking to the vacuole in Arabidopsis thaliana. We show that the partial loss-of-function mutant alix-1 displays reduced vacuolar degradation of PHT1;1. ALIX derivatives containing the alix-1 mutation showed reduced interaction with SNF7, providing a simple molecular explanation for impaired cargo trafficking in alix-1 mutants. In fact, the alix-1 mutation also hampered vacuolar sorting of the brassinosteroid receptor BRI1. We also show that alix-1 displays altered vacuole morphogenesis, implying a new role for ALIX proteins in vacuolar biogenesis, likely acting as part of ESCRT-III complexes. In line with a presumed broad target spectrum, the alix-1 mutation is pleiotropic, leading to reduced plant growth and late flowering, with stronger alix mutations being lethal, indicating that ALIX participates in diverse processes in plants essential for their life. PMID:26342016

  6. Two high-affinity ligand binding states of uterine estrogen receptor distinguished by modulation of hydrophobic environment

    SciTech Connect

    Hutchens, T.W.; Li, C.M.; Zamah, N.M.; Besch, P.K.

    1987-02-10

    The steroid binding function of soluble (cytosolic) estrogen receptors from calf uteri was evaluated under conditions known to modify the extent of hydrophobic interaction with receptor-associated proteins. Receptor preparations were equilibrated into 6 M urea buffers and control buffers by chromatography through small columns of Sephadex G-25 or by dialysis at 0.6 /sup 0/C. Equilibrium dissociation constants (K/sub d/) and binding capacities (n) of experimental and control receptor preparations were determined by 13-point Scatchard analyses using concentrations of 17..beta..-(/sup 3/H)estradiol from 0.05 to 10 nM. Nonspecific binding was determined at each concentration by parallel incubations with a 200-fold molar excess of the receptor-specific competitor diethylstilbestrol. The control receptor population was consistently found to be a single class of binding sites with a high affinity for estradiol which was unaffected by G-25 chromatography, by dialysis, by dilution, or by the presence of 0.4 M KCl. However, equilibration into 6 M urea induced a discrete (10-fold) reduction in receptor affinity to reveal a second, thermodynamically stable, high-affinity binding state. The presence of 0.4 M KCl did not significantly influence the discrete change in receptor affinity induced by urea. The effects of urea on both receptor affinity and binding capacity were reversible, suggesting a lack of covalent modification. These results demonstrate nonenzymatic means by which not only the binding capacity but also the affinity of receptor for estradiol can be reversibly controlled, suggesting that high concentrations of urea might be more effectively utilized during the physicochemical characterization and purification of steroid receptor proteins.

  7. Sequential class switching is required for the generation of high affinity IgE antibodies

    PubMed Central

    Xiong, Huizhong; Dolpady, Jayashree; Wabl, Matthias; Curotto de Lafaille, Maria A.

    2012-01-01

    IgE antibodies with high affinity for their antigens can be stably cross-linked at low concentrations by trace amounts of antigen, whereas IgE antibodies with low affinity bind their antigens weakly. In this study, we find that there are two distinct pathways to generate high and low affinity IgE. High affinity IgE is generated through sequential class switching (μ→γ→ε) in which an intermediary IgG phase is necessary for the affinity maturation of the IgE response, where the IgE inherits somatic hypermutations and high affinity from the IgG1 phase. In contrast, low affinity IgE is generated through direct class switching (μ→ε) and is much less mutated. Mice deficient in IgG1 production cannot produce high affinity IgE, even after repeated immunizations. We demonstrate that a small amount of high affinity IgE can cause anaphylaxis and is pathogenic. Low affinity IgE competes with high affinity IgE for binding to Fcε receptors and prevents anaphylaxis and is thus beneficial. PMID:22249450

  8. Investigation of a protein complex network

    NASA Astrophysics Data System (ADS)

    Mashaghi, A. R.; Ramezanpour, A.; Karimipour, V.

    2004-09-01

    The budding yeast Saccharomyces cerevisiae is the first eukaryote whose genome has been completely sequenced. It is also the first eukaryotic cell whose proteome (the set of all proteins) and interactome (the network of all mutual interactions between proteins) has been analyzed. In this paper we study the structure of the yeast protein complex network in which weighted edges between complexes represent the number of shared proteins. It is found that the network of protein complexes is a small world network with scale free behavior for many of its distributions. However we find that there are no strong correlations between the weights and degrees of neighboring complexes. To reveal non-random features of the network we also compare it with a null model in which the complexes randomly select their proteins. Finally we propose a simple evolutionary model based on duplication and divergence of proteins.

  9. Complementary Proteomic Analysis of Protein Complexes

    PubMed Central

    Greco, Todd M.; Miteva, Yana; Conlon, Frank L.; Cristea, Ileana M.

    2013-01-01

    Proteomic characterization of protein complexes leverages the versatile platform of liquid chromatography-tandem mass spectrometry to elucidate molecular and cellular signaling processes underlying the dynamic regulation of macromolecular assemblies. Here, we describe a complementary proteomic approach optimized for immunoisolated protein complexes. As the relative complexity, abundance, and physiochemical properties of proteins can vary significantly between samples, we have provided (1) complementary sample preparation workflows, (2) detailed steps for HPLC and mass spectrometric method development, and (3) a bioinformatic workflow that provides confident peptide/protein identification paired with unbiased functional gene ontology analysis. This protocol can also be extended for characterization of larger complexity samples from whole cell or tissue Xenopus proteomes. PMID:22956100

  10. Interaction proteomics of synapse protein complexes

    PubMed Central

    Klemmer, Patricia; Smit, August B.

    2010-01-01

    The brain integrates complex types of information, and executes a wide range of physiological and behavioral processes. Trillions of tiny organelles, the synapses, are central to neuronal communication and information processing in the brain. Synaptic transmission involves an intricate network of synaptic proteins that forms the molecular machinery underlying transmitter release, activation, and modulation of transmitter receptors and signal transduction cascades. These processes are dynamically regulated and underlie neuroplasticity, crucial to learning and memory formation. In recent years, interaction proteomics has increasingly been used to elucidate the constituents of synaptic protein complexes. Unlike classic hypothesis-based assays, interaction proteomics detects both known and novel interactors without bias. In this trend article, we focus on the technical aspects of recent proteomics to identify synapse protein complexes, and the complementary methods used to verify the protein–protein interaction. Moreover, we discuss the experimental feasibility of performing global analysis of the synapse protein interactome. PMID:20361179

  11. GHB receptor targets in the CNS: focus on high-affinity binding sites.

    PubMed

    Bay, Tina; Eghorn, Laura F; Klein, Anders B; Wellendorph, Petrine

    2014-01-15

    γ-Hydroxybutyric acid (GHB) is an endogenous compound in the mammalian brain with both low- and high-affinity receptor targets. GHB is used clinically in the treatment of symptoms of narcolepsy and alcoholism, but also illicitly abused as the recreational drug Fantasy. Major pharmacological effects of exogenous GHB are mediated by GABA subtype B (GABAB) receptors that bind GHB with low affinity. The existence of GHB high-affinity binding sites has been known for more than three decades, but the uncovering of their molecular identity has only recently begun. This has been prompted by the generation of molecular tools to selectively study high-affinity sites. These include both genetically modified GABAB knock-out mice and engineered selective GHB ligands. Recently, certain GABA subtype A (GABAA) receptor subtypes emerged as high-affinity GHB binding sites and potential physiological mediators of GHB effects. In this research update, a description of the various reported receptors for GHB is provided, including GABAB receptors, certain GABAA receptor subtypes and other reported GHB receptors. The main focus will thus be on the high-affinity binding targets for GHB and their potential functional roles in the mammalian brain.

  12. Discovery of host-viral protein complexes during infection

    PubMed Central

    Rowles, Daniell L.; Terhune, Scott S.; Cristea, Ileana M.

    2014-01-01

    Summary Viruses have co-evolved with their hosts, developing effective approaches for hijacking and manipulating host cellular processes. Therefore, for their efficient replication and spread, viruses depend on dynamic and temporally-regulated interactions with host proteins. The rapid identification of host proteins targeted by viral proteins during infection provides significant insights into mechanisms of viral protein function. The resulting discoveries often lead to unique and innovative hypotheses on viral protein function. Here, we describe a robust method for identifying virus-host protein interactions and protein complexes, which we have successfully utilized to characterize spatial-temporal protein interactions during infections with either DNA or RNA viruses, including human cytomegalovirus (HCMV), herpes simplex virus type 1 (HSV-1), pseudorabies virus (PRV), human immunodeficiency virus (HIV-1), Sindbis, and West Nile virus (WNV). This approach involves cryogenic cell lysis, rapid immunoaffinity purification targeting a virus or host protein, followed by identification of associated proteins using mass spectrometry. Like most proteomic approaches, this methodology has evolved over the past few years and continues to evolve. We are presenting here the updated approaches for each step, and discuss alternative strategies allowing for the protocol to be optimized for different biological systems. PMID:23996249

  13. Mathematical modeling of the low and high affinity arabinose transport systems in Escherichia coli.

    PubMed

    Yildirim, Necmettin

    2012-04-01

    A mathematical model was developed for the low and high affinity arabinose transport systems in E. coli. The model is a system of three ordinary differential equations and takes the dynamics of mRNAs for the araE and araFGH proteins and the internal arabinose into account. Special attention was paid to estimate the model parameters from the literature. Our analysis and simulations suggest that the high affinity transport system helps the low affinity transport system to respond to high concentration of extracellular arabinose faster, whereas the high affinity transport system responds to a small amount of extracellular arabinose. Steady state analysis of the model also predicts that there is a regime for the extracellular concentration of arabinose where the arabinose system can show bistable behavior.

  14. Architecture of high-affinity unnatural-base DNA aptamers toward pharmaceutical applications.

    PubMed

    Matsunaga, Ken-ichiro; Kimoto, Michiko; Hanson, Charlotte; Sanford, Michael; Young, Howard A; Hirao, Ichiro

    2015-01-01

    We present a remodeling method for high-affinity unnatural-base DNA aptamers to augment their thermal stability and nuclease resistance, for use as drug candidates targeting specific proteins. Introducing a unique mini-hairpin DNA provides robust stability to unnatural-base DNA aptamers generated by SELEX using genetic alphabet expansion, without reducing their high affinity. By this method, >80% of the remodeled DNA aptamer targeting interferon-γ (KD of 33 pM) survived in human serum at 37 °C after 3 days under our experimental conditions, and sustainably inhibited the biological activity of interferon-γ. PMID:26690672

  15. SORPTION OF LEAD ON A HIGH AFFINITY OXIDE: MACROSCOPIC AND MICROSCOPIC STUDIES

    EPA Science Inventory

    Sorption of lead (Pb) was investigated on an innovative metal oxide compound using macroscopic and microscopic techniques. The objective of this study was to elucidate the sorption mechanism of Pb on the high-affinity engineered oxide with time at pH 6 employing batch methods an...

  16. SORPTION OF LEAD ON A HIGH AFFINITY OXIDE: MACROSCOPIC AND MICROSCOPIC STUDIES (ABSTRACT)

    EPA Science Inventory

    Sorption of lead (Pb) was investigated on an innovative metal oxide compound using macroscopic and microscopic techniques. The objective of this study was to elucidate the sorption mechanism of Pb on the high-affinity engineered oxide with time at pH 6 employing batch methods an...

  17. Prioritizing protein complexes implicated in human diseases by network optimization

    PubMed Central

    2014-01-01

    Background The detection of associations between protein complexes and human inherited diseases is of great importance in understanding mechanisms of diseases. Dysfunctions of a protein complex are usually defined by its member disturbance and consequently result in certain diseases. Although individual disease proteins have been widely predicted, computational methods are still absent for systematically investigating disease-related protein complexes. Results We propose a method, MAXCOM, for the prioritization of candidate protein complexes. MAXCOM performs a maximum information flow algorithm to optimize relationships between a query disease and candidate protein complexes through a heterogeneous network that is constructed by combining protein-protein interactions and disease phenotypic similarities. Cross-validation experiments on 539 protein complexes show that MAXCOM can rank 382 (70.87%) protein complexes at the top against protein complexes constructed at random. Permutation experiments further confirm that MAXCOM is robust to the network structure and parameters involved. We further analyze protein complexes ranked among top ten for breast cancer and demonstrate that the SWI/SNF complex is potentially associated with breast cancer. Conclusions MAXCOM is an effective method for the discovery of disease-related protein complexes based on network optimization. The high performance and robustness of this approach can facilitate not only pathologic studies of diseases, but also the design of drugs targeting on multiple proteins. PMID:24565064

  18. Crystallization and preliminary X-ray diffraction analysis of a high-affinity phosphate-binding protein endowed with phosphatase activity from Pseudomonas aeruginosa PAO1.

    PubMed

    Djeghader, Ahmed; Gotthard, Guillaume; Suh, Andrew; Gonzalez, Daniel; Scott, Ken; Chabriere, Eric; Elias, Mikael

    2013-10-01

    In prokaryotes, phosphate starvation induces the expression of numerous phosphate-responsive genes, such as the pst operon including the high-affinity phosphate-binding protein (PBP or pstS) and alkaline phosphatases such as PhoA. This response increases the cellular inorganic phosphate import efficiency. Notably, some Pseudomonas species secrete, via a type-2 secretion system, a phosphate-binding protein dubbed LapA endowed with phosphatase activity. Here, the expression, purification, crystallization and X-ray data collection at 0.87 Å resolution of LapA are described. Combined with biochemical and enzymatic characterization, the structure of this intriguing phosphate-binding protein will help to elucidate the molecular origin of its phosphatase activity and to decipher its putative role in phosphate uptake.

  19. A Protein Complex Map of Trypanosoma brucei

    PubMed Central

    Mehta, Vaibhav; Najafabadi, Hamed S.; Moshiri, Houtan; Jardim, Armando; Salavati, Reza

    2016-01-01

    The functions of the majority of trypanosomatid-specific proteins are unknown, hindering our understanding of the biology and pathogenesis of Trypanosomatida. While protein-protein interactions are highly informative about protein function, a global map of protein interactions and complexes is still lacking for these important human parasites. Here, benefiting from in-depth biochemical fractionation, we systematically interrogated the co-complex interactions of more than 3354 protein groups in procyclic life stage of Trypanosoma brucei, the protozoan parasite responsible for human African trypanosomiasis. Using a rigorous methodology, our analysis led to identification of 128 high-confidence complexes encompassing 716 protein groups, including 635 protein groups that lacked experimental annotation. These complexes correlate well with known pathways as well as for proteins co-expressed across the T. brucei life cycle, and provide potential functions for a large number of previously uncharacterized proteins. We validated the functions of several novel proteins associated with the RNA-editing machinery, identifying a candidate potentially involved in the mitochondrial post-transcriptional regulation of T. brucei. Our data provide an unprecedented view of the protein complex map of T. brucei, and serve as a reliable resource for further characterization of trypanosomatid proteins. The presented results in this study are available at: www.TrypsNetDB.org. PMID:26991453

  20. Three Recombinant Engineered Antibodies against Recombinant Tags with High Affinity and Specificity.

    PubMed

    Zhao, Hongyu; Shen, Ao; Xiang, Yang K; Corey, David P

    2016-01-01

    We describe three recombinant engineered antibodies against three recombinant epitope tags, constructed with divalent binding arms to recognize divalent epitopes and so achieve high affinity and specificity. In two versions, an epitope is inserted in tandem into a protein of interest, and a homodimeric antibody is constructed by fusing a high-affinity epitope-binding domain to a human or mouse Fc domain. In a third, a heterodimeric antibody is constructed by fusing two different epitope-binding domains which target two different binding sites in GFP, to polarized Fc fragments. These antibody/epitope pairs have affinities in the low picomolar range and are useful tools for many antibody-based applications.

  1. Three Recombinant Engineered Antibodies against Recombinant Tags with High Affinity and Specificity

    PubMed Central

    Zhao, Hongyu; Shen, Ao; Xiang, Yang K.; Corey, David P.

    2016-01-01

    We describe three recombinant engineered antibodies against three recombinant epitope tags, constructed with divalent binding arms to recognize divalent epitopes and so achieve high affinity and specificity. In two versions, an epitope is inserted in tandem into a protein of interest, and a homodimeric antibody is constructed by fusing a high-affinity epitope-binding domain to a human or mouse Fc domain. In a third, a heterodimeric antibody is constructed by fusing two different epitope-binding domains which target two different binding sites in GFP, to polarized Fc fragments. These antibody/epitope pairs have affinities in the low picomolar range and are useful tools for many antibody-based applications. PMID:26943906

  2. "DAKLI": a multipurpose ligand with high affinity and selectivity for dynorphin (kappa opioid) binding sites.

    PubMed Central

    Goldstein, A; Nestor, J J; Naidu, A; Newman, S R

    1988-01-01

    We describe a synthetic ligand, "DAKLI" (Dynorphin A-analogue Kappa LIgand), related to the opioid peptide dynorphin A. A single reactive amino group at the extended carboxyl terminus permits various reporter groups to be attached, such as 125I-labeled Bolton-Hunter reagent, fluorescein isothiocyanate, or biotin. These derivatives have high affinity and selectivity for the dynorphin (kappa opioid) receptor. An incidental finding is that untreated guinea pig brain membranes have saturable avidin binding sites. PMID:2902630

  3. Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging.

    PubMed

    Maute, Roy L; Gordon, Sydney R; Mayer, Aaron T; McCracken, Melissa N; Natarajan, Arutselvan; Ring, Nan Guo; Kimura, Richard; Tsai, Jonathan M; Manglik, Aashish; Kruse, Andrew C; Gambhir, Sanjiv S; Weissman, Irving L; Ring, Aaron M

    2015-11-24

    Signaling through the immune checkpoint programmed cell death protein-1 (PD-1) enables tumor progression by dampening antitumor immune responses. Therapeutic blockade of the signaling axis between PD-1 and its ligand programmed cell death ligand-1 (PD-L1) with monoclonal antibodies has shown remarkable clinical success in the treatment of cancer. However, antibodies have inherent limitations that can curtail their efficacy in this setting, including poor tissue/tumor penetrance and detrimental Fc-effector functions that deplete immune cells. To determine if PD-1:PD-L1-directed immunotherapy could be improved with smaller, nonantibody therapeutics, we used directed evolution by yeast-surface display to engineer the PD-1 ectodomain as a high-affinity (110 pM) competitive antagonist of PD-L1. In contrast to anti-PD-L1 monoclonal antibodies, high-affinity PD-1 demonstrated superior tumor penetration without inducing depletion of peripheral effector T cells. Consistent with these advantages, in syngeneic CT26 tumor models, high-affinity PD-1 was effective in treating both small (50 mm(3)) and large tumors (150 mm(3)), whereas the activity of anti-PD-L1 antibodies was completely abrogated against large tumors. Furthermore, we found that high-affinity PD-1 could be radiolabeled and applied as a PET imaging tracer to efficiently distinguish between PD-L1-positive and PD-L1-negative tumors in living mice, providing an alternative to invasive biopsy and histological analysis. These results thus highlight the favorable pharmacology of small, nonantibody therapeutics for enhanced cancer immunotherapy and immune diagnostics. PMID:26604307

  4. Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging

    PubMed Central

    Maute, Roy L.; Gordon, Sydney R.; Mayer, Aaron T.; McCracken, Melissa N.; Natarajan, Arutselvan; Ring, Nan Guo; Kimura, Richard; Tsai, Jonathan M.; Manglik, Aashish; Kruse, Andrew C.; Gambhir, Sanjiv S.; Weissman, Irving L.; Ring, Aaron M.

    2015-01-01

    Signaling through the immune checkpoint programmed cell death protein-1 (PD-1) enables tumor progression by dampening antitumor immune responses. Therapeutic blockade of the signaling axis between PD-1 and its ligand programmed cell death ligand-1 (PD-L1) with monoclonal antibodies has shown remarkable clinical success in the treatment of cancer. However, antibodies have inherent limitations that can curtail their efficacy in this setting, including poor tissue/tumor penetrance and detrimental Fc-effector functions that deplete immune cells. To determine if PD-1:PD-L1–directed immunotherapy could be improved with smaller, nonantibody therapeutics, we used directed evolution by yeast-surface display to engineer the PD-1 ectodomain as a high-affinity (110 pM) competitive antagonist of PD-L1. In contrast to anti–PD-L1 monoclonal antibodies, high-affinity PD-1 demonstrated superior tumor penetration without inducing depletion of peripheral effector T cells. Consistent with these advantages, in syngeneic CT26 tumor models, high-affinity PD-1 was effective in treating both small (50 mm3) and large tumors (150 mm3), whereas the activity of anti–PD-L1 antibodies was completely abrogated against large tumors. Furthermore, we found that high-affinity PD-1 could be radiolabeled and applied as a PET imaging tracer to efficiently distinguish between PD-L1–positive and PD-L1–negative tumors in living mice, providing an alternative to invasive biopsy and histological analysis. These results thus highlight the favorable pharmacology of small, nonantibody therapeutics for enhanced cancer immunotherapy and immune diagnostics. PMID:26604307

  5. EF5 Is the High-Affinity Mg(2+) Site in ALG-2.

    PubMed

    Tanner, John J; Frey, Benjamin B; Pemberton, Travis; Henzl, Michael T

    2016-09-13

    The penta-EF-hand (PEF) protein ALG-2 (apoptosis-linked gene 2) has been implicated in several important physiological processes, including endoplasmic reticulum-Golgi vesicular transport and endosomal biogenesis/transport. ALG-2 was recently shown to harbor a metal ion-binding site with a high affinity for Mg(2+) and a low affinity for Ca(2+). We herein present the X-ray structure of Mg(2+)-bound ALG-2des23(wt). Although the C(α) trace is nearly indistinguishable from that of the Ca(2+)-free protein, the orientation of the C-terminal helix differs in the two structures. Consistent with that observation, replacement of the +x ligand in EF5, D169, with alanine eliminates high-affinity Mg(2+) binding. It also eliminates the low-affinity Ca(2+) site and lowers the affinity of the remaining Ca(2+)-binding sites, EF3 and EF1. The coordination environment in EF5 approaches ideal Mg(2+) octahedral geometry. The ligand array, consisting of three carboxylates (+x, +y, +z), a backbone carbonyl (-y), and two water molecules (-x, -z), may offer a recipe for a high-affinity, high-selectivity Mg(2+)-binding site. Sequence data for other PEF proteins indicate that select calpain large subunits, notably CAPN1 and CAPN8, may also possess a high-affinity Mg(2+)-binding site. In Mg(2+)-bound ALG-2, the carbonyl of F188 and the C-terminal carboxylate of V191 interact with the ε-ammonium group of K137 in the opposing subunit, suggesting that Mg(2+) binding could have an impact on dimerization. Interestingly, EF1 and EF3 are also occupied in the crystal, despite having modest affinity for Mg(2+). The results of a calorimetry-based analysis indicate that their Mg(2+) binding constants are 2 orders of magnitude lower than that determined for EF5. PMID:27541325

  6. Selective high-affinity polydentate ligands and methods of making such

    SciTech Connect

    DeNardo, Sally; DeNardo, Gerald; Balhorn, Rodney

    2013-09-17

    This invention provides polydentate selective high affinity ligands (SHALs) that can be used in a variety of applications in a manner analogous to the use of antibodies. SHALs typically comprise a multiplicity of ligands that each binds different regions on the target molecule. The ligands are joined directly or through a linker thereby forming a polydentate moiety that typically binds the target molecule with high selectivity and avidity.

  7. Novel Ubiquitin-derived High Affinity Binding Proteins with Tumor Targeting Properties*

    PubMed Central

    Lorey, Susan; Fiedler, Erik; Kunert, Anja; Nerkamp, Jörg; Lange, Christian; Fiedler, Markus; Bosse-Doenecke, Eva; Meysing, Maren; Gloser, Manja; Rundfeldt, Chris; Rauchhaus, Una; Hänssgen, Ilka; Göttler, Thomas; Steuernagel, Arnd; Fiedler, Ulrike; Haupts, Ulrich

    2014-01-01

    Targeting effector molecules to tumor cells is a promising mode of action for cancer therapy and diagnostics. Binding proteins with high affinity and specificity for a tumor target that carry effector molecules such as toxins, cytokines, or radiolabels to their intended site of action are required for these applications. In order to yield high tumor accumulation while maintaining low levels in healthy tissues and blood, the half-life of such conjugates needs to be in an optimal range. Scaffold-based binding molecules are small proteins with high affinity and short systemic circulation. Due to their low molecular complexity, they are well suited for combination with effector molecules as well as half-life extension technologies yielding therapeutics with half-lives adapted to the specific therapy. We have identified ubiquitin as an ideal scaffold protein due to its outstanding biophysical and biochemical properties. Based on a dimeric ubiquitin library, high affinity and specific binding molecules, so-called Affilin® molecules, have been selected against the extradomain B of fibronectin, a target almost exclusively expressed in tumor tissues. Extradomain B-binding molecules feature high thermal and serum stability as well as strong in vitro target binding and in vivo tumor accumulation. Application of several half-life extension technologies results in molecules of largely unaffected affinity but significantly prolonged in vivo half-life and tumor retention. Our results demonstrate the utility of ubiquitin as a scaffold for the generation of high affinity binders in a modular fashion, which can be combined with effector molecules and half-life extension technologies. PMID:24474690

  8. High affinity binding of (/sup 3/H)neurotensin of rat uterus

    SciTech Connect

    Pettibone, D.J.; Totaro, J.A.

    1987-11-01

    (/sup 3/H)Neurotensin (NT) was found to bind specifically and with high affinity to crude membranes prepared from rat uterus. Scatchard analysis of saturation binding studies indicated that (/sup 3/H)NT apparently binds to two sites (high affinity Kd 0.5 nM; low affinity Kd 9 nM) with the density of high affinity sites (41 fmoles/mg prot.) being about one-third that of the low affinity sites (100 fmoles/mg prot.). In competition studies, NT and various fragments inhibited (/sup 3/H)NT binding with the following potencies (approximately IC50): NT 8-13 (0.4 nM), NT 1-13 (4 nM), NT 9-13 (130 nM), NT 1-11, NT 1-8 (greater than 100 microM). Quantitatively similar results were obtained using brain tissue. These findings raise the possibility of a role for NT in uterine function.

  9. ELISA-mimic screen for synthetic polymer nanoparticles with high affinity to target proteins.

    PubMed

    Yonamine, Yusuke; Hoshino, Yu; Shea, Kenneth J

    2012-09-10

    Synthetic polymer nanoparticles (NPs) that display high affinity to protein targets have significant potential for medical and biotechnological applications as protein capture agents or functional replacements of antibodies ("plastic antibodies"). In this study, we modified an immunological assay (enzyme-linked immunosorbent assay: ELISA) into a high-throughput screening method to select nanoparticles with high affinity to target proteins. Histone and fibrinogen were chosen as target proteins to demonstrate this concept. The selection process utilized a biotinylated NP library constructed with combinations of functional monomers. The screen identified NPs with distinctive functional group compositions that exhibited high affinity to either histone or fibrinogen. The variation of protein affinity with changes in the nature and amount of functional groups in the NP provided chemical insight into the principle determinants of protein-NP binding. The NP affinity was semiquantified using the ELISA-mimic assay by varying the NP concentrations. The screening results were found to correlate with solution-based assay results. This screening system utilizing a biotinylated NP is a general approach to optimize functional monomer compositions and can be used to rapidly search for synthetic polymers with high (or low) affinity for target biological macromolecules. PMID:22813352

  10. Single-experiment displacement assay for quantifying high-affinity binding by isothermal titration calorimetry.

    PubMed

    Krainer, Georg; Keller, Sandro

    2015-04-01

    Isothermal titration calorimetry (ITC) is the gold standard for dissecting the thermodynamics of a biomolecular binding process within a single experiment. However, reliable determination of the dissociation constant (KD) from a single titration is typically limited to the range 100 μM>KD>1 nM. Interactions characterized by a lower KD can be assessed indirectly by so-called competition or displacement assays, provided that a suitable competitive ligand is available whose KD falls within the directly accessible window. However, this protocol is limited by the fact that it necessitates at least two titrations to characterize one high-affinity inhibitor, resulting in considerable consumption of both sample material and time. Here, we introduce a fast and efficient ITC displacement assay that allows for the simultaneous characterization of both a high-affinity ligand and a moderate-affinity ligand competing for the same binding site on a receptor within a single experiment. The protocol is based on a titration of the high-affinity ligand into a solution containing the moderate-affinity ligand bound to the receptor present in excess. The resulting biphasic binding isotherm enables accurate and precise determination of KD values and binding enthalpies (ΔH) of both ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation, explore its potential and limitations with the aid of simulations and statistical analyses, and elaborate on potential applications to protein-inhibitor interactions.

  11. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates

    PubMed Central

    Noy-Porat, Tal; Rosenfeld, Ronit; Ariel, Naomi; Epstein, Eyal; Alcalay, Ron; Zvi, Anat; Kronman, Chanoch; Ordentlich, Arie; Mazor, Ohad

    2016-01-01

    Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit) was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (koff < 1 × 10−7 s−1) that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication. PMID:26950154

  12. Quality Control of a Cytoplasmic Protein Complex

    PubMed Central

    Scazzari, Mario; Amm, Ingo; Wolf, Dieter H.

    2015-01-01

    For the assembly of protein complexes in the cell, the presence of stoichiometric amounts of the respective protein subunits is of utmost importance. A surplus of any of the subunits may trigger unspecific and harmful protein interactions and has to be avoided. A stoichiometric amount of subunits must finally be reached via transcriptional, translational, and/or post-translational regulation. Synthesis of saturated 16 and 18 carbon fatty acids is carried out by fatty acid synthase: in yeast Saccharomyces cerevisiae, a 2.6-MDa molecular mass assembly containing six protomers each of two different subunits, Fas1 (β) and Fas2 (α). The (α)6(β)6 complex carries six copies of all eight enzymatic activities required for fatty acid synthesis. The FAS1 and FAS2 genes in yeast are unlinked and map on two different chromosomes. Here we study the fate of the α-subunit of the complex, Fas2, when its partner, the β-subunit Fas1, is absent. Individual subunits of fatty acid synthase are proteolytically degraded when the respective partner is missing. Elimination of Fas2 is achieved by the proteasome. Here we show that a ubiquitin transfer machinery is required for Fas2 elimination. The major ubiquitin ligase targeting the superfluous Fas2 subunit to the proteasome is Ubr1. The ubiquitin-conjugating enzymes Ubc2 and Ubc4 assist the degradation process. The AAA-ATPase Cdc48 and the Hsp70 chaperone Ssa1 are crucially involved in the elimination of Fas2. PMID:25564609

  13. Membrane protein complexes catalyze both 4- and 3-hydroxylation of cinnamic acid derivatives in monolignol biosynthesis.

    PubMed

    Chen, Hsi-Chuan; Li, Quanzi; Shuford, Christopher M; Liu, Jie; Muddiman, David C; Sederoff, Ronald R; Chiang, Vincent L

    2011-12-27

    The hydroxylation of 4- and 3-ring carbons of cinnamic acid derivatives during monolignol biosynthesis are key steps that determine the structure and properties of lignin. Individual enzymes have been thought to catalyze these reactions. In stem differentiating xylem (SDX) of Populus trichocarpa, two cinnamic acid 4-hydroxylases (PtrC4H1 and PtrC4H2) and a p-coumaroyl ester 3-hydroxylase (PtrC3H3) are the enzymes involved in these reactions. Here we present evidence that these hydroxylases interact, forming heterodimeric (PtrC4H1/C4H2, PtrC4H1/C3H3, and PtrC4H2/C3H3) and heterotrimeric (PtrC4H1/C4H2/C3H3) membrane protein complexes. Enzyme kinetics using yeast recombinant proteins demonstrated that the enzymatic efficiency (V(max)/k(m)) for any of the complexes is 70-6,500 times greater than that of the individual proteins. The highest increase in efficiency was found for the PtrC4H1/C4H2/C3H3-mediated p-coumaroyl ester 3-hydroxylation. Affinity purification-quantitative mass spectrometry, bimolecular fluorescence complementation, chemical cross-linking, and reciprocal coimmunoprecipitation provide further evidence for these multiprotein complexes. The activities of the recombinant and SDX plant proteins demonstrate two protein-complex-mediated 3-hydroxylation paths in monolignol biosynthesis in P. trichocarpa SDX; one converts p-coumaric acid to caffeic acid and the other converts p-coumaroyl shikimic acid to caffeoyl shikimic acid. Cinnamic acid 4-hydroxylation is also mediated by the same protein complexes. These results provide direct evidence for functional involvement of membrane protein complexes in monolignol biosynthesis.

  14. Principles of assembly reveal a periodic table of protein complexes.

    PubMed

    Ahnert, Sebastian E; Marsh, Joseph A; Hernández, Helena; Robinson, Carol V; Teichmann, Sarah A

    2015-12-11

    Structural insights into protein complexes have had a broad impact on our understanding of biological function and evolution. In this work, we sought a comprehensive understanding of the general principles underlying quaternary structure organization in protein complexes. We first examined the fundamental steps by which protein complexes can assemble, using experimental and structure-based characterization of assembly pathways. Most assembly transitions can be classified into three basic types, which can then be used to exhaustively enumerate a large set of possible quaternary structure topologies. These topologies, which include the vast majority of observed protein complex structures, enable a natural organization of protein complexes into a periodic table. On the basis of this table, we can accurately predict the expected frequencies of quaternary structure topologies, including those not yet observed. These results have important implications for quaternary structure prediction, modeling, and engineering. PMID:26659058

  15. Principles of assembly reveal a periodic table of protein complexes.

    PubMed

    Ahnert, Sebastian E; Marsh, Joseph A; Hernández, Helena; Robinson, Carol V; Teichmann, Sarah A

    2015-12-11

    Structural insights into protein complexes have had a broad impact on our understanding of biological function and evolution. In this work, we sought a comprehensive understanding of the general principles underlying quaternary structure organization in protein complexes. We first examined the fundamental steps by which protein complexes can assemble, using experimental and structure-based characterization of assembly pathways. Most assembly transitions can be classified into three basic types, which can then be used to exhaustively enumerate a large set of possible quaternary structure topologies. These topologies, which include the vast majority of observed protein complex structures, enable a natural organization of protein complexes into a periodic table. On the basis of this table, we can accurately predict the expected frequencies of quaternary structure topologies, including those not yet observed. These results have important implications for quaternary structure prediction, modeling, and engineering.

  16. Immersion freezing of ice nucleating active protein complexes

    NASA Astrophysics Data System (ADS)

    Hartmann, S.; Augustin, S.; Clauss, T.; Voigtländer, J.; Niedermeier, D.; Wex, H.; Stratmann, F.

    2012-08-01

    Biological particles, e.g. bacteria and their Ice Nucleating Active (INA) protein complexes, might play an important role for the ice formation in atmospheric mixed-phase clouds. Therefore, the immersion freezing behavior of INA protein complexes generated from a SnomaxTM solution/suspension was investigated as function of temperature in a range of -5 °C to -38 °C at the Leipzig Aerosol Cloud Interaction Simulator (LACIS). The immersion freezing of droplets containing small numbers of INA protein complexes occurs in a temperature range of -7 °C and -10 °C. The experiments performed in the lower temperature range, where all droplets freeze which contain at least one INA protein complex, are used to determine the average number of INA protein complexes present, assuming that the INA protein complexes are Poisson distributed over the droplet ensemble. Knowing the average number of INA protein complexes, the heterogeneous ice nucleation rate and rate coefficient of a single INA protein complex is determined by using the newly-developed CHESS model (stoCHastic model of idEntical poiSSon distributed ice nuclei). Therefore, we assume the ice nucleation process to be of stochastic nature, and a parameterization of the INA protein complex's nucleation rate. Analyzing the results of immersion freezing experiments from literature (SnomaxTM and Pseudomonas syringae bacteria), to results gained in this study, demonstrates that first, a similar temperature dependence of the heterogeneous ice nucleation rate for a single INA protein complex was found in all experiments, second, the shift of the ice fraction curves to higher temperatures can be explained consistently by a higher average number of INA protein complexes being present in the droplet ensemble, and finally the heterogeneous ice nucleation rate of one single INA protein complex might be also applicable for intact Pseudomonas syringae bacteria cells. The results obtained in this study allow a new perspective on the

  17. High-affinity antibodies to the 1,4-dihydropyridine Ca2+-channel blockers

    SciTech Connect

    Campbell, K.P.; Sharp, A.; Strom, M.; Kahl, S.D.

    1986-05-01

    Antibodies with high affinity and specificity for the 1,4-dihydropyridine Ca2+-channel blockers have been produced in rabbits by immunization with dihydropyridine-protein conjugates. Anti-dihydropyridine antibodies were found to specifically bind (/sup 3/H)nitrendipine, (/sup 3/H)-nimodipine, (/sup 3/H)nisoldipine, and (/sup 3/H)PN 200-110 (all 1,4-dihydropyridine Ca2+-channel blockers) with high affinity, while (/sup 3/H)verapamil, (/sup 3/H)diltiazem, and (/sup 3/H)trifluoperazine were not recognized. The average dissociation constant of the (/sup 3/H)nitrendipine-antibody complex was 0.06 (+/- 0.02) X 10(-9) M for an antiserum studied in detail and ranged from 0.01 to 0.24 X 10(-9) M for all antisera. Inhibition of (/sup 3/H)nitrendipine binding was specific for the 1,4-dihydropyridine Ca2+-channel modifiers and the concentrations required for half-maximal inhibition ranged between 0.25 and 0.90 nM. Structurally unrelated Ca2+-channel blockers, calmodulin antagonists, inactive metabolites of nitrendipine, and UV-inactivated nisoldipine did not modify (/sup 3/H)nitrendipine binding to the anti-dihydropyridine antibodies. Dihydropyridines without a bulky substituent in the 4-position of the heterocycle were able to displace (/sup 3/H)nitrendipine binding, but the concentrations required for half-maximal inhibition were greater than 800 nM. In summary, anti-dihydropyridine antibodies have been shown to have high affinity and specificity for the 1,4-dihydropyridine Ca2+-channel blockers and to exhibit dihydropyridine binding properties similar to the membrane receptor for the 1,4-dihydropyridine Ca2+-channel blockers.

  18. High affinity nanobodies against human epidermal growth factor receptor selected on cells by E. coli display

    PubMed Central

    Salema, Valencio; Mañas, Carmen; Cerdán, Lidia; Piñero-Lambea, Carlos; Marín, Elvira; Roovers, Rob C.; Van Bergen en Henegouwen, Paul M.P.; Fernández, Luis Ángel

    2016-01-01

    ABSTRACT Most therapeutic antibodies (Abs) target cell surface proteins on tumor and immune cells. Cloning of Ab gene libraries in E. coli and their display on bacteriophages is commonly used to select novel therapeutic Abs binding target antigens, either purified or expressed on cells. However, the sticky nature of bacteriophages renders phage display selections on cells challenging. We previously reported an E. coli display system for expression of VHHs (i.e., nanobodies, Nbs) on the surface of bacteria and selection of high-affinity clones by magnetic cell sorting (MACS). Here, we demonstrate that E. coli display is also an attractive method for isolation of Nbs against cell surface antigens, such as the epidermal growth factor receptor (EGFR), upon direct selection and screening of Ab libraries on live cells. We employ a whole cell-based strategy using a VHH library obtained by immunization with human tumor cells over-expressing EGFR (i.e., A431), and selection of bacterial clones bound to murine fibroblast NIH-3T3 cells transfected with human EGFR, after depletion of non-specific clones on untransfected cells. This strategy resulted in the isolation of high-affinity Nbs binding distinct epitopes of EGFR, including Nbs competing with the ligand, EGF, as characterized by flow cytometry of bacteria displaying the Nbs and binding assays with purified Nbs using surface plasmon resonance. Hence, our study demonstrates that E. coli display of VHH libraries and selection on cells enables efficient isolation and characterization of high-affinity Nbs against cell surface antigens. PMID:27472381

  19. High-affinity nasal extraction of vinyl acetate vapor is carboxylesterase dependent.

    PubMed

    Bogdanffy, M S; Manning, L A; Sarangapani, R

    1999-10-01

    Vinyl acetate induces nasal tumors in rats, but not mice. Species differences in airflow patterns, physiology, and biochemistry complicate extrapolation of nasal dosimetry from rats to humans. Physiologically based pharmacokinetic modeling of vinyl acetate dosimetry in rats suggested the presence of a saturable metabolic removal pathway in rat nasal mucus. We explored the possibility that this pathway is either a cytochrome P-450 2E1 (CYP2E1) or high-affinity carboxylesterase. Nasal extraction of vinyl acetate vapor (150 ppm) was measured in the surgically isolated nasal cavity of anesthetized rats. Vinyl acetate (150 ppm) was extracted with 73% efficiency in controls. Pretreatment of rats with the CYP2E1 inhibitor diallyl sulfide (DAS) had no effect on extraction, despite significantly reducing CYP2E1 activity. Pretreatment with bis(p-nitrophenyl) phosphate (BNPP), a carboxylesterase inhibitor, reduced extraction to approximately 41%. Acetaldehyde production was similarly unaffected by DAS but was reduced to 55% of control by BNPP. Rat nasal mucus carboxylesterase activity had a K(m) value (32 microM) similar, within a factor of 2, to the value predicted by the physiologically based model, although V(max) was significantly lower than the model prediction. Histochemical observations support the inference that the high-affinity carboxylesterase is bound to the luminal plasma membrane of nasal tissue and is not readily released by nasal lavage, providing an explanation for the low V(max) of the lavage enzyme. This high-affinity isoenzyme could be important in the removal of odorants from the sensory cell-rich nasal olfactory epithelium.

  20. Characterization of specific high affinity receptors for human tumor necrosis factor on mouse fibroblasts

    SciTech Connect

    Hass, P.E.; Hotchkiss, A.; Mohler, M.; Aggarwal, B.B.

    1985-10-05

    Mouse L-929 fibroblasts, an established line of cells, are very sensitive to lysis by human lymphotoxin (hTNF-beta). Specific binding of a highly purified preparation of hTNF-beta to these cells was examined. Recombinant DNA-derived hTNF-beta was radiolabeled with (TH)propionyl succinimidate at the lysine residues of the molecule to a specific activity of 200 microCi/nmol of protein. (TH)hTNF-beta was purified by high performance gel permeation chromatography and the major fraction was found to be monomeric by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The labeled hTNF-beta was fully active in causing lysis of L-929 fibroblasts and bound specifically to high affinity binding sites on these cells. Scatchard analysis of the binding data revealed the presence of a single class of high affinity receptors with an apparent Kd of 6.7 X 10(-11) M and a capacity of 3200 binding sites/cell. Unlabeled recombinant DNA-derived hTNF-beta was found to be approximately 5-fold more effective competitive inhibitor of binding than the natural hTNF-beta. The binding of hTNF-beta to these mouse fibroblasts was also correlated with the ultimate cell lysis. Neutralizing polyclonal antibodies to hTNF-beta efficiently inhibited the binding of (TH)hTNF-beta to the cells. The authors conclude that the specific high affinity binding site is the receptor for hTNF-beta and may be involved in lysis of cells.

  1. High-affinity benzodiazepine receptor ligands among benzodiazepines and betacarbolines with different intrinsic activity

    SciTech Connect

    Yliniemelae, A.; Gynther, J. ); Konschin, H.; Tylli, H. ); Rouvinen, J. )

    1989-01-01

    Structural and electrostatic features of diazepam, flumazenil, and methyl betacarboline-3-carboxylate (BCCM) have been investigated using the molecular superimposition method. These high-affinity benzodiazepine (BZ) receptor ligands are structurally unrelated and they have different intrinsic activity. These ligands are superimposed in such a way that common structural and electrostatic features essential for the high receptor binding affinity overlap. In addition to this binding pharmacophore, there are roughly three separate binding zones in the BZ receptor, one for each class of ligands. The intrinsic activity of BZ receptor ligands depends on the molecular structures and the way the ligand approaches the receptor.

  2. Diacylglycerol kinase η1 is a high affinity isozyme for diacylglycerol.

    PubMed

    Komenoi, Suguru; Takemura, Fumika; Sakai, Hiromichi; Sakane, Fumio

    2015-05-01

    Diacylglycerol kinase (DGK) η plays important roles in various patho-physiological events such as oncogenesis. In this study, we performed an enzymological characterization of DGKη splice variant 1 (DGKη1). The Km value for diacylglycerol was 0.14 mol%. Intriguingly, the Km value of DGKη1 for diacylglycerol was at least 9-fold lower than those of other DGK isozymes including DGKα, indicating that DGKη1 is a high affinity isozyme for diacylglycerol. Therefore, DGKη1 is a unique DGK isozyme, which may function at particular membrane sites where only low concentrations of diacylglycerol are supplied.

  3. Choline uptake in Agrobacterium tumefaciens by the high-affinity ChoXWV transporter.

    PubMed

    Aktas, Meriyem; Jost, Kathinka A; Fritz, Christiane; Narberhaus, Franz

    2011-10-01

    Agrobacterium tumefaciens is a facultative phytopathogen that causes crown gall disease. For successful plant transformation A. tumefaciens requires the membrane lipid phosphatidylcholine (PC), which is produced via the methylation and the PC synthase (Pcs) pathways. The latter route is dependent on choline. Although choline uptake has been demonstrated in A. tumefaciens, the responsible transporter(s) remained elusive. In this study, we identified the first choline transport system in A. tumefaciens. The ABC-type choline transporter is encoded by the chromosomally located choXWV operon (ChoX, binding protein; ChoW, permease; and ChoV, ATPase). The Cho system is not critical for growth and PC synthesis. However, [14C]choline uptake is severely reduced in A. tumefaciens choX mutants. Recombinant ChoX is able to bind choline with high affinity (equilibrium dissociation constant [KD] of ≈2 μM). Since other quaternary amines are bound by ChoX with much lower affinities (acetylcholine, KD of ≈80 μM; betaine, KD of ≈470 μM), the ChoXWV system functions as a high-affinity transporter with a preference for choline. Two tryptophan residues (W40 and W87) located in the predicted ligand-binding pocket are essential for choline binding. The structural model of ChoX built on Sinorhizobium meliloti ChoX resembles the typical structure of substrate binding proteins with a so-called "Venus flytrap mechanism" of substrate binding. PMID:21803998

  4. Mechanism of high affinity inhibition of the human urate transporter URAT1

    PubMed Central

    Tan, Philip K.; Ostertag, Traci M.; Miner, Jeffrey N.

    2016-01-01

    Gout is caused by elevated serum urate levels, which can be treated using inhibitors of the uric acid transporter, URAT1. We exploited affinity differences between the human and rat transporters to map inhibitor binding sites in URAT1. Human-rat transporter chimeras revealed that human URAT1 serine-35, phenylalanine-365 and isoleucine-481 are necessary and sufficient to provide up to a 100-fold increase in affinity for inhibitors. Moreover, serine-35 and phenylalanine-365 are important for high-affinity interaction with the substrate urate. A novel URAT1 binding assay provides support for direct interaction with these amino acids; thus, current clinically important URAT1 inhibitors likely bind the same site in URAT1. A structural model suggests that these three URAT1 residues are in close proximity potentially projecting within the channel. Our results indicate that amino acids from several transmembrane segments functionally cooperate to form a high-affinity URAT1 inhibitor binding site that, when occupied, prevents substrate interactions. PMID:27713539

  5. High affinity binding of (/sup 3/H)cocaine to rat liver microsomes

    SciTech Connect

    El-Maghrabi, E.A.; Calligaro, D.O.; Eldefrawi, M.E.

    1988-01-01

    )/sup 3/H)cocaine bound reversible, with high affinity and stereospecificity to rat liver microsomes. Little binding was detected in the lysosomal, mitochondrial and nuclear fractions. The binding kinetics were slow and the kinetically calculated K/sub D/ was 2 nM. Induction of mixed function oxidases by phenobarbital did not produce significant change in (/sup 3/H)cocaine binding. On the other hand, chronic administration of cocaine reduced (/sup 3/H)cocaine binding drastically. Neither treatment affected the affinity of the liver binding protein for cocaine. Microsomes from mouse and human livers had less cocaine-binding protein and lower affinity for cocaine than those from rat liver. Binding of (/sup 3/H)cocaine to rat liver microsomes was insensitive to monovalent cations and > 10 fold less sensitive to biogenic amines than the cocaine receptor in rat striatum. However, the liver protein had higher affinity for cocaine and metabolites except for norcocaine. Amine uptake inhibitors displaced (/sup 3/H)cocaine binding to liver with a different rank order of potency than their displacement of (/sup 3/H)cocaine binding to striatum. This high affinity (/sup 3/H)cocaine binding protein in liver is not likely to be monooxygenase, but may have a role in cocaine-induced hepatotoxicity

  6. The human organic cation transporter OCT1 mediates high affinity uptake of the anticancer drug daunorubicin

    PubMed Central

    Andreev, Emil; Brosseau, Nicolas; Carmona, Euridice; Mes-Masson, Anne-Marie; Ramotar, Dindial

    2016-01-01

    Anthracyclines such as daunorubicin are anticancer agents that are transported into cells, and exert cytotoxicity by blocking DNA metabolism. Although there is evidence for active uptake of anthracyclines into cells, the specific transporter involved in this process has not been identified. Using the high-grade serous ovarian cancer cell line TOV2223G, we show that OCT1 mediated the high affinity (Km ~ 5 μM) uptake of daunorubicin into the cells, and that micromolar amounts of choline completely abolished the drug entry. OCT1 downregulation by shRNA impaired daunorubicin uptake into the TOV2223G cells, and these cells were significantly more resistant to the drug in comparison to the control shRNA. Transfection of HEK293T cells, which accommodated the ectopic expression of OCT1, with a plasmid expressing OCT1-EYFP showed that the transporter was predominantly localized to the plasma membrane. These transfected cells exhibited an increase in the uptake of daunorubicin in comparison to control cells transfected with an empty EYFP vector. Furthermore, a variant of OCT1, OCT1-D474C-EYFP, failed to enhance daunorubicin uptake. This is the first report demonstrating that human OCT1 is involved in the high affinity transport of anthracyclines. We postulate that OCT1 defects may contribute to the resistance of cancer cells treated with anthracyclines. PMID:26861753

  7. Screening of high-affinity scFvs from a ribosome displayed library using BIAcore biosensor.

    PubMed

    Yuan, Qing; Wang, Zhongkang; Nian, Siji; Yin, Youping; Chen, Gang; Xia, Yuxian

    2009-02-01

    An experimental protocol was developed to screen high-affinity single-chain Fv antibody fragments (scFvs) from a Xanthomonas axonopodis pv. citri (Xac) immunized ribosome display library using BIAcore biosensor. The screening methods involved immobilizing antigen [lipopolysaccharides (LPS) of Xac] on sensor chip HPA and then unpurified expression products of scFvs flowing over the immobilized sensor chip. The affinity-improved scFvs were selected based on dissociation rate constants (k (d)). Thirty-five enzyme-linked immunosorbent assay-positive scFvs were analyzed by BIAcore, and three of those (scFv A1, B2, and C5) with lower k (d) were screened. To demonstrate the accuracy of the screening method, the three scFvs were expressed in Escherichia coli HB2151 and purified. The purified scFvs were subsequently further identified according to association rate and affinity constants. The results showed that the three scFvs (A1, B2, and C5) had high affinity for LPS of Xac (3.51 x 10(-11), 1.13 x 10(-10), 5.06 x 10(-10) M, respectively). Furthermore, the scFv B2 was highly specific for LPS of Xac and had no any cross-reactions with bovine serum albumin and LPS from Xac-related bacteria. This provided evidence that the information from the BIAcore screening assay could be accurate. PMID:18574567

  8. Genetic evidence of a high-affinity cyanuric acid transport system in Pseudomonas sp. ADP.

    PubMed

    Platero, Ana I; Santero, Eduardo; Govantes, Fernando

    2014-03-01

    The Pseudomonas sp. ADP plasmid pADP-1 encodes the activities involved in the hydrolytic degradation of the s-triazine herbicide atrazine. Here, we explore the presence of a specific transport system for the central intermediate of the atrazine utilization pathway, cyanuric acid, in Pseudomonas sp. ADP. Growth in fed-batch cultures containing limiting cyanuric acid concentrations is consistent with high-affinity transport of this substrate. Acquisition of the ability to grow at low cyanuric acid concentrations upon conjugal transfer of pADP1 to the nondegrading host Pseudomonas putida KT2442 suggests that all activities required for this phenotype are encoded in this plasmid. Co-expression of the pADP1-borne atzDEF and atzTUVW genes, encoding the cyanuric acid utilization pathway and the subunits of an ABC-type solute transport system, in P. putida KT2442 was sufficient to promote growth at cyanuric acid concentrations as low as 50 μM in batch culture. Taken together, our results strongly suggest that the atzTUVW gene products are involved in high-affinity transport of cyanuric acid.

  9. Synergistic effect of high-affinity binding and flow preconditioning on endothelial cell adhesion.

    PubMed

    Mathur, Anshu B; Truskey, George A; Reichert, William M

    2003-01-01

    The current study examined whether the combined introduction of high-affinity avidin-biotin bonds and fibronectin-integrin bonds (i.e., dual ligand treatment) would further augment the adhesion of flow-preconditioned endothelial cells to model substrates via contributions to the actin cytoskeleton and the formation of focal contacts. Human umbilical vein endothelial cells (HUVEC) were grown under static conditions or exposed to a flow-preconditioning regimen for 24 h. Cell retention was determined by exposure to 75 dynes/cm(2). The combination of flow preconditioning and the dual ligand treatment yielded higher cell retention under flow compared to the cells adherent via fibronectin-integrin bonds only. This increase in adhesion strength correlated with a greater focal contact area. Elongation of the HUVEC occurred after exposure to flow preconditioning; however, orientation of dual ligand adherent cells was restricted due to the presence of the high-affinity ligand. Flow-preconditioned cells showed increased stress fiber formation compared to nonconditioned cells although the stress fibers per cell for flow-preconditioned cells were the same on both the ligand systems employed. The results indicate that enhanced adhesion strength is due to a combination of increased focal contact area, stress fiber formation, and cell alignment. PMID:12483708

  10. Humanization of high-affinity antibodies targeting glypican-3 in hepatocellular carcinoma

    PubMed Central

    Zhang, Yi-Fan; Ho, Mitchell

    2016-01-01

    Glypican-3 (GPC3) is a cell-surface heparan sulfate proteoglycan highly expressed in hepatocellular carcinoma (HCC). We have generated a group of high-affinity mouse monoclonal antibodies targeting GPC3. Here, we report the humanization and testing of these antibodies for clinical development. We compared the affinity and cytotoxicity of recombinant immunotoxins containing mouse single-chain variable regions fused with a Pseudomonas toxin. To humanize the mouse Fvs, we grafted the combined KABAT/IMGT complementarity determining regions (CDR) into a human IgG germline framework. Interestingly, we found that the proline at position 41, a non-CDR residue in heavy chain variable regions (VH), is important for humanization of mouse antibodies. We also showed that two humanized anti-GPC3 antibodies (hYP7 and hYP9.1b) in the IgG format induced antibody-dependent cell-mediated cytotoxicity and complement-dependent-cytotoxicity in GPC3-positive cancer cells. The hYP7 antibody was tested and showed inhibition of HCC xenograft tumor growth in nude mice. This study successfully humanizes and validates high affinity anti-GPC3 antibodies and sets a foundation for future development of these antibodies in various clinical formats in the treatment of liver cancer. PMID:27667400

  11. Only high-affinity receptors for interleukin 2 mediate internalization of ligand

    SciTech Connect

    Weissman, A.M.; Harford, J.B.; Svetlik, P.B.; Leonard, W.L.; Depper, J.M.; Waldmann, T.A.; Greene, W.C.; Klausner, R.D.

    1986-03-01

    Interleukin 2 (IL-2) receptors are expressed on activated T cells and in select T-cell leukemias. Recently, it has been demonstrated that at least two classes of receptor for IL-2 exist with markedly different affinities for ligand. All known biological actions of IL-2 have been correlated with occupancy of high-affinity sites; the function of the low-affinity sites remains unknown. Receptor-mediated endocytosis is the primary means of internalization of cell-surface receptors and their ligands. The internalization of IL-2 bound to high- and low-affinity receptor sites was studied in a human T-cell lymphotrophic virus type 1 (HTLV-1)-infected human T-cell leukemia cell line and in a cloned murine cytotoxic T-cell line (CTLL). Internalization of IL-2 occurred only when bound to high-affinity sites. In addition, an anti-receptor antibody (anti-Tac), which binds equally well to high- and low-affinity sites, demonstrated no detectable internalization. The implications of these findings as they relate to IL-2 receptor structure and function are discussed.

  12. High-affinity receptors for bombesin-like peptides in normal guinea pig lung membranes

    SciTech Connect

    Lach, E.; Trifilieff, A.; Landry, Y.; Gies, J.P. )

    1991-01-01

    The binding of the radiolabeled bombesin analogue ({sup 125}I-Tyr{sup 4})bombesin to guinea-pig lung membranes was investigated. Binding of ({sup 125}I-Tyr{sup 4})bombesin was specific, saturable, reversible and linearly related to the protein concentration. Scatchard analysis of equilibrium binding data at 25C indicated the presence of a single class of non-interacting binding sites for bombesin (B{sub max} = 7.7 fmol/mg protein). The value of the equilibrium dissociation constant (K{sub D} = 90 pM) agrees with a high-affinity binding site. Bombesin and structurally related peptides such as ({sup 125}I-Tyr{sup 4})bombesin, neuromedin B and neuromedin C inhibited the binding of ({sup 125}I-Tyr{sup 4})bombesin in an order of potencies as follows: ({sup 125}I-Tyr{sup 4})bombesin {gt} bombesin {ge} neuromedin C {much gt} neuromedin B. These results indicate that guinea-pig lung membranes possess a single class of bombesin receptors with a high affinity for bombesin and a lower one for neuromedin B.

  13. In Vivo Neutralization of α-Cobratoxin with High-Affinity Llama Single-Domain Antibodies (VHHs) and a VHH-Fc Antibody

    PubMed Central

    Richard, Gabrielle; Meyers, Ashley J.; McLean, Michael D.; Arbabi-Ghahroudi, Mehdi; MacKenzie, Roger; Hall, J. Christopher

    2013-01-01

    Small recombinant antibody fragments (e.g. scFvs and VHHs), which are highly tissue permeable, are being investigated for antivenom production as conventional antivenoms consisting of IgG or F(ab’)2 antibody fragments do not effectively neutralize venom toxins located in deep tissues. However, antivenoms composed entirely of small antibody fragments may have poor therapeutic efficacy due to their short serum half-lives. To increase serum persistence and maintain tissue penetration, we prepared low and high molecular mass antivenom antibodies. Four llama VHHs were isolated from an immune VHH-displayed phage library and were shown to have high affinity, in the low nM range, for α-cobratoxin (α–Cbtx), the most lethal component of Naja kaouthia venom. Subsequently, our highest affinity VHH (C2) was fused to a human Fc fragment to create a VHH2-Fc antibody that would offer prolonged serum persistence. After in planta (Nicotiana benthamiana) expression and purification, we show that our VHH2-Fc antibody retained high affinity binding to α–Cbtx. Mouse α–Cbtx challenge studies showed that our highest affinity VHHs (C2 and C20) and the VHH2-Fc antibody effectively neutralized lethality induced by α–Cbtx at an antibody:toxin molar ratio as low as ca. 0.75×:1. Further research towards the development of an antivenom therapeutic involving these anti-α-Cbtx VHHs and VHH2-Fc antibody molecules should involve testing them as a combination, to determine whether they maintain tissue penetration capability and low immunogenicity, and whether they exhibit improved serum persistence and therapeutic efficacy. PMID:23894495

  14. Identification of Signaling Protein Complexes by Parallel Affinity Precipitation Coupled with Mass Spectrometry

    PubMed Central

    Lu, Heng; Lin, Qishan; Zhao, Jihe

    2014-01-01

    Protein–protein interactions play a pivotal role in both inter- and intra-cellular signaling. Identification of signaling protein complexes can thus shed important new insights into cell communications. We developed a parallel affinity precipitation protocol to overcome the disadvantages of the tandem affinity purification procedure, such as the potential disruption of target protein conformation, subcellular localization or function by epitope tags, the potential need of large amounts of cell culture or generation of stable cell lines, and relatively long duration the two-step precipitation takes. This new simplified assay of protein interaction is quick, economic and specific. This paper describes the details in the design and method of the assay. PMID:24839392

  15. A nitrogen-dependent switch in the high affinity ammonium transport in Medicago truncatula.

    PubMed

    Straub, Daniel; Ludewig, Uwe; Neuhäuser, Benjamin

    2014-11-01

    Ammonium transporters (AMTs) are crucial for the high affinity primary uptake and translocation of ammonium in plants. In the model legume Medicago truncatula, the genomic set of AMT-type ammonium transporters comprises eight members. Only four genes were abundantly expressed in young seedlings, both in roots and shoots. While the expression of all AMTs in the shoot was not affected by the nitrogen availability, the dominating MtAMT1;1 gene was repressed by nitrogen in roots, despite that cellular nitrogen concentrations were far above deficiency levels. A contrasting de-repression by nitrogen was observed for MtAMT1;4 and MtAMT2;1, which were both expressed at intermediate level. Weak expression was found for MtAMT1;2 and MtAMT2;3, while the other AMTs were not detected in young seedlings. When expressed from their endogenous promoters, translational fusion proteins of MtAMT1;1 and MtAMT2;1 with green fluorescent protein were co-localized in the plasma membrane of rhizodermal cells, but also detected in cortical root layers. Both transporter proteins similarly functionally complemented a yeast strain that is deficient in high affinity ammonium transport, both at acidic and neutral pH. The uptake into yeast mediated by these transporters saturated with Km AMT1;1 = 89 µM and Km AMT2;1 = 123 µM, respectively. When expressed in oocytes, MtAMT1;1 mediated much larger (15)N-ammonium uptake than MtAMT2;1, but NH4 (+) currents were only recorded for MtAMT1;1. These currents saturated with a voltage-dependent Km = 90 µM at -80 mV. The cellular localization and regulation of the AMTs suggests that MtAMT1;1 encodes the major high affinity ammonium transporter gene in low nitrogen grown young M. truncatula roots and despite the similar localization and substrate affinity, MtAMT2;1 appears functionally distinct and more important at higher nitrogen supply. PMID:25164101

  16. Cross-linking approach to affinity capture of protein complexes from chaotrope-solubilized cell lysates.

    PubMed

    Alloza, Iraide; Martens, Erik; Hawthorne, Susan; Vandenbroeck, Koen

    2004-01-01

    Affinity capture methods are widely used for isolation and analysis of protein complexes. Short peptide tags fused to the protein of interest normally facilitate straightforward purification and detection of interacting proteins. We investigated the suitability of applying C-terminally hexahistidine-tagged interleukin-12 (IL-12) alpha- and beta-chains as "bait" proteins for cocapturing novel binding partners using heterologous recombinant human embryonic kidney-293 (HEK-293) cell lines. The beta-chain, but not the alpha-chain, extracted from cell lysates was capable of binding to the Ni(2+)-nitrilotriacetic acid affinity resin under nondenaturing conditions. Retention of the alpha-chain on this matrix was dependent on treatment of cell lysates with high concentrations of chaotropes such as urea. Since under these conditions any noncovalent protein associations are destroyed, prior cross-linking of proteins interacting with the alpha-chain in intact cells was required. The use of the thiol-cleavable cross-linker 3,3'-dithiobis(succinimidyl proprionate) facilitated dissociation of alpha-chain-binding proteins by means of dithiothreitol following purification. Using this approach we were able to demonstrate a strong interaction between the endoplasmic reticulum chaperone calreticulin (CRT) and the IL-12 alpha-chain that was confirmed in a reciprocal anti-CRT immunoprecipitation assay. The assay presented here provides a simple approach to exposing concealed hexahistidine tags while retaining native noncovalent protein interactions and should be generally applicable in a range of pull-down or affinity capture methods aiming at analysis of protein complexes. PMID:14654056

  17. A Bacillus megaterium System for the Production of Recombinant Proteins and Protein Complexes.

    PubMed

    Biedendieck, Rebekka

    2016-01-01

    For many years the Gram-positive bacterium Bacillus megaterium has been used for the production and secretion of recombinant proteins. For this purpose it was systematically optimized. Plasmids with different inducible promoter systems, with different compatible origins, with small tags for protein purification and with various specific signals for protein secretion were combined with genetically improved host strains. Finally, the development of appropriate cultivation conditions for the production strains established this organism as a bacterial cell factory even for large proteins. Along with the overproduction of individual proteins the organism is now also used for the simultaneous coproduction of up to 14 recombinant proteins, multiple subsequently interacting or forming protein complexes. Some of these recombinant strains are successfully used for bioconversion or the biosynthesis of valuable components including vitamins. The titers in the g per liter scale for the intra- and extracellular recombinant protein production prove the high potential of B. megaterium for industrial applications. It is currently further enhanced for the production of recombinant proteins and multi-subunit protein complexes using directed genetic engineering approaches based on transcriptome, proteome, metabolome and fluxome data. PMID:27165321

  18. Co-expression of RNA–protein complexes in Escherichia coli and applications to RNA biology

    PubMed Central

    Ponchon, Luc; Catala, Marjorie; Seijo, Bili; El Khouri, Marguerite; Dardel, Frédéric; Nonin-Lecomte, Sylvie; Tisné, Carine

    2013-01-01

    RNA has emerged as a major player in many cellular processes. Understanding these processes at the molecular level requires homogeneous RNA samples for structural, biochemical and pharmacological studies. We previously devised a generic approach that allows efficient in vivo expression of recombinant RNA in Escherichia coli. In this work, we have extended this method to RNA/protein co-expression. We have engineered several plasmids that allow overexpression of RNA–protein complexes in E. coli. We have investigated the potential of these tools in many applications, including the production of nuclease-sensitive RNAs encapsulated in viral protein pseudo-particles, the co-production of non-coding RNAs with chaperone proteins, the incorporation of a post-transcriptional RNA modification by co-production with the appropriate modifying enzyme and finally the production and purification of an RNA–His-tagged protein complex by nickel affinity chromatography. We show that this last application easily provides pure material for crystallographic studies. The new tools we report will pave the way to large-scale structural and molecular investigations of RNA function and interactions with proteins. PMID:23804766

  19. Interaction network containing conserved and essential protein complexes in Escherichia coli

    NASA Astrophysics Data System (ADS)

    Butland, Gareth; Peregrín-Alvarez, José Manuel; Li, Joyce; Yang, Wehong; Yang, Xiaochun; Canadien, Veronica; Starostine, Andrei; Richards, Dawn; Beattie, Bryan; Krogan, Nevan; Davey, Michael; Parkinson, John; Greenblatt, Jack; Emili, Andrew

    2005-02-01

    Proteins often function as components of multi-subunit complexes. Despite its long history as a model organism, no large-scale analysis of protein complexes in Escherichia coli has yet been reported. To this end, we have targeted DNA cassettes into the E. coli chromosome to create carboxy-terminal, affinity-tagged alleles of 1,000 open reading frames (~ 23% of the genome). A total of 857 proteins, including 198 of the most highly conserved, soluble non-ribosomal proteins essential in at least one bacterial species, were tagged successfully, whereas 648 could be purified to homogeneity and their interacting protein partners identified by mass spectrometry. An interaction network of protein complexes involved in diverse biological processes was uncovered and validated by sequential rounds of tagging and purification. This network includes many new interactions as well as interactions predicted based solely on genomic inference or limited phenotypic data. This study provides insight into the function of previously uncharacterized bacterial proteins and the overall topology of a microbial interaction network, the core components of which are broadly conserved across Prokaryota.

  20. A Bacillus megaterium System for the Production of Recombinant Proteins and Protein Complexes.

    PubMed

    Biedendieck, Rebekka

    2016-01-01

    For many years the Gram-positive bacterium Bacillus megaterium has been used for the production and secretion of recombinant proteins. For this purpose it was systematically optimized. Plasmids with different inducible promoter systems, with different compatible origins, with small tags for protein purification and with various specific signals for protein secretion were combined with genetically improved host strains. Finally, the development of appropriate cultivation conditions for the production strains established this organism as a bacterial cell factory even for large proteins. Along with the overproduction of individual proteins the organism is now also used for the simultaneous coproduction of up to 14 recombinant proteins, multiple subsequently interacting or forming protein complexes. Some of these recombinant strains are successfully used for bioconversion or the biosynthesis of valuable components including vitamins. The titers in the g per liter scale for the intra- and extracellular recombinant protein production prove the high potential of B. megaterium for industrial applications. It is currently further enhanced for the production of recombinant proteins and multi-subunit protein complexes using directed genetic engineering approaches based on transcriptome, proteome, metabolome and fluxome data.

  1. Practical strategies for the evaluation of high-affinity protein/nucleic acid interactions.

    PubMed

    Altschuler, Sarah E; Lewis, Karen A; Wuttke, Deborah S

    2013-01-01

    The quantitative evaluation of binding interactions between proteins and nucleic acids is highly sensitive to a variety of experimental conditions. Optimization of these conditions is critical for obtaining high quality, reproducible data, particularly in the context of very high affinity interactions. Here, we discuss the practical considerations involved in optimizing the apparent binding constant of an interaction as measured by two common quantitative assays, electrophoretic mobility shift assay and double-filter binding when measuring extremely tight protein/nucleic acid interactions with sub-nanomolar binding affinities. We include specific examples from two telomere end-binding protein systems, Schizo -saccharomyces pombe Pot1 and Saccharomyces cerevisiae Cdc13, to demonstrate potential experimental pitfalls and some useful strategies for optimization.

  2. Practical strategies for the evaluation of high-affinity protein/nucleic acid interactions

    PubMed Central

    Altschuler, Sarah E.; Lewis, Karen A.; Wuttke, Deborah S.

    2014-01-01

    The quantitative evaluation of binding interactions between proteins and nucleic acids is highly sensitive to a variety of experimental conditions. Optimization of these conditions is critical for obtaining high quality, reproducible data, particularly in the context of very high affinity interactions. Here, we discuss the practical considerations involved in optimizing the apparent binding constant of an interaction as measured by two common quantitative assays, electrophoretic mobility shift assay and double-filter binding when measuring extremely tight protein/nucleic acid interactions with sub-nanomolar binding affinities. We include specific examples from two telomere end-binding protein systems, Schizo -saccharomyces pombe Pot1 and Saccharomyces cerevisiae Cdc13, to demonstrate potential experimental pitfalls and some useful strategies for optimization. PMID:25197549

  3. Neutrophil recruitment limited by high-affinity bent β2 integrin binding ligand in cis

    PubMed Central

    Fan, Zhichao; McArdle, Sara; Marki, Alex; Mikulski, Zbigniew; Gutierrez, Edgar; Engelhardt, Britta; Deutsch, Urban; Ginsberg, Mark; Groisman, Alex; Ley, Klaus

    2016-01-01

    Neutrophils are essential for innate immunity and inflammation and many neutrophil functions are β2 integrin-dependent. Integrins can extend (E+) and acquire a high-affinity conformation with an ‘open' headpiece (H+). The canonical switchblade model of integrin activation proposes that the E+ conformation precedes H+, and the two are believed to be structurally linked. Here we show, using high-resolution quantitative dynamic footprinting (qDF) microscopy combined with a homogenous conformation-reporter binding assay in a microfluidic device, that a substantial fraction of β2 integrins on human neutrophils acquire an unexpected E−H+ conformation. E−H+ β2 integrins bind intercellular adhesion molecules (ICAMs) in cis, which inhibits leukocyte adhesion in vitro and in vivo. This endogenous anti-inflammatory mechanism inhibits neutrophil aggregation, accumulation and inflammation. PMID:27578049

  4. Mu/sub 1/: A very high affinity subtype of enkephalin binding sites in rat brain

    SciTech Connect

    Lutz, R.A.; Cruciani, R.A.; Munson, P.J.; Rodbard, D.

    1985-06-10

    Displacement studies of (/sup 3/H)-(D-Ala/sup 2/-MePhe/sup 4/-Gly-ol/sup 5/)-enkephalin ((/sup 3/H)-DAGO) and (/sup 3/H)-(D-Ala/sup 2/-D-Leu/sup 5/)-enkephalin ((/sup 3/H)-DADL) by the corresponding unlabeled ligands show that there are at least three classes of sites which bind these enkephalin analogs with high affinity. Using computer modeling, the introduction of the third site significantly improved the goodness of fit in ten consecutive experiments. These sites appear to correspond to the ..mu.., delta, and ..mu../sub 1/ sites, with mean dissociation constants of 11, 1.3 and 0.9 nM for DADL and 2.5, 300 and 0.3 nM for DAGO, respectively. 15 reference, 3 figures, 1 table.

  5. Structure-based Design of Peptides with High Affinity and Specificity to HER2 Positive Tumors

    PubMed Central

    Geng, Lingling; Wang, Zihua; Yang, Xiaoliang; Li, Dan; Lian, Wenxi; Xiang, Zhichu; Wang, Weizhi; Bu, Xiangli; Lai, Wenjia; Hu, Zhiyuan; Fang, Qiaojun

    2015-01-01

    To identify peptides with high affinity and specificity against human epidermal growth factor receptor 2 (HER2), a series of peptides were designed based on the structure of HER2 and its Z(HER2:342) affibody. By using a combination protocol of molecular dynamics modeling, MM/GBSA binding free energy calculations, and binding free energy decomposition analysis, two novel peptides with 27 residues, pep27 and pep27-24M, were successfully obtained. Immunocytochemistry and flow cytometry analysis verified that both peptides can specifically bind to the extracellular domain of HER2 protein at cellular level. The Surface Plasmon Resonance imaging (SPRi) analysis showed that dissociation constants (KD) of these two peptides were around 300 nmol/L. Furthermore, fluorescence imaging of peptides against nude mice xenografted with SKBR3 cells indicated that both peptides have strong affinity and high specificity to HER2 positive tumors. PMID:26284145

  6. Protein unfolding as a switch from self-recognition to high-affinity client binding

    PubMed Central

    Groitl, Bastian; Horowitz, Scott; Makepeace, Karl A. T.; Petrotchenko, Evgeniy V.; Borchers, Christoph H.; Reichmann, Dana; Bardwell, James C. A.; Jakob, Ursula

    2016-01-01

    Stress-specific activation of the chaperone Hsp33 requires the unfolding of a central linker region. This activation mechanism suggests an intriguing functional relationship between the chaperone's own partial unfolding and its ability to bind other partially folded client proteins. However, identifying where Hsp33 binds its clients has remained a major gap in our understanding of Hsp33's working mechanism. By using site-specific Fluorine-19 nuclear magnetic resonance experiments guided by in vivo crosslinking studies, we now reveal that the partial unfolding of Hsp33's linker region facilitates client binding to an amphipathic docking surface on Hsp33. Furthermore, our results provide experimental evidence for the direct involvement of conditionally disordered regions in unfolded protein binding. The observed structural similarities between Hsp33's own metastable linker region and client proteins present a possible model for how Hsp33 uses protein unfolding as a switch from self-recognition to high-affinity client binding. PMID:26787517

  7. Preorganized Peptide Scaffolds as Mimics of Phosphorylated Proteins Binding Sites with a High Affinity for Uranyl.

    PubMed

    Starck, Matthieu; Sisommay, Nathalie; Laporte, Fanny A; Oros, Stéphane; Lebrun, Colette; Delangle, Pascale

    2015-12-01

    Cyclic peptides with two phosphoserines and two glutamic acids were developed to mimic high-affinity binding sites for uranyl found in proteins such as osteopontin, which is believed to be a privileged target of this ion in vivo. These peptides adopt a β-sheet structure that allows the coordination of the latter amino acid side chains in the equatorial plane of the dioxo uranyl cation. Complementary spectroscopic and analytical methods revealed that these cyclic peptides are efficient uranyl chelating peptides with a large contribution from the phosphorylated residues. The conditional affinity constants were measured by following fluorescence tryptophan quenching and are larger than 10(10) at physiological pH. These compounds are therefore promising models for understanding uranyl chelation by proteins, which is relevant to this actinide ion toxicity. PMID:26583259

  8. Further characterization of the subunits of the receptor with high affinity for immunoglobulin E

    SciTech Connect

    Alcaraz, G.; Kinet, J.P.; Liu, T.Y.; Metzger, H.

    1987-05-05

    The ..cap alpha.., ..beta.., ..gamma.. subunits of the receptor with high affinity for immunoglobulin E were isolated and their compositions assessed by direct amino acid analysis and by incorporation of radioactive precursors. The compositions show no unusual features other than a rather high content of tryptophan in the ..cap alpha.. chain as assessed from the incorporation studies. The results combined with future sequence data will permit unambiguous determination of the multiplicity of the chains in the receptor. Chymotryptic peptide maps of the extrinsically iodinated subunits show several similar peptides, particularly for ..cap alpha.. and ..beta... However, these putative homologies were not apparent when tryptic maps of the biosynthetically ((/sup 3/H)leucine) labeled subunits were analyzed.

  9. Molecular editing of cellular responses by the high-affinity receptor for IgE.

    PubMed

    Suzuki, Ryo; Leach, Sarah; Liu, Wenhua; Ralston, Evelyn; Scheffel, Jörg; Zhang, Weiguo; Lowell, Clifford A; Rivera, Juan

    2014-02-28

    Cellular responses elicited by cell surface receptors differ according to stimulus strength. We investigated how the high-affinity receptor for immunoglobulin E (IgE) modulates the response of mast cells to a high- or low-affinity stimulus. Both high- and low-affinity stimuli elicited similar receptor phosphorylation; however, differences were observed in receptor cluster size, mobility, distribution, and the cells' effector responses. Low-affinity stimulation increased receptor association with the Src family kinase Fgr and shifted signals from the adapter LAT1 to the related adapter LAT2. LAT1-dependent calcium signals required for mast cell degranulation were dampened, but the role of LAT2 in chemokine production was enhanced, altering immune cell recruitment at the site of inflammation. These findings uncover how receptor discrimination of stimulus strength can be interpreted as distinct in vivo outcomes.

  10. Antibody Binding Selectivity: Alternative Sets of Antigen Residues Entail High-Affinity Recognition

    PubMed Central

    Nominé, Yves; Choulier, Laurence; Travé, Gilles; Vernet, Thierry; Altschuh, Danièle

    2015-01-01

    Understanding the relationship between protein sequence and molecular recognition selectivity remains a major challenge. The antibody fragment scFv1F4 recognizes with sub nM affinity a decapeptide (sequence 6TAMFQDPQER15) derived from the N-terminal end of human papilloma virus E6 oncoprotein. Using this decapeptide as antigen, we had previously shown that only the wild type amino-acid or conservative replacements were allowed at positions 9 to 12 and 15 of the peptide, indicating a strong binding selectivity. Nevertheless phenylalanine (F) was equally well tolerated as the wild type glutamine (Q) at position 13, while all other amino acids led to weaker scFv binding. The interfaces of complexes involving either Q or F are expected to diverge, due to the different physico-chemistry of these residues. This would imply that high-affinity binding can be achieved through distinct interfacial geometries. In order to investigate this point, we disrupted the scFv–peptide interface by modifying one or several peptide positions. We then analyzed the effect on binding of amino acid changes at the remaining positions, an altered susceptibility being indicative of an altered role in complex formation. The 23 starting variants analyzed contained replacements whose effects on scFv1F4 binding ranged from minor to drastic. A permutation analysis (effect of replacing each peptide position by all other amino acids except cysteine) was carried out on the 23 variants using the PEPperCHIP® Platform technology. A comparison of their permutation patterns with that of the wild type peptide indicated that starting replacements at position 11, 12 or 13 modified the tolerance to amino-acid changes at the other two positions. The interdependence between the three positions was confirmed by SPR (Biacore® technology). Our data demonstrate that binding selectivity does not preclude the existence of alternative high-affinity recognition modes. PMID:26629896

  11. LNP 906, the first high-affinity photoaffinity ligand selective for I1 imidazoline receptors

    PubMed Central

    Dragan, Urosevic; Stephan, Schann; Jean-Daniel, Ehrhardt; Pascal, Bousquet; Hugues, Greney

    2004-01-01

    The hypotensive effect of imidazoline-like drugs, such as clonidine, was attributed both to α2-adrenergic receptors and nonadrenergic imidazoline receptors, which are divided into I1, I2 and I3 subtypes. We have recently synthesized a derivative of (2-(2-chloro-4-iodo-phenylamino)-5-methyl-pyrroline (LNP 911), the first high-affinity and selective ligand for I1 receptors (I1R), with a photoactivable function (LNP 906). This work aims to test whether this derivative retained the binding properties of LNP 911 and bound irreversibly to I1R. Binding studies showed that LNP 906 exhibited nanomolar affinity for I1R and was selective for I1R over I2 receptors and α2-adrenergic receptors (α2Ars). Upon exposure to u.v. light, LNP 906 irreversibly blocked the binding of [125I]-paraiodoclonidine (PIC) to I1R, time- and dose-dependently, on PC12 cell membranes and interacted with I1R in a reversible and competitive manner in the absence of light. Pharmacological studies showed that this blockade was prevented by the concomitant presence of rilmenidine (a well-known I1 agonist), but not by rauwolscine (an α2 antagonist). Finally, LNP 906 clearly antagonized the decrease in forskolin-stimulated cAMP level induced by rilmenidine, but not by melatonin. These results indicate that LNP 906 is the first high-affinity and selective photoaffinity ligand for I1R and that it behaves as an I1R antagonist. PMID:15178642

  12. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

    SciTech Connect

    Wang, Deng-Liang; Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan; Yang, Hai-Tao; Wang, Jiang-Jie; Yao, Pei-Sen; Pan, Ru-Jun; Yang, Chaoyong James; Kang, De-Zhi

    2014-10-31

    Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K{sub d} 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K{sub d} 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.

  13. High affinity binding of an engineered, modular peptide to bone tissue.

    PubMed

    Brounts, Sabrina H; Lee, Jae Sung; Weinberg, Sean; Lan Levengood, Sheeny K; Smith, Everett L; Murphy, William L

    2013-05-01

    Bone grafting procedures have become common due in part to a global trend of population aging. Native bone graft is a popular choice when compared to various synthetic bone graft substitutes, owing to superior biological activity. Nonetheless, the insufficient ability of bone allograft to induce new bone formation and the insufficient remodeling of native bone grafts call for osteoinductive factors during bone repair, exemplified by recombinant human bone morphogenetic protein 2 (rhBMP2). We previously developed a modular bone morphogenetic peptide (mBMP) to address complications associated with the clinical use of rhBMP2 as a bone graft substitute. The mBMP is designed to strongly bind to hydroxyapatite, the main inorganic component of bone and teeth, and to provide pro-osteogenic properties analogous to rhBMP2. Our previous in vivo animal studies showed that mBMP bound to hydroxyapatite-coated orthopedic implants with high affinity and stimulated new bone formation. In this study, we demonstrate specific binding of mBMP to native bone grafts. The results show that mBMP binds with high affinity to both cortical and trabecular bones, and that the binding is dependent on the mBMP concentration and incubation time. Importantly, efficient mBMP binding is also achieved in an ex vivo bone bioreactor where bone tissue is maintained viable for several weeks. In addition, mBMP binding can be localized with spatial control on native bone tissue via simple methods, such as dip-coating, spotting, and direct writing. Taken together with the pro-osteogenic activity of mBMP established in previous bone repair models, these results suggest that mBMP may promote bone healing when coated on native bone grafts in a clinically compatible manner.

  14. Concurrent low- and high-affinity sulfate reduction kinetics in marine sediment

    NASA Astrophysics Data System (ADS)

    Harder Tarpgaard, Irene; Røy, Hans; Jørgensen, Bo Barker

    Bacterial sulfate reduction in marine sediments generally occurs in the presence of high millimolar concentrations of sulfate. Published data indicate that low sulfate concentrations may limit sulfate reduction rates below 0.2-2 mM. Yet, high sulfate reduction rates occur in the 1-100 μM range in freshwater sediments and at the sulfate-methane transition in marine sediments. Through a combination of 35S-tracer experiments, including initial velocity experiments and time course experiments, we searched for different sulfate affinities in the mixed community of sulfate reducers in a marine sediment. We supported the radiotracer experiments with a highly sensitive ion chromatographic technique for sulfate with a detection limit of 0.15 μM SO 42- in marine pore water. Our results showed that high and low affinities for sulfate co-occur and that the applied experimental approach may determine the observed apparent half saturation constant, Km. Our experimental and model data both show that sulfate reduction in the studied marine sediment could be explained by two dominating affinities for sulfate: a low affinity with a mean half saturation constant, Km, of 430 μM SO 42- and a high affinity with a mean Km of 2.6 μM SO 42-. The high-affinity sulfate reduction was thermodynamically un-constrained down to <1 μM SO 42-, both in our experiments and under in situ conditions. The reduction of radio-labeled sulfate was partly reversible due to concurrent re-oxidation of sulfide by Fe(III) and possibly due to a reversibility of the enzymatic pathway of sulfate reduction. A literature survey of apparent Km values for sediments and pure cultures is presented and discussed.

  15. Mass Spectrometry of Protein Complexes: From Origins to Applications

    NASA Astrophysics Data System (ADS)

    Mehmood, Shahid; Allison, Timothy M.; Robinson, Carol V.

    2015-04-01

    Now routine is the ability to investigate soluble and membrane protein complexes in the gas phase of a mass spectrometer while preserving folded structure and ligand-binding properties. Several recent transformative developments have occurred to arrive at this point. These include advances in mass spectrometry instrumentation, particularly with respect to resolution; the ability to study intact membrane protein complexes released from detergent micelles; and the use of protein unfolding in the gas phase to obtain stability parameters. Together, these discoveries are providing unprecedented information on the compositional heterogeneity of biomacromolecules, the unfolding trajectories of multidomain proteins, and the stability imparted by ligand binding to both soluble and membrane-embedded protein complexes. We review these recent breakthroughs, highlighting the challenges that had to be overcome and the physicochemical insight that can now be gained from studying proteins and their assemblies in the gas phase.

  16. A high-affinity estrogen-binding protein in rat placental trophoblast.

    PubMed

    McCormack, S A; Glasser, S R

    1976-09-01

    A high-affinity, low-capacity estradiol-binding molecule (RE) has been demonstrated in the basal zone trophoblast (BZT) of the pregnant rat. On day 11 of pregnancy (day 0 = first sperm-positive day) RE is present in BZT cytosol, where it has a ka of 1.2 X 10(6)M-1 sec-1, t1/2 = 12.7 min, at 20 C. The Kd, under similar conditions, consists of 2 components, 1.3 X 10(-4) sec-1, t1/2 = 90 min, and 5.9 X 10(-5) sec-1, t1/2 = 196 min. When one uses the faster component, the equilibrium constant, Kd, obtained from kd/ka is 1.1 X 10(-10)M, in close agreement with that obtained from Scatchard analysis of specific estradiol (E2) binding at 20 C. On day 11 there were approximately 12,000 sites/cell in BZT cytosol. Scatchard analysis of nuclear RE on day 11 indicated a Kd of 1.85 X 10(-10)M and approximately 21,000 sites/nucleus, but, in day 15 BZT, nuclear RE was undetectable. Neither cytosol nor nuclei prepared from placental labyrinthine zone (LZT) tissue (fetal placenta) showed evidence of high-affinity, low-capacity E2 binding. Sucrose density gradient analysis on 5-20% linear gradients showed the cytosol RE to be approximately 4S whether in high or low-salt conditions. When measured against binding by 3H-labeled estradiol (*E2), the cytosol BTZ RE was competed for strongly (80-90%) by estrone, estriol, diethylstilbestrol, and estradiol-17alpha at 200 times excess. Nafoxidine-HCl, also at 200X excess, competed to approximately 50%. Corticosterone, progesterone, testosterone, dehydroepiandrosterone, and pregnenolone did not compete. The hormone specificity of nuclear BZT RE was similar to that of the comparable cytosol RE with the exception that nafoxidine did not compete. This was probably due to differences in kinetics, nafoxidine requiring a longer time to reach equilibrium than the other estrogens. The size of the nuclear RE by sucrose density gradient analysis was approximately 2S by KCl extraction (which was inefficient) or 4S by trypsin extraction. We conclude that

  17. Expression of high-affinity IL-4 receptors on human melanoma, ovarian and breast carcinoma cells.

    PubMed Central

    Obiri, N I; Siegel, J P; Varricchio, F; Puri, R K

    1994-01-01

    It has previously been shown that murine sarcoma cells express high-affinity IL-4 receptors (IL-4R) which are internalized after binding to the ligand (Puri et al., Cancer Res 1991; 51:3011-7). We have also reported that human renal cell carcinoma cells express high-affinity IL-4R, and IL-4 inhibits tumour growth in vitro (Obiri et al., J Clin Invest 1993; 91:88). In this study we investigated the expression and function of IL-4R on other human solid tumours. Human melanoma, ovarian carcinoma and breast carcinoma cell lines were assessed for the cell surface expression of IL-4R by radio-ligand receptor binding and for IL-4R gene expression by Northern blot analysis. Primary cultures of mesothelioma and neurofibrosarcoma cells were similarly investigated. Human melanoma, ovarian carcinoma and breast carcinoma cell lines expressed IL-4R on their cell surface with a dissociation constant (Kd) of 140-549 pM. These tumour lines expressed a single 4 kb species of mRNA for IL-4R. Similarly, primary cultures of mesothelioma and neurofibrosarcoma cells were positive for the IL-4R mRNA by Northern blot analysis. Fresh, non-cultured mesothelioma and neurofibrosarcoma tumour sections were also positive for the presence of IL-4R as determined by immunohistochemistry of frozen sections using anti-IL-4R antibody. In order to study possible functions of IL-4R, we evaluated the effects of IL-4 on cell growth and its effect on MHC antigen expression in the presence or absence of interferon-gamma (IFN-gamma). In tissue culture, IL-4 reduced the growth of tumour cell lines and primary cell cultures studied. IL-4 had very little effect on MHC class I antigen expression on ovarian, breast and melanoma cell lines; however, MHC class II (HLA-DR) expression was enhanced on melanoma and breast carcinoma cells. IL-4 also enhanced the IFN-gamma-induced class II expression on melanoma and breast carcinoma cells. Taken together, our observations indicate that IL-4R are expressed on a variety of

  18. New Synthesis and Tritium Labeling of a Selective Ligand for Studying High-affinity γ-Hydroxybutyrate (GHB) Binding Sites

    PubMed Central

    Vogensen, Stine B.; Marek, Aleš; Bay, Tina; Wellendorph, Petrine; Kehler, Jan; Bundgaard, Christoffer; Frølund, Bente; Pedersen, Martin H.F.; Clausen, Rasmus P.

    2013-01-01

    3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [3H]-1 is described. The radiosynthesis employs in situ generated lithium trimethoxyborotritide. Screening of 1 against different CNS targets establishes a high selectivity and we demonstrate in vivo brain penetration. In vitro characterization of [3H]-1 binding shows high specificity to the high-affinity GHB binding sites. PMID:24053696

  19. Molecular evolutionary analysis of the high-affinity K+ transporter gene family in angiosperms.

    PubMed

    Yang, P; Hua, C; Zhou, F; Zhang, B-J; Cai, X-N; Chen, Q-Z; Wang, R-L

    2016-07-15

    The high-affinity K(+) transporter (HKT) family comprises a group of multifunctional cation transporters widely distributed in organisms ranging from Bacteria to Eukarya. In angiosperms, the HKT family consists primarily of nine types, whose evolutionary relationships are not fully understood. The available sequences from 31 plant species were used to perform a comprehensive evolutionary analysis, including an examination of selection pressure and estimating phylogenetic tree and gene duplication events. Our results show that a gene duplication in the HKT1;5/HKT1;4 cluster might have led to the divergence of the HKT1;5 and HKT1;4 subfamilies. Additionally, maximum likelihood analysis revealed that the HKT family has undergone a strong purifying selection. An analysis of the amino acids provided strong statistical evidence for a functional divergence between subfamilies 1 and 2. Our study was the first to provide evidence of this functional divergence between these two subfamilies. Analysis of co-evolution in HKT identified 25 co-evolved groups. These findings expanded our understanding of the evolutionary mechanisms driving functional diversification of HKT proteins.

  20. High-affinity cannabinoid binding site in brain: A possible marijuana receptor

    SciTech Connect

    Nye, J.S.

    1988-01-01

    The mechanism by which delta{sup 9} tetrahydrocannabinol (delta{sup 9}THC), the major psychoactive component of marijuana or hashish, produces its potent psychological and physiological effects is unknown. To find receptor binding sites for THC, we designed a water-soluble analog for use as a radioligand. 5{prime}-Trimethylammonium-delta{sup 8}THC (TMA) is a positively charged analog of delta-{sup 8}THC modified on the 5{prime} carbon, a portion of the molecule not important for its psychoactivity. We have studied the binding of ({sup 3}H)-5{prime}-trimethylammonium-delta-{sup 8}THC (({sup 3}H)TMA) to rat neuronal membranes. ({sup 3}H)TMA binds saturably and reversibly to brain membranes with high affinity to apparently one class of sites. Highest binding site density occurs in brain, but several peripheral organs also display specific binding. Detergent solubilizes the sites without affecting their pharmacologial properties. Molecular sieve chromatography reveals a bimodal peak of ({sup 3}H)TMA binding activity of approximately 60,000 daltons apparent molecular weight.

  1. Molecular evolutionary analysis of the high-affinity K+ transporter gene family in angiosperms.

    PubMed

    Yang, P; Hua, C; Zhou, F; Zhang, B-J; Cai, X-N; Chen, Q-Z; Wang, R-L

    2016-01-01

    The high-affinity K(+) transporter (HKT) family comprises a group of multifunctional cation transporters widely distributed in organisms ranging from Bacteria to Eukarya. In angiosperms, the HKT family consists primarily of nine types, whose evolutionary relationships are not fully understood. The available sequences from 31 plant species were used to perform a comprehensive evolutionary analysis, including an examination of selection pressure and estimating phylogenetic tree and gene duplication events. Our results show that a gene duplication in the HKT1;5/HKT1;4 cluster might have led to the divergence of the HKT1;5 and HKT1;4 subfamilies. Additionally, maximum likelihood analysis revealed that the HKT family has undergone a strong purifying selection. An analysis of the amino acids provided strong statistical evidence for a functional divergence between subfamilies 1 and 2. Our study was the first to provide evidence of this functional divergence between these two subfamilies. Analysis of co-evolution in HKT identified 25 co-evolved groups. These findings expanded our understanding of the evolutionary mechanisms driving functional diversification of HKT proteins. PMID:27525850

  2. New Regulatory Roles of Galectin-3 in High-Affinity IgE Receptor Signaling

    PubMed Central

    Bambouskova, Monika; Polakovicova, Iva; Halova, Ivana; Goel, Gautam; Draberova, Lubica; Bugajev, Viktor; Doan, Aivi; Utekal, Pavol; Gardet, Agnes; Xavier, Ramnik J.

    2016-01-01

    Aggregation of the high-affinity receptor for IgE (FcεRI) in mast cells initiates activation events that lead to degranulation and release of inflammatory mediators. To better understand the signaling pathways and genes involved in mast cell activation, we developed a high-throughput mast cell degranulation assay suitable for RNA interference experiments using lentivirus-based short hairpin RNA (shRNA) delivery. We tested 432 shRNAs specific for 144 selected genes for effects on FcεRI-mediated mast cell degranulation and identified 15 potential regulators. In further studies, we focused on galectin-3 (Gal3), identified in this study as a negative regulator of mast cell degranulation. FcεRI-activated cells with Gal3 knockdown exhibited upregulated tyrosine phosphorylation of spleen tyrosine kinase and several other signal transduction molecules and enhanced calcium response. We show that Gal3 promotes internalization of IgE-FcεRI complexes; this may be related to our finding that Gal3 is a positive regulator of FcεRI ubiquitination. Furthermore, we found that Gal3 facilitates mast cell adhesion and motility on fibronectin but negatively regulates antigen-induced chemotaxis. The combined data indicate that Gal3 is involved in both positive and negative regulation of FcεRI-mediated signaling events in mast cells. PMID:26929198

  3. High-affinity transport of L-glutamine by a plasma membrane preparation from rat brain.

    PubMed

    Roon, R J; Shofner, S A; Koerner, J F

    1989-10-01

    Plasma membrane vesicles prepared from rat brain contain a saturable, high-affinity transport system for L-glutamine that exhibits the following characteristics: (1) The rate of L-glutamine transport is linear up to 200 micrograms/mL membrane protein. (2) Transport of [3H]-L-glutamine is linear with time for at least 10 min, is significantly reduced by lowering the assay temperature to 4 degrees C, and is essentially abolished by the addition of excess unlabeled L-glutamine. (3) The transport rate is optimal in the range of pH 7.4-8.2. (4) The system exhibits a Km for L-glutamine of approximately 1.7 microM and a Vmax of approximately 46 pmol/(min.mg of protein). (5) The system is not highly dependent upon the addition of monovalent or divalent cations. (6) Inhibitor studies reveal that the amino acid amides exhibit the highest affinity for the system and that there is a high specificity for the L-isomers.

  4. Identification of a high affinity nucleocapsid protein binding element from the bovine leukemia virus genome.

    PubMed

    Yildiz, F Zehra; Babalola, Kathlene; Summers, Michael F

    2013-02-01

    Retroviral genome recognition is mediated by interactions between the nucleocapsid (NC) domain of the virally encoded Gag polyprotein and cognate RNA packaging elements that, for most retroviruses, appear to reside primarily within the 5'-untranslated region (5'-UTR) of the genome. Recent studies suggest that a major packaging determinant of bovine leukemia virus (BLV), a member of the human T-cell leukemia virus (HTLV)/BLV family and a non-primate animal model for HTLV-induced leukemogenesis, resides within the gag open reading frame. We have prepared and purified the recombinant BLV NC protein and conducted electrophoretic mobility shift and isothermal titration calorimetry studies with RNA fragments corresponding to these proposed packaging elements. The gag-derived RNAs did not exhibit significant affinity for NC, suggesting an alternate role in packaging. However, an 83-nucleotide fragment of the 5'-UTR that resides just upstream of the gag start codon binds NC stoichiometrically and with high affinity (K(d)=136±21 nM). These nucleotides were predicted to form tandem hairpin structures, and studies with smaller fragments indicate that the NC binding site resides exclusively within the distal hairpin (residues G369-U399, K(d)=67±8 nM at physiological ionic strength). Unlike all other structurally characterized retroviral NC binding RNAs, this fragment is not expected to contain exposed guanosines, suggesting that RNA binding may be mediated by a previously uncharacterized mechanism.

  5. Characterization of a high affinity cocaine binding site in rat brain

    SciTech Connect

    Calligaro, D.; Eldefrawi, M.

    1986-03-05

    Binding of (/sup 3/H)cocaine to synaptic membranes from whole rat brain was reversible and saturable. Nonlinear regression analysis of binding isotherms indicated two binding affinities: one with k/sub d/ = 16 nM, B/sub max/ = 0.65 pmoles/mg protein and the other with K/sub d/ = 660 nM, B/sub max/ = 5.1 pmoles/mg protein. The high-affinity binding of (/sup 3/H)cocaine was sensitive to the actions of trypsin and chymotrypsin but not carboxypeptidase, and was eliminated by exposure of the membranes to 95/sup 0/C for 5 min. Specific binding at 2 nM was higher at pH 8.8 than at pH 7.0. Binding of (/sup 3/H)cocaine (15 nM) was inhibited by increasing concentrations of Na/sup +/ ions. Several cocaine analogues, neurotransmitter uptake inhibitors and local anesthetics displaced specific (/sup 3/H)cocaine binding at 2 nM with various potencies. The cocaine analogue (-)-norcocaine was the most potent (IC/sub 50/ = 10 nM), while the local anesthetic tetracaine was the least potent in inhibiting (/sup 3/H)cocaine binding. Several biogenic amine uptake inhibitors, including tricyclic antidepressants and phencyclidine, had IC/sub 50/ values below ..mu..M concentrations.

  6. Conformation-Dependent High-Affinity Potent Ricin-Neutralizing Monoclonal Antibodies

    PubMed Central

    Hu, Wei-Gang; Yin, Junfei; Chau, Damon; Hu, Charles Chen; Lillico, Dustin; Yu, Justin; Negrych, Laurel M.; Cherwonogrodzky, John W.

    2013-01-01

    Ricin is a potential biothreat agent with no approved antidote available for ricin poisoning. The aim of this study was to develop potent antibody-based antiricin antidotes. Four strong ricin resistant hybridoma clones secreting antiricin monoclonal antibodies (mAbs) were developed. All four mAbs are bound to conformational epitopes of ricin toxin B (RTB) with high affinity (KD values from 2.55 to 36.27 nM). RTB not only triggers cellular uptake of ricin, but also facilitates transport of the ricin toxin A (RTA) from the endoplasmic reticulum to the cytosol, where RTA exerts its toxic activity. The four mAbs were found to have potent ricin-neutralizing capacities and synergistic effects among them as determined by an in vitro neutralization assay. In vivo protection assay demonstrated that all four mAbs had strong efficacy against ricin challenges. D9 was found to be exceptionally effective. Intraperitoneal (i.p.) administration of D9, at a dose of 5 μg, 6 weeks before or 6 hours after an i.p. challenge with 5 × LD50 of ricin was able to protect or rescue 100% of the mice, indicating that mAb D9 is an excellent candidate to be developed as a potent antidote against ricin poisoning for both prophylactic and therapeutic purposes. PMID:23484120

  7. Single-molecule dissection of the high-affinity cohesin–dockerin complex

    PubMed Central

    Stahl, Stefan W.; Nash, Michael A.; Fried, Daniel B.; Slutzki, Michal; Barak, Yoav; Bayer, Edward A.; Gaub, Hermann E.

    2012-01-01

    Cellulose-degrading enzyme systems are of significant interest from both a scientific and technological perspective due to the diversity of cellulase families, their unique assembly and substrate binding mechanisms, and their potential applications in several key industrial sectors, notably cellulose hydrolysis for second-generation biofuel production. Particularly fascinating are cellulosomes, the multimodular extracellular complexes produced by numerous anaerobic bacteria. Using single-molecule force spectroscopy, we analyzed the mechanical stability of the intermolecular interfaces between the cohesin and the dockerin modules responsible for self-assembly of the cellulosomal components into the multienzyme complex. The observed cohesin–dockerin rupture forces (>120 pN) are among the highest reported for a receptor–ligand system to date. Using an atomic force microscope protocol that quantified single-molecule binding activity, we observed force-induced dissociation of calcium ions from the duplicated loop–helix F-hand motif located within the dockerin module, which in the presence of EDTA resulted in loss of affinity to the cohesin partner. A cohesin amino acid mutation (D39A) that eliminated hydrogen bonding with the dockerin’s critically conserved serine residues reduced the observed rupture forces. Consequently, no calcium loss occurred and dockerin activity was maintained throughout multiple forced dissociation events. These results offer insights at the single-molecule level into the stability and folding of an exquisite class of high-affinity protein–protein interactions that dictate fabrication and architecture of cellulose-degrading molecular machines. PMID:23188794

  8. Bimodal high-affinity association of Brd4 with murine leukemia virus integrase and mononucleosomes

    PubMed Central

    Larue, Ross C.; Plumb, Matthew R.; Crowe, Brandon L.; Shkriabai, Nikoloz; Sharma, Amit; DiFiore, Julia; Malani, Nirav; Aiyer, Sriram S.; Roth, Monica J.; Bushman, Frederic D.; Foster, Mark P.; Kvaratskhelia, Mamuka

    2014-01-01

    The importance of understanding the molecular mechanisms of murine leukemia virus (MLV) integration into host chromatin is highlighted by the development of MLV-based vectors for human gene-therapy. We have recently identified BET proteins (Brd2, 3 and 4) as the main cellular binding partners of MLV integrase (IN) and demonstrated their significance for effective MLV integration at transcription start sites. Here we show that recombinant Brd4, a representative of the three BET proteins, establishes complementary high-affinity interactions with MLV IN and mononucleosomes (MNs). Brd4(1–720) but not its N- or C-terminal fragments effectively stimulate MLV IN strand transfer activities in vitro. Mass spectrometry- and NMR-based approaches have enabled us to map key interacting interfaces between the C-terminal domain of BRD4 and the C-terminal tail of MLV IN. Additionally, the N-terminal fragment of Brd4 binds to both DNA and acetylated histone peptides, allowing it to bind tightly to MNs. Comparative analyses of the distributions of various histone marks along chromatin revealed significant positive correlations between H3- and H4-acetylated histones, BET protein-binding sites and MLV-integration sites. Our findings reveal a bimodal mechanism for BET protein-mediated MLV integration into select chromatin locations. PMID:24520112

  9. Bimodal high-affinity association of Brd4 with murine leukemia virus integrase and mononucleosomes.

    PubMed

    Larue, Ross C; Plumb, Matthew R; Crowe, Brandon L; Shkriabai, Nikoloz; Sharma, Amit; DiFiore, Julia; Malani, Nirav; Aiyer, Sriram S; Roth, Monica J; Bushman, Frederic D; Foster, Mark P; Kvaratskhelia, Mamuka

    2014-04-01

    The importance of understanding the molecular mechanisms of murine leukemia virus (MLV) integration into host chromatin is highlighted by the development of MLV-based vectors for human gene-therapy. We have recently identified BET proteins (Brd2, 3 and 4) as the main cellular binding partners of MLV integrase (IN) and demonstrated their significance for effective MLV integration at transcription start sites. Here we show that recombinant Brd4, a representative of the three BET proteins, establishes complementary high-affinity interactions with MLV IN and mononucleosomes (MNs). Brd4(1-720) but not its N- or C-terminal fragments effectively stimulate MLV IN strand transfer activities in vitro. Mass spectrometry- and NMR-based approaches have enabled us to map key interacting interfaces between the C-terminal domain of BRD4 and the C-terminal tail of MLV IN. Additionally, the N-terminal fragment of Brd4 binds to both DNA and acetylated histone peptides, allowing it to bind tightly to MNs. Comparative analyses of the distributions of various histone marks along chromatin revealed significant positive correlations between H3- and H4-acetylated histones, BET protein-binding sites and MLV-integration sites. Our findings reveal a bimodal mechanism for BET protein-mediated MLV integration into select chromatin locations. PMID:24520112

  10. Evolved Streptavidin Mutants Reveal Key Role of Loop Residue in High-affinity Binding

    SciTech Connect

    M Magalhaes; C Melo Czekster; R Guan; V Malashkevich; S Almo; M Levy

    2011-12-31

    We have performed a detailed analysis of streptavidin variants with altered specificity towards desthiobiotin. In addition to changes in key residues which widen the ligand binding pocket and accommodate the more structurally flexible desthiobiotin, the data revealed the role of a key, non-active site mutation at the base of the flexible loop (S52G) which slows dissociation of this ligand by approximately sevenfold. Our data suggest that this mutation results in the loss of a stabilizing contact which keeps this loop open and accessible in the absence of ligand. When this mutation was introduced into the wild-type protein, destabilization of the opened loop conferred a {approx}10-fold decrease in both the on-rate and off-rate for the ligand biotin-4-fluoroscein. A similar effect was observed when this mutation was added to a monomeric form of this protein. Our results provide key insight into the role of the streptavidin flexible loop in ligand binding and maintaining high affinity interactions.

  11. Coordinated transporter activity shapes high-affinity iron acquisition in cyanobacteria

    PubMed Central

    Kranzler, Chana; Lis, Hagar; Finkel, Omri M; Schmetterer, Georg; Shaked, Yeala; Keren, Nir

    2014-01-01

    Iron bioavailability limits biological activity in many aquatic and terrestrial environments. Broad scale genomic meta-analyses indicated that within a single organism, multiple iron transporters may contribute to iron acquisition. Here, we present a functional characterization of a cyanobacterial iron transport pathway that utilizes concerted transporter activities. Cyanobacteria are significant contributors to global primary productivity with high iron demands. Certain cyanobacterial species employ a siderophore-mediated uptake strategy; however, many strains possess neither siderophore biosynthesis nor siderophore transport genes. The unicellular, planktonic, freshwater cyanobacterium, Synechocystis sp. PCC 6803, employs an alternative to siderophore-based uptake-reduction of Fe(III) species before transport through the plasma membrane. In this study, we combine short-term radioactive iron uptake and reduction assays with a range of disruption mutants to generate a working model for iron reduction and uptake in Synechocystis sp. PCC 6803. We found that the Fe(II) transporter, FeoB, is the major iron transporter in this organism. In addition, we uncovered a link between a respiratory terminal oxidase (Alternate Respiratory Terminal Oxidase) and iron reduction - suggesting a coupling between these two electron transfer reactions. Furthermore, quantitative RNA transcript analysis identified a function for subunits of the Fe(III) transporter, FutABC, in modulating reductive iron uptake. Collectively, our results provide a molecular basis for a tightly coordinated, high-affinity iron transport system. PMID:24088625

  12. High-affinity capture of proteins by diamond nanoparticles for mass spectrometric analysis.

    PubMed

    Kong, X L; Huang, L C L; Hsu, C-M; Chen, W-H; Han, C-C; Chang, H-C

    2005-01-01

    Carboxylated/oxidized diamond nanoparticles (nominal size 100 nm) exhibit exceptionally high affinity for proteins through both hydrophilic and hydrophobic forces. The affinity is so high that proteins in dilute solution can be easily captured by diamonds, simply separated by centrifugation, and directly analyzed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). No preseparation of the adsorbed molecules from diamonds is required for the mass spectrometric analysis. Compared to conventional MALDI-TOF-MS, an enhancement in detection sensitivity by more than 2 orders of magnitude is achieved for dilute solution containing cytochrome c, myoglobin, and albumin because of preconcentration of the probed molecules. The lowest concentration detectable is 100 pM for a 1-mL solution. Aside from the enhanced sensitivity, the overall performance of this technique does not show any sign of deterioration for highly contaminated protein solutions, and furthermore, no significant peak broadening and band shift were observed in the mass spectra. The promise of this new method for clinical proteomics research is demonstrated with an application to human blood serum.

  13. High Affinity Antibodies against Influenza Characterize the Plasmablast Response in SLE Patients After Vaccination

    PubMed Central

    Kaur, Kaval; Zheng, Nai-Ying; Smith, Kenneth; Huang, Min; Li, Lie; Pauli, Noel T.; Henry Dunand, Carole J.; Lee, Jane-Hwei; Morrissey, Michael; Wu, Yixuan; Joachims, Michelle L.; Munroe, Melissa E.; Lau, Denise; Qu, Xinyan; Krammer, Florian; Wrammert, Jens; Palese, Peter; Ahmed, Rafi; James, Judith A.; Wilson, Patrick C.

    2015-01-01

    Breakdown of B cell tolerance is a cardinal feature of systemic lupus erythematosus (SLE). Increased numbers of autoreactive mature naïve B cells have been described in SLE patients and autoantibodies have been shown to arise from autoreactive and non-autoreactive precursors. How these defects, in the regulation of B cell tolerance and selection, influence germinal center (GC) reactions that are directed towards foreign antigens has yet to be investigated. Here, we examined the characteristics of post-GC foreign antigen-specific B cells from SLE patients and healthy controls by analyzing monoclonal antibodies generated from plasmablasts induced specifically by influenza vaccination. We report that many of the SLE patients had anti-influenza antibodies with higher binding affinity and neutralization capacity than those from controls. Although overall frequencies of autoreactivity in the influenza-specific plasmablasts were similar for SLE patients and controls, the variable gene repertoire of influenza-specific plasmablasts from SLE patients was altered, with increased usage of JH6 and long heavy chain CDR3 segments. We found that high affinity anti-influenza antibodies generally characterize the plasmablast responses of SLE patients with low levels of autoreactivity; however, certain exceptions were noted. The high-avidity antibody responses in SLE patients may also be correlated with cytokines that are abnormally expressed in lupus. These findings provide insights into the effects of dysregulated immunity on the quality of antibody responses following influenza vaccination and further our understanding of the underlying abnormalities of lupus. PMID:25951191

  14. Regulation of a high-affinity diamine transport system in Trypanosoma cruzi epimastigotes.

    PubMed Central

    Le Quesne, S A; Fairlamb, A H

    1996-01-01

    Trypanosoma cruzi epimastigotes take up exogenous [3H]putrescine and [3H]cadaverine by a rapid, high-affinity, transport system that exhibits saturable kinetics (putrescine K(m) 2.0 microM, V(max) 3.3 nmol/min per 10(8) cells; cadaverine K(m) 13.4 microM, V(max) 3.9 nmol/min per 10(8) cells). Putrescine transport is temperature dependent and requires the presence of a membrane potential and thiol groups for activity. Its activity is altered in response to extracellular putrescine levels and as the cells proceed through the growth cycle. This transporter shows high specificity for the diamines putrescine and cadaverine, but low specificity for the polyamines spermidine and spermine. The existence of rapid diamine/polyamine transport systems whose activity can be adjusted in response to the growth conditions is of particular importance, as they seem unable to synthesize their own putrescine [Hunter, Le Quesne and Fairlamb (1994) Eur. J. Biochem. 226, 1019-1027]. PMID:8687391

  15. Development of high-affinity single chain Fv against foot-and-mouth disease virus.

    PubMed

    Jung, Joon-Goo; Jeong, Gu Min; Yim, Sung Sun; Jeong, Ki Jun

    2016-03-01

    Foot-and-mouth disease (FMD) is caused by the FMD virus (FMDV) and results in severe economic losses in livestock farming. For rapid FMD diagnostic and therapeutic purposes, an effective antibody against FMDV is needed. Here, we developed a high-affinity antibody against FMDV by FACS-based high throughput screening of a random library. With the FITC-conjugated VP1 epitope of FMDV and high-speed FACS sorting, we screened the synthetic antibody (scFv) library in which antibody variants are displayed in the periplasm of Escherichia coli. After three rounds of sorting, we isolated one antibody fragment (#138-scFv) against the VP1 epitope of FMDV. Next, to improve its affinity, a mutation library of #138-scFV was constructed by error-prone PCR and screened by FACS. After three rounds of sorting, we isolated one antibody (AM-32 scFv), which has a higher binding affinity (KD=42.7nM) than that of the original #138-scFv. We also confirmed that it specifically binds to whole inactivated FMDV. PMID:26827774

  16. Comparison of biosensor platforms in the evaluation of high affinity antibody-antigen binding kinetics.

    PubMed

    Yang, Danlin; Singh, Ajit; Wu, Helen; Kroe-Barrett, Rachel

    2016-09-01

    The acquisition of reliable kinetic parameters for the characterization of biomolecular interactions is an important component of the drug discovery and development process. While several benchmark studies have explored the variability of kinetic rate constants obtained from multiple laboratories and biosensors, a direct comparison of these instruments' performance has not been undertaken, and systematic factors contributing to data variability from these systems have not been discussed. To address these questions, a panel of ten high-affinity monoclonal antibodies was simultaneously evaluated for their binding kinetics against the same antigen on four biosensor platforms: GE Healthcare's Biacore T100, Bio-Rad's ProteOn XPR36, ForteBio's Octet RED384, and Wasatch Microfluidics's IBIS MX96. We compared the strengths and weaknesses of these systems and found that despite certain inherent systematic limitations in instrumentation, the rank orders of both the association and dissociation rate constants were highly correlated between these instruments. Our results also revealed a trade-off between data reliability and sample throughput. Biacore T100, followed by ProteOn XPR36, exhibited excellent data quality and consistency, whereas Octet RED384 and IBIS MX96 demonstrated high flexibility and throughput with compromises in data accuracy and reproducibility. Our results support the need for a "fit-for-purpose" approach in instrument selection for biosensor studies. PMID:27365220

  17. Stable U(IV) complexes form at high-affinity mineral surface sites.

    PubMed

    Latta, Drew E; Mishra, Bhoopesh; Cook, Russell E; Kemner, Kenneth M; Boyanov, Maxim I

    2014-01-01

    Uranium (U) poses a significant contamination hazard to soils, sediments, and groundwater due to its extensive use for energy production. Despite advances in modeling the risks of this toxic and radioactive element, lack of information about the mechanisms controlling U transport hinders further improvements, particularly in reducing environments where U(IV) predominates. Here we establish that mineral surfaces can stabilize the majority of U as adsorbed U(IV) species following reduction of U(VI). Using X-ray absorption spectroscopy and electron imaging analysis, we find that at low surface loading, U(IV) forms inner-sphere complexes with two metal oxides, TiO2 (rutile) and Fe3O4 (magnetite) (at <1.3 U nm(-2) and <0.037 U nm(-2), respectively). The uraninite (UO2) form of U(IV) predominates only at higher surface loading. U(IV)-TiO2 complexes remain stable for at least 12 months, and U(IV)-Fe3O4 complexes remain stable for at least 4 months, under anoxic conditions. Adsorbed U(IV) results from U(VI) reduction by Fe(II) or by the reduced electron shuttle AH2QDS, suggesting that both abiotic and biotic reduction pathways can produce stable U(IV)-mineral complexes in the subsurface. The observed control of high-affinity mineral surface sites on U(IV) speciation helps explain the presence of nonuraninite U(IV) in sediments and has important implications for U transport modeling.

  18. Evidence that synaptosomal high-affinity carriers for amino acid neurotransmitters are glycosylated

    SciTech Connect

    Zaleska, M.M.; Erecinska, M.

    1987-05-01

    The effect of removal of surface sialic acid from synaptosomes on the high-affinity, Na/sup +/-dependent uptake systems for amino acid neurotransmitters was evaluated. Synaptosomes from rat forebrain were preincubated with neuraminidase from Vibrio cholerae for 20 min at 34/sup 0/. After washing and resuspension, their ability to transport /sup 14/C-GABA and the acidic amino acid, /sup 3/H-aspartate was studied. Pretreatment with neuraminidase resulted in a concentration-dependent inhibition of the uptake of both amino acids while the influx of /sup 3/H-L-leucine was unaffected. Inhibition was a function of the amount of sialic acid released from membranes. The analysis of the kinetic parameters of amino acid uptake revealed that inhibition resulted from a decrease of Vmax without any change in the Km. Neither the synaptosomal energy levels nor the internal concentration of potassium ions was affected by the pretreatment with neuraminidase. The maximum accumulation ratios for both amino acids remained largely unaltered. It is concluded that the GABA and acidic amino acid transporters are glycosylated and that sialic acid is involved in the operation of carrier proteins directly and not through modification of the driving forces responsible for amino acid transport.

  19. A 45-amino acid scaffold mined from the Protein Data Bank for high affinity ligand engineering

    PubMed Central

    Kruziki, Max A.; Bhatnagar, Sumit; Woldring, Daniel R.; Duong, Vandon T.; Hackel, Benjamin J.

    2015-01-01

    Summary Small protein ligands can provide superior physiological distribution versus antibodies and improved stability, production, and specific conjugation. Systematic evaluation of the Protein Data Bank identified a scaffold to push the limits of small size and robust evolution of stable, high-affinity ligands: 45-residue T7 phage gene 2 protein (Gp2) contains an α-helix opposite a β-sheet with two adjacent loops amenable to mutation. De novo ligand discovery from 108 mutants and directed evolution towards four targets yielded target-specific binders with affinities as strong as 200 ±100 pM, Tm’s from 65 ±3 °C to 80 ±1 °C, and retained activity after thermal denaturation. For cancer targeting, a Gp2 domain for epidermal growth factor receptor was evolved with 18 ±8 nM affinity, receptor-specific binding, and high thermal stability with refolding. The efficiency of evolving new binding function and the size, affinity, specificity, and stability of evolved domains render Gp2 a uniquely effective ligand scaffold. PMID:26165154

  20. The High-Affinity E. Coli Methionine ABC Transporter: Structure And Allosteric Regulation

    SciTech Connect

    Kadaba, N.S.; Kaiser, J.T.; Johnson, E.; Lee, A.; Rees, D.C.

    2009-05-18

    The crystal structure of the high-affinity Escherichia coli MetNI methionine uptake transporter, a member of the adenosine triphosphate (ATP)-binding cassette (ABC) family, has been solved to 3.7 angstrom resolution. The overall architecture of MetNI reveals two copies of the adenosine triphosphatase (ATPase) MetN in complex with two copies of the transmembrane domain MetI, with the transporter adopting an inward-facing conformation exhibiting widely separated nucleotide binding domains. Each MetI subunit is organized around a core of five transmembrane helices that correspond to a subset of the helices observed in the larger membrane-spanning subunits of the molybdate (ModBC) and maltose (MalFGK) ABC transporters. In addition to the conserved nucleotide binding domain of the ABC family, MetN contains a carboxyl-terminal extension with a ferredoxin-like fold previously assigned to a conserved family of regulatory ligand-binding domains. These domains separate the nucleotide binding domains and would interfere with their association required for ATP binding and hydrolysis. Methionine binds to the dimerized carboxyl-terminal domain and is shown to inhibit ATPase activity. These observations are consistent with an allosteric regulatory mechanism operating at the level of transport activity, where increased intracellular levels of the transported ligand stabilize an inward-facing, ATPase-inactive state of MetNI to inhibit further ligand translocation into the cell.

  1. Intra-clonal competition inhibits the formation of high affinity antibody secreting cells1

    PubMed Central

    Le, Thuc-vy L.; Kim, Tea Hyun; Chaplin, David D.

    2010-01-01

    Protective immunity requires a diverse, polyclonal B cell repertoire. We demonstrate that affinity maturation of the humoral response to a hapten is impaired when pre-existing clonally restricted cells recognizing the hapten are dominant in the B cell repertoire. B1- 8i+/− mice, which feature a high frequency of B cells with nitrophenyl (NP) binding specificity, respond to NP-haptenated proteins with the production of NP-specific antibodies, but affinity maturation is impaired due to insufficient generation of high affinity antibody producing cells. We manipulated the frequency of NP-specific B cells by adoptive transfer of B1-8 B cells into naïve, wild-type recipients. Remarkably, when 104 B1-8 B cells were transferred, these cells supported efficient affinity maturation and plasma cell differentiation. In contrast, when 106 B1-8 cells were transferred, affinity maturation did not occur. These data indicate that restricting the frequency of clonally related B cells is required to support affinity maturation. PMID:18941192

  2. High-Affinity PEGylated Polyacridine Peptide Polyplexes Mediate Potent In Vivo Gene Expression

    PubMed Central

    Kizzire, Koby; Khargharia, Sanjib; Rice, Kevin G.

    2012-01-01

    PEGylated polyacridine peptides bind to plasmid DNA with high affinity to form unique polyplexes that possess a long circulatory half-life and are hydrodynamically (HD)-stimulated to produce efficient gene expression in the liver of mice. We previously demonstrated that (Acr-Lys)6-Cys-PEG5kDa stabilizes a 1 μg pGL3 dose for up to 1 hr in the circulation, resulting in HD-stimulated (saline only) gene expression in the liver, equivalent in magnitude to direct-HD dosing of 1 μg of pGL3 (Fernandez C.A. et al. Gene Therapy 2011). In the present study we report that increasing the spacing of Acr with either 4 or 5 Lys residues, dramatically increases the stability of PEGylated polyacridine peptide polyplexes in the circulation allowing maximal HD-stimulated expression for up to 5 hrs post-DNA administration. Co-administration of a decoy dose of 9 μg of non-expressing DNA polyplex with 1 μg of pGL3 polyplex further extended the HD-stimulated expression to 9 hrs. This structure-activity relationship study defines the PEGylated polyacridine peptide requirements for maintaining fully transfection competent plasmid DNA in the circulation for 5 hrs and provides an understanding as to why polyplexes or lipoplexes prepared with PEI, chitosan or Lipofectamine are inactive within 5 min following i.v. dosing. PMID:22786534

  3. In silico design of high-affinity ligands for the immobilization of inulinase.

    PubMed

    Holyavka, M G; Kondratyev, M S; Samchenko, A A; Kabanov, A V; Komarov, V M; Artyukhov, V G

    2016-04-01

    Using computer modeling, virtual screening of high-affinity ligands for immobilization of inulinase - an enzyme that cleaves inulin and fructose-containing polymers to fructose - has been performed. The inulinase molecule from Aspergillus ficuum (pdb: 3SC7) taken from the database of protein structures was used as a protein model and the target for flexible docking. The set of ligands studied included simple sugars (activators, inhibitors, products of enzymatic catalysis), as well as high-molecular weight compounds (polycation and polyanion exchange resins, glycoproteins, phenylalanine-proline peptide, polylactate, and caffeine). Based on the comparative analysis of the values of the total energy and the localization of ligand binding sites, we made several assumptions concerning the mechanisms of interaction of the suggested matrices for the immobilization of enzyme molecules and the structural features of such complexes. It was also assumed that the candidates for immobilization agents meeting the industrial requirements may be glycoproteins, for which we propose an additional incorporation of cysteine residues into their structure, aimed to create disulfide «anchors» to the surface. PMID:26945599

  4. Identification of high-affinity calmodulin-binding proteins in rat liver

    SciTech Connect

    Hanley, R.M.; Dedman, J.R.; Shenolikar, S.

    1987-03-01

    The Ca/sup 2 +/-dependent binding of (/sup 125/I) calmodulin (CaM) to hepatic proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was utilized to identify CaM binding or acceptor proteins or CAPs. Two proteins of apparent molecular weight of 60,000 (CAP-60) and 45,000 (CAP-45) comprised > 80% of the Ca/sup 2 +/-dependent CaM binding in rat liver cytosol. CAP-60 and CAP-45 were partially purified by a variety of chromatographic steps, including affinity chromatography on CaM Sepharose. CAP-60 possessed a native molecular size of 400,000, indicating it to be the CaM-binding subunit of a larger oligomeric complex. In contrast, CAP-45 was monomeric as judged by gel filtration. Neither CAP-60 nor CAP-45 possessed chromatographic properties consistent with known CaM-dependent enzymes reported in the literature. Two-dimensional peptide mapping provided convincing evidence that CAP-60 and CAP-45 were unrelated to other well-characterized CAPs, namely Ca/sup 2 +/ (CaM)-dependent protein kinase II, calcineurin, or the CaM-dependent cyclic nucleotide phosphodiesterase. The relative abundance and high affinity for CaM could suggest that these novel target proteins, CAP-60 and CAP-45, represent a dominant pathway for CaM action in the mammalian liver.

  5. Rapid and efficient cancer cell killing mediated by high-affinity death receptor homotrimerizing TRAIL variants

    PubMed Central

    Reis, C R; van der Sloot, A M; Natoni, A; Szegezdi, E; Setroikromo, R; Meijer, M; Sjollema, K; Stricher, F; Cool, R H; Samali, A; Serrano, L; Quax, W J

    2010-01-01

    The tumour necrosis factor family member TNF-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in a variety of cancer cells through the activation of death receptors 4 (DR4) and 5 (DR5) and is considered a promising anticancer therapeutic agent. As apoptosis seems to occur primarily via only one of the two death receptors in many cancer cells, the introduction of DR selectivity is thought to create more potent TRAIL agonists with superior therapeutic properties. By use of a computer-aided structure-based design followed by rational combination of mutations, we obtained variants that signal exclusively via DR4. Besides an enhanced selectivity, these TRAIL-DR4 agonists show superior affinity to DR4, and a high apoptosis-inducing activity against several TRAIL-sensitive and -resistant cancer cell lines in vitro. Intriguingly, combined treatment of the DR4-selective variant and a DR5-selective TRAIL variant in cancer cell lines signalling by both death receptors leads to a significant increase in activity when compared with wild-type rhTRAIL or each single rhTRAIL variant. Our results suggest that TRAIL induced apoptosis via high-affinity and rapid-selective homotrimerization of each DR represent an important step towards an efficient cancer treatment. PMID:21368856

  6. Cutting Edge: Resident Memory CD8 T Cells Express High-Affinity TCRs.

    PubMed

    Frost, Elizabeth L; Kersh, Anna E; Evavold, Brian D; Lukacher, Aron E

    2015-10-15

    Tissue-resident memory T (TRM) cells serve as vanguards of antimicrobial host defense in nonlymphoid tissues, particularly at barrier epithelia and in organs with nonrenewable cell types (e.g., brain). In this study, we asked whether an augmented ability to sense Ag complemented their role as early alarms of pathogen invasion. Using mouse polyomavirus, we show that brain-resident mouse polyomavirus-specific CD8 T cells, unlike memory cells in the spleen, progressively increase binding to MHC class I tetramers and CD8 coreceptor expression. Using the two-dimensional micropipette adhesion-frequency assay, we show that TRM cells in brain, as well as in kidney, express TCRs with up to 20-fold higher affinity than do splenic memory T cells, whereas effector cells express TCRs of similar high affinity in all organs. Together, these data demonstrate that TRM cells retain high TCR affinity, which endows them with the high Ag sensitivity needed for front-line defense against infectious agents. PMID:26371252

  7. Expression of high affinity folate receptor in breast cancer brain metastasis.

    PubMed

    Leone, José Pablo; Bhargava, Rohit; Theisen, Brian K; Hamilton, Ronald L; Lee, Adrian V; Brufsky, Adam M

    2015-10-01

    High affinity folate receptor (HFR) can be overexpressed in breast cancer and is associated with poor prognosis, however the expression in breast cancer brain metastases (BCBM) is unknown. The aim of this study was to analyze the rate of HFR expression in BCBM and its role in the prognosis of this high-risk cohort. We analyzed 19 brain metastasis (BM) and 13 primary tumors (PT) from a total of 25 patients. HFR status was assessed by immunohistochemistry. Median follow-up was 4.2 years (range 0.6-18.5). HFR was positive in 4/19 BM (21.1%) and in 1/13 PT (7.7%). Positive samples had low H-scores (range 1-50). 56% of patients had apocrine differentiation. OS was similar between patients with positive HFR (median OS 48 months) and negative HFR (median OS 69 months) (P = 0.25); and between patients with apocrine differentiation (median OS 63 months) and those without apocrine differentiation (median OS 69 months) (P = 0.49). To the best of our knowledge, this is the first analysis of HFR expression in BCBM. While previous studies associated the presence of HFR with worse prognosis; in our cohort HFR was positive in only 21.1% of BM with low levels of positivity. Neither HFR nor apocrine features had impact in OS.

  8. Conformation-dependent high-affinity potent ricin-neutralizing monoclonal antibodies.

    PubMed

    Hu, Wei-Gang; Yin, Junfei; Chau, Damon; Hu, Charles Chen; Lillico, Dustin; Yu, Justin; Negrych, Laurel M; Cherwonogrodzky, John W

    2013-01-01

    Ricin is a potential biothreat agent with no approved antidote available for ricin poisoning. The aim of this study was to develop potent antibody-based antiricin antidotes. Four strong ricin resistant hybridoma clones secreting antiricin monoclonal antibodies (mAbs) were developed. All four mAbs are bound to conformational epitopes of ricin toxin B (RTB) with high affinity (KD values from 2.55 to 36.27 nM). RTB not only triggers cellular uptake of ricin, but also facilitates transport of the ricin toxin A (RTA) from the endoplasmic reticulum to the cytosol, where RTA exerts its toxic activity. The four mAbs were found to have potent ricin-neutralizing capacities and synergistic effects among them as determined by an in vitro neutralization assay. In vivo protection assay demonstrated that all four mAbs had strong efficacy against ricin challenges. D9 was found to be exceptionally effective. Intraperitoneal (i.p.) administration of D9, at a dose of 5 μ g, 6 weeks before or 6 hours after an i.p. challenge with 5 × LD50 of ricin was able to protect or rescue 100% of the mice, indicating that mAb D9 is an excellent candidate to be developed as a potent antidote against ricin poisoning for both prophylactic and therapeutic purposes. PMID:23484120

  9. A high-affinity, radioiodinatable neuropeptide FF analogue incorporating a photolabile p-(4-hydroxybenzoyl)phenylalanine.

    PubMed

    Bray, Lauriane; Moulédous, Lionel; Tafani, Jean A M; Germanier, Maryse; Zajac, Jean-Marie

    2014-05-15

    A new radioiodinated photoaffinity compound, [(125)I]YE(Bpa)WSLAAPQRFNH2, derived from a peptide present in the rat neuropeptide FF (NPFF) precursor was synthesized, and its binding characteristics were investigated on a neuroblastoma clone, SH-SY5Y, stably expressing rat NPFF2 receptors tagged with the T7 epitope. The binding of the probe was saturable and revealed a high-affinity interaction (KD=0.24nM) with a single class of binding sites. It was also able to affinity label NPFF2 receptor in a specific and efficient manner given that 38% of the bound radioligand at saturating concentration formed a wash-resistant binding after ultraviolet (UV) irradiation. Photoaffinity labeling with [(125)I]YE(Bpa)WSLAAPQRFamide showed two molecular forms of NPFF2 receptor with apparent molecular weights of 140 and 95kDa in a 2:1 ratio. The comparison of the results between photoaffinity labeling and Western blot analysis suggests that all receptor forms bind the probe irreversibly with the same efficiency. On membranes of mouse olfactory bulb, only the high molecular weight form of NPFF2 receptor is observed. [(125)I]YE(Bpa)WSLAAPQRFamide is an excellent radioiodinated peptidic ligand for direct and selective labeling of NPFF2 receptors in vitro.

  10. Consequences of inducing intrinsic disorder in a high-affinity protein-protein interaction.

    PubMed

    Papadakos, Grigorios; Sharma, Amit; Lancaster, Lorna E; Bowen, Rebecca; Kaminska, Renata; Leech, Andrew P; Walker, Daniel; Redfield, Christina; Kleanthous, Colin

    2015-04-29

    The kinetic and thermodynamic consequences of intrinsic disorder in protein-protein recognition are controversial. We address this by inducing one partner of the high-affinity colicin E3 rRNase domain-Im3 complex (K(d) ≈ 10(-12) M) to become an intrinsically disordered protein (IDP). Through a variety of biophysical measurements, we show that a single alanine mutation at Tyr507 within the hydrophobic core of the isolated colicin E3 rRNase domain causes the enzyme to become an IDP (E3 rRNase(IDP)). E3 rRNase(IDP) binds stoichiometrically to Im3 and forms a structure that is essentially identical to the wild-type complex. However, binding of E3 rRNase(IDP) to Im3 is 4 orders of magnitude weaker than that of the folded rRNase, with thermodynamic parameters reflecting the disorder-to-order transition on forming the complex. Critically, pre-steady-state kinetic analysis of the E3 rRNase(IDP)-Im3 complex demonstrates that the decrease in affinity is mostly accounted for by a drop in the electrostatically steered association rate. Our study shows that, notwithstanding the advantages intrinsic disorder brings to biological systems, this can come at severe kinetic and thermodynamic cost. PMID:25856265

  11. Immunoprecipitation and Characterization of Membrane Protein Complexes from Yeast

    ERIC Educational Resources Information Center

    Parra-Belky, Karlett; McCulloch, Kathryn; Wick, Nicole; Shircliff, Rebecca; Croft, Nicolas; Margalef, Katrina; Brown, Jamie; Crabill, Todd; Jankord, Ryan; Waldo, Eric

    2005-01-01

    In this undergraduate biochemistry laboratory experiment, the vacuolar ATPase protein complex is purified from yeast cell extracts by doing immunoprecipitations under nondenaturing conditions. Immunoprecipitations are performed using monoclonal antibodies to facilitate data interpretation, and subunits are separated on the basis of their molecular…

  12. Determining the Composition and Stability of Protein Complexes Using an Integrated Label-Free and Stable Isotope Labeling Strategy

    PubMed Central

    Greco, Todd M.; Guise, Amanda J.; Cristea, Ileana M.

    2016-01-01

    In biological systems, proteins catalyze the fundamental reactions that underlie all cellular functions, including metabolic processes and cell survival and death pathways. These biochemical reactions are rarely accomplished alone. Rather, they involve a concerted effect from many proteins that may operate in a directed signaling pathway and/or may physically associate in a complex to achieve a specific enzymatic activity. Therefore, defining the composition and regulation of protein complexes is critical for understanding cellular functions. In this chapter, we describe an approach that uses quantitative mass spectrometry (MS) to assess the specificity and the relative stability of protein interactions. Isolation of protein complexes from mammalian cells is performed by rapid immunoaffinity purification, and followed by in-solution digestion and high-resolution mass spectrometry analysis. We employ complementary quantitative MS workflows to assess the specificity of protein interactions using label-free MS and statistical analysis, and the relative stability of the interactions using a metabolic labeling technique. For each candidate protein interaction, scores from the two workflows can be correlated to minimize nonspecific background and profile protein complex composition and relative stability. PMID:26867737

  13. Expression, purification, and immobilization of recombinant tamavidin 2 fusion proteins.

    PubMed

    Takakura, Yoshimitsu; Oka, Naomi; Tsunashima, Masako

    2014-01-01

    Tamavidin 2 is a fungal avidin-like protein that binds biotin with high affinity. Unlike avidin or streptavidin, tamavidin 2 in soluble form is produced at high levels in Escherichia coli. In this chapter, we describe a method for immobilization and purification of recombinant proteins with the use of tamavidin 2 as an affinity tag. The protein fused to tamavidin 2 is tightly immobilized and simultaneously purified on biotinylated magnetic microbeads without loss of activity. PMID:24943317

  14. Measurements of relative binding of cohesin and dockerin mutants using an advanced ELISA technique for high-affinity interactions.

    PubMed

    Slutzki, Michal; Barak, Yoav; Reshef, Dan; Schueler-Furman, Ora; Lamed, Raphael; Bayer, Edward A

    2012-01-01

    The cellulosome is a large bacterial extracellular multienzyme complex able to degrade crystalline cellulosic substrates. The complex contains catalytic and noncatalytic subunits, interconnected by high-affinity cohesin-dockerin interactions. In this chapter, we introduce an optimized method for comparative binding among different cohesins or cohesin mutants to the dockerin partner. This assay offers advantages over other methods (such as ELISA, cELIA, SPR, and ITC) for particularly high-affinity binding interactions. In this approach, the high-affinity interaction of interest occurs in the liquid phase during the equilibrated binding step, whereas the interaction with the immobilized phase is used only for detection of the unbound dockerins that remain in the solution phase. Once equilibrium conditions are reached, the change in free energy of binding (ΔΔG(binding)), as well as the affinity constant of mutants, can be estimated against the known affinity constant of the wild-type interaction. In light of the above, we propose this method as a preferred alternative for the relative quantification of high-affinity protein interactions. PMID:22608739

  15. Glycan:glycan interactions: High affinity biomolecular interactions that can mediate binding of pathogenic bacteria to host cells

    PubMed Central

    Day, Christopher J.; Tran, Elizabeth N.; Semchenko, Evgeny A.; Tram, Greg; Hartley-Tassell, Lauren E.; Ng, Preston S. K.; King, Rebecca M.; Ulanovsky, Rachel; McAtamney, Sarah; Apicella, Michael A.; Tiralongo, Joe; Morona, Renato; Korolik, Victoria; Jennings, Michael P.

    2015-01-01

    Cells from all domains of life express glycan structures attached to lipids and proteins on their surface, called glycoconjugates. Cell-to-cell contact mediated by glycan:glycan interactions have been considered to be low-affinity interactions that precede high-affinity protein–glycan or protein–protein interactions. In several pathogenic bacteria, truncation of surface glycans, lipooligosaccharide (LOS), or lipopolysaccharide (LPS) have been reported to significantly reduce bacterial adherence to host cells. Here, we show that the saccharide component of LOS/LPS have direct, high-affinity interactions with host glycans. Glycan microarrays reveal that LOS/LPS of four distinct bacterial pathogens bind to numerous host glycan structures. Surface plasmon resonance was used to determine the affinity of these interactions and revealed 66 high-affinity host–glycan:bacterial–glycan pairs with equilibrium dissociation constants (KD) ranging between 100 nM and 50 µM. These glycan:glycan affinity values are similar to those reported for lectins or antibodies with glycans. Cell assays demonstrated that glycan:glycan interaction-mediated bacterial adherence could be competitively inhibited by either host cell or bacterial glycans. This is the first report to our knowledge of high affinity glycan:glycan interactions between bacterial pathogens and the host. The discovery of large numbers of glycan:glycan interactions between a diverse range of structures suggests that these interactions may be important in all biological systems. PMID:26676578

  16. Glycan:glycan interactions: High affinity biomolecular interactions that can mediate binding of pathogenic bacteria to host cells.

    PubMed

    Day, Christopher J; Tran, Elizabeth N; Semchenko, Evgeny A; Tram, Greg; Hartley-Tassell, Lauren E; Ng, Preston S K; King, Rebecca M; Ulanovsky, Rachel; McAtamney, Sarah; Apicella, Michael A; Tiralongo, Joe; Morona, Renato; Korolik, Victoria; Jennings, Michael P

    2015-12-29

    Cells from all domains of life express glycan structures attached to lipids and proteins on their surface, called glycoconjugates. Cell-to-cell contact mediated by glycan:glycan interactions have been considered to be low-affinity interactions that precede high-affinity protein-glycan or protein-protein interactions. In several pathogenic bacteria, truncation of surface glycans, lipooligosaccharide (LOS), or lipopolysaccharide (LPS) have been reported to significantly reduce bacterial adherence to host cells. Here, we show that the saccharide component of LOS/LPS have direct, high-affinity interactions with host glycans. Glycan microarrays reveal that LOS/LPS of four distinct bacterial pathogens bind to numerous host glycan structures. Surface plasmon resonance was used to determine the affinity of these interactions and revealed 66 high-affinity host-glycan:bacterial-glycan pairs with equilibrium dissociation constants (K(D)) ranging between 100 nM and 50 µM. These glycan:glycan affinity values are similar to those reported for lectins or antibodies with glycans. Cell assays demonstrated that glycan:glycan interaction-mediated bacterial adherence could be competitively inhibited by either host cell or bacterial glycans. This is the first report to our knowledge of high affinity glycan:glycan interactions between bacterial pathogens and the host. The discovery of large numbers of glycan:glycan interactions between a diverse range of structures suggests that these interactions may be important in all biological systems. PMID:26676578

  17. Rate of chase-promoted hydrolysis of ATP in the high affinity catalytic site of beef heart mitochondrial ATPase

    SciTech Connect

    Penefsky, H.S.

    1988-05-05

    Incubation of (..gamma..-/sup 32/P)ATP with a molar excess of the soluble, homogeneous ATPase from beef heart mitochondria (F/sub 1/) results in binding of substrate primarily in a single, very high affinity catalytic site and in a slow rate of hydrolysis characteristic of single site catalysis. Subsequent addition of millimolar concentrations of nonradioactive ATP as a cold chase, sufficient to fill catalytic sites on the enzyme, results in an acceleration of hydrolysis of bound radioactive ATP of as much as 10/sup 6/-fold, that is to V/sub max/ rates. For this reason, it was proposed that the high affinity catalytic site is a normal catalytic site on the molecule. This paper shows, in experiments with a rapid mixing-chemical quench apparatus, that hydrolysis of ATP bound in the high affinity catalytic site is accelerated to V/sub max/ rates following addition of 5 ..mu..M ATP as a cold chase. Hydrolysis of bound ATP appears to precede that of the chase. The weight of the available evidence continues to support the original suggestion that the high affinity catalytic site of beef heart F/sub 1/ is a normal catalytic site.

  18. Targeting Protein-Protein Interactions with Trimeric Ligands: High Affinity Inhibitors of the MAGUK Protein Family

    PubMed Central

    Nissen, Klaus B.; Haugaard-Kedström, Linda M.; Wilbek, Theis S.; Nielsen, Line S.; Åberg, Emma; Kristensen, Anders S.; Bach, Anders; Jemth, Per; Strømgaard, Kristian

    2015-01-01

    PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins. PMID:25658767

  19. Water channel in the binding site of a high affinity anti-methotrexate antibody.

    PubMed

    Gayda, Susan; Longenecker, Kenton L; Manoj, Sharmila; Judge, Russell A; Saldana, Sylvia C; Ruan, Qiaoqiao; Swift, Kerry M; Tetin, Sergey Y

    2014-06-17

    In the present study, we report the structure of the free and drug-bound Fab fragment of a high affinity anti-methotrexate antibody and perform a thermodynamic analysis of the binding process. The anti-methotrexate Fab fragment features a remarkably rigid tunnel-like binding site that extends into a water channel serving as a specialized route to move solvent out and into the site upon ligand binding and dissociation. This new finding in antibody structure-function relationships directly relates to the fast association (1 × 10⁷ M⁻¹ s⁻¹) and slow dissociation (4 × 10⁻⁵ s⁻¹) rates determined for mAb ADD056, resulting in a very strong binding with a K(D) ~ 3.6 pM at 20 °C. As follows from the X-ray data analysis, the methotrexate-antibody complex is stabilized by an extended network of hydrogen bonds and stacking interactions. The analysis also shows structural involvement of the CDR H3 in formation of the water channel revealing another important role of this hypervariable region. This suggests a new direction in natural affinity maturation and opens a new possibility in antibody engineering. Methotrexate is a widely used therapeutic agent for many malignant diseases and inflammatory disorders. Unfortunately, it may also interfere with central aspects of metabolism and thereby cause inevitable side effects. Therefore, methotrexate therapy requires careful monitoring of drug blood levels, which is traditionally done by immunoassays. An understanding of the structure-function properties of antibodies selected for drug monitoring substantiates the performance and robustness of such tests.

  20. High-Affinity Accumulation of a Maytansinoid in Cells via Weak Tubulin Interaction

    PubMed Central

    Goldmacher, Victor S.; Audette, Charlene A.; Guan, Yinghua; Sidhom, Eriene-Heidi; Shah, Jagesh V.; Whiteman, Kathleen R.; Kovtun, Yelena V.

    2015-01-01

    The microtubule-targeting maytansinoids accumulate in cells and induce mitotic arrest at 250- to 1000-fold lower concentrations than those required for their association with tubulin or microtubules. To identify the mechanisms of this intracellular accumulation and exceptional cytotoxicity of maytansinoids we studied interaction of a highly cytotoxic maytansinoid, S-methyl DM1 and several other maytansinoids with cells. S-methyl DM1 accumulated inside the cells with a markedly higher apparent affinity than to tubulin or microtubules. The apparent affinities of maytansinoids correlated with their cytotoxicities. The number of intracellular binding sites for S-methyl DM1 in MCF7 cells was comparable to the number of tubulin molecules per cell (~ 4–6 × 107 copies). Efflux of 3 [H]-S-methyl DM1 from cells was enhanced in the presence of an excess of non-labeled S-methyl DM1, indicating that re-binding of 3 [H]-S-methyl DM1 to intracellular binding sites contributed to its intracellular retention. Liposomes loaded with non-polymerized tubulin recapitulated the apparent high-affinity association of S-methyl DM1 to cells. We propose a model for the intracellular accumulation of maytansinoids in which molecules of the compounds diffuse into a cell and associate with tubulin. Affinities of maytansinoids for individual tubulin molecules are weak, but the high intracellular concentration of tubulin favors, after dissociation of a compound-tubulin complex, their re-binding to a tubulin molecule, or to a tip of a microtubule in the same cell, over their efflux. As a result, a significant fraction of microtubule tips is occupied with a maytansinoid when added to cells at sub-nanomolar concentrations, inducing mitotic arrest and cell death. PMID:25671541

  1. Devices and approaches for generating specific high-affinity nucleic acid aptamers

    NASA Astrophysics Data System (ADS)

    Szeto, Kylan; Craighead, Harold G.

    2014-09-01

    High-affinity and highly specific antibody proteins have played a critical role in biological imaging, medical diagnostics, and therapeutics. Recently, a new class of molecules called aptamers has emerged as an alternative to antibodies. Aptamers are short nucleic acid molecules that can be generated and synthesized in vitro to bind to virtually any target in a wide range of environments. They are, in principal, less expensive and more reproducible than antibodies, and their versatility creates possibilities for new technologies. Aptamers are generated using libraries of nucleic acid molecules with random sequences that are subjected to affinity selections for binding to specific target molecules. This is commonly done through a process called Systematic Evolution of Ligands by EXponential enrichment, in which target-bound nucleic acids are isolated from the pool, amplified to high copy numbers, and then reselected against the desired target. This iterative process is continued until the highest affinity nucleic acid sequences dominate the enriched pool. Traditional selections require a dozen or more laborious cycles to isolate strongly binding aptamers, which can take months to complete and consume large quantities of reagents. However, new devices and insights from engineering and the physical sciences have contributed to a reduction in the time and effort needed to generate aptamers. As the demand for these new molecules increases, more efficient and sensitive selection technologies will be needed. These new technologies will need to use smaller samples, exploit a wider range of chemistries and techniques for manipulating binding, and integrate and automate the selection steps. Here, we review new methods and technologies that are being developed towards this goal, and we discuss their roles in accelerating the availability of novel aptamers.

  2. Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124

    SciTech Connect

    Auld, Douglas S.; Lovell, Scott; Thorne, Natasha; Lea, Wendy A.; Maloney, David J.; Shen, Min; Rai, Ganesha; Battaile, Kevin P.; Thomas, Craig J.; Simeonov, Anton; Hanzlik, Robert P.; Inglese, James

    2010-04-07

    Firefly luciferase (FLuc), an ATP-dependent bioluminescent reporter enzyme, is broadly used in chemical biology and drug discovery assays. PTC124 Ataluren; (3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid) discovered in an FLuc-based assay targeting nonsense codon suppression, is an unusually potent FLuc-inhibitor. Paradoxically, PTC124 and related analogs increase cellular FLuc activity levels by posttranslational stabilization. In this study, we show that FLuc inhibition and stabilization is the result of an inhibitory product formed during the FLuc-catalyzed reaction between its natural substrate, ATP, and PTC124. A 2.0 {angstrom} cocrystal structure revealed the inhibitor to be the acyl-AMP mixed-anhydride adduct PTC124-AMP, which was subsequently synthesized and shown to be a high-affinity multisubstrate adduct inhibitor (MAI; KD = 120 pM) of FLuc. Biochemical assays, liquid chromatography/mass spectrometry, and near-attack conformer modeling demonstrate that formation of this novel MAI is absolutely dependent upon the precise positioning and reactivity of a key meta-carboxylate of PTC124 within the FLuc active site. We also demonstrate that the inhibitory activity of PTC124-AMP is relieved by free coenzyme A, a component present at high concentrations in luciferase detection reagents used for cell-based assays. This explains why PTC124 can appear to increase, instead of inhibit, FLuc activity in cell-based reporter gene assays. To our knowledge, this is an unusual example in which the 'off-target' effect of a small molecule is mediated by an MAI mechanism.

  3. Integrin alphaVbeta6 is a high-affinity receptor for coxsackievirus A9.

    PubMed

    Heikkilä, Outi; Susi, Petri; Stanway, Glyn; Hyypiä, Timo

    2009-01-01

    Coxsackievirus A9 (CAV9), a member of the genus Enterovirus in the family Picornaviridae, possesses an integrin-binding arginine-glycine-aspartic acid (RGD) motif in the C terminus of VP1 capsid protein. CAV9 has been shown to utilize integrins alphaVbeta3 and alphaVbeta6 as primary receptors for cell attachment. While CAV9 RGD-mutants (RGE and RGDdel) are capable of infecting rhabdomyosarcoma (RD) cell line, they grow very poorly in an epithelial lung carcinoma cell line (A549). In this study, the relationships between CAV9 infectivity in A549 and RD cells, receptor expression and integrin binding were analysed. A549 cells were shown to express both integrins alphaVbeta3 and alphaVbeta6, whereas alphaVbeta6 expression was not detected on the RD cells. Native CAV9 but not RGE and RGDdel mutants bound efficiently to immobilized alphaVbeta3 and alphaVbeta6. Adhesion of CAV9 but not RGE/RGDdel to A549 cells was also significantly higher than to RD cells. In contrast, no affinity or adhesion of bacterially produced VP1 proteins to the integrins or to the cells was detected. Function-blocking antibodies against alphaV-integrins blocked CAV9 but not CAV9-RGDdel infectivity, indicating that the viruses use different internalization routes; this may explain the differential infection kinetics of CAV9 and RGDdel. In an affinity assay, soluble alphaVbeta6, but not alphaVbeta3, bound to immobilized CAV9. Similarly, only soluble alphaVbeta6 blocked virus infectivity. These data suggest that CAV9 binding to alphaVbeta6 is a high-affinity interaction, which may indicate its importance in clinical infections; this remains to be determined.

  4. High affinity of acid phosphatase encoded by PHO3 gene in Saccharomyces cerevisiae for thiamin phosphates.

    PubMed

    Nosaka, K

    1990-02-01

    The enzymatic properties of acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) encoded by PHO3 gene in Saccharomyces cerevisiae, which is repressed by thiamin and has thiamin-binding activity at pH 5.0, were investigated to study physiological functions. The following results led to the conclusion that thiamin-repressible acid phosphatase physiologically catalyzes the hydrolysis of thiamin phosphates in the periplasmic space of S. cerevisiae, thus participating in utilization of the thiamin moiety of the phosphates by yeast cells: (a) thiamin-repressible acid phosphatase showed Km values of 1.6 and 1.7 microM at pH 5.0 for thiamin monophosphate and thiamin pyrophosphate, respectively. These Km values were 2-3 orders of magnitude lower than those (0.61 and 1.7 mM) for p-nitrophenyl phosphate; (b) thiamin exerted remarkable competitive inhibition in the hydrolysis of thiamin monophosphate (Ki 2.2 microM at pH 5.0), whereas the activity for p-nitrophenyl phosphate was slightly affected by thiamin; (c) the inhibitory effect of inorganic phosphate, which does not repress the thiamin-repressible enzyme, on the hydrolysis of thiamin monophosphate was much smaller than that of p-nitrophenyl phosphate. Moreover, the modification of thiamin-repressible acid phosphatase of S. cerevisiae with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide resulted in the complete loss of thiamin-binding activity and the Km value of the modified enzyme for thiamin monophosphate increased nearly to the value of the native enzyme for p-nitrophenyl phosphate. These results also indicate that the high affinity of the thiamin-repressible acid phosphatase for thiamin phosphates is due to the thiamin-binding properties of this enzyme.

  5. KW-3902, a selective high affinity antagonist for adenosine A1 receptors.

    PubMed Central

    Nonaka, H.; Ichimura, M.; Takeda, M.; Kanda, T.; Shimada, J.; Suzuki, F.; Kase, H.

    1996-01-01

    1. We demonstrate that 8-(noradamantan-3-yl)-1,3-dipropylxanthine (KW-3902) is a very potent and selective adenosine A1 receptor antagonist, assessed by radioligand binding and cyclic AMP response in cells. 2. In rat forebrain adenosine A1 receptors labelled with [3H]-cyclohexyladenosine (CHA), KW-3902 had a Ki value of 0.19 nM, whereas it showed a Ki value of 170 nM in rat striatal A2A receptors labelled with [3H]-2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoad enosine (CGS21680), indicating 890 fold A1 receptor selectivity versus the A2A receptor. KW-3902 at 10 microM showed no effect on recombinant rat A3 receptors expressed on CHO cells. 3. Saturation studies with [3H]-KW-3902 revealed that it bound with high affinity (Kd = 77 pM) and limited capacity (Bmax = 470 fmol mg-1 of protein) to a single class of recognition sites. A high positive correlation was observed between the pharmacological profile of adenosine ligands inhibiting the binding of [3H]-KW-3902 and that of [3H]-CHA. 4. KW-3902 showed potent A1 antagonism against the inhibition of forskolin-induced cyclic AMP accumulation in DDT1 MF-2 cells by the A1-selective agonist, cyclopentyladenosine with a dissociation constant (KB value) of 0.34 nM. KW-3902 antagonized 5'-N-ethylcarboxamidoadenosine-elicited cyclic AMP accumulation via A2B receptors with a KB value of 52 nM. 5. KW-3902 exhibited marked species-dependent differences in the binding affinities. The highest affinity was for the rat A1 receptor (ki = 0.19 nM) and these values for guinea-pig and dog A1 receptors were 1.3 and 10 nM, respectively. PMID:8732272

  6. Cloning of the outer membrane high-affinity Fe(III)-pyochelin receptor of Pseudomonas aeruginosa.

    PubMed Central

    Ankenbauer, R G

    1992-01-01

    Pseudomonas aeruginosa produces the phenolic siderophore pyochelin under iron-limiting conditions. In this study, an Fe(III)-pyochelin transport-negative (Fpt-) strain, IA613, was isolated and characterized. 55Fe(III)-pyochelin transport assays determined that no Fe(III)-pyochelin associated with the Fpt- IA613 cells while a significant amount associated with KCN-poisoned Fpt+ cells. A P. aeruginosa genomic library was constructed in the IncP cosmid pLAFR1. The genomic library was mobilized into IA613, and a recombinant cosmid, pCC41, which complemented the Fpt- phenotype of IA613, was isolated. pCC41 contained a 28-kb insert of P. aeruginosa DNA, and the Fpt(-)-complementing region was localized to a 3.6-kb BamHI-EcoRI fragment by deletion and subcloning of the insert. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of IA613 revealed that it lacked a 75-kDa outer membrane protein present in Fpt+ strains. IA613 strains bearing plasmid pRML303, which carries the 3.6-kb BamHI-EcoRI fragment of pCC41, expressed the 75-kDa outer membrane protein and demonstrated a 55Fe(III)-pyochelin transport phenotype identical to that of a wild-type Fpt+ strain. Minicell analysis demonstrated that the 3.6-kb BamHI-EcoRI fragment of pCC41 encoded a protein of approximately 75 kDa. The results presented here and in a previous report (D. E. Heinrichs, L. Young, and K. Poole, Infect. Immun. 59:3680-3684, 1991) lead to the conclusion that the 75-kDa outer membrane protein is the high-affinity receptor for Fe(III)-pyochelin in P. aeruginosa. Images PMID:1320609

  7. Early Signs of Pathological Cognitive Aging in Mice Lacking High-Affinity Nicotinic Receptors

    PubMed Central

    Konsolaki, Eleni; Tsakanikas, Panagiotis; Polissidis, Alexia V.; Stamatakis, Antonios; Skaliora, Irini

    2016-01-01

    In order to address pathological cognitive decline effectively, it is critical to adopt early preventive measures in individuals considered at risk. It is therefore essential to develop approaches that identify such individuals before the onset of irreversible dementia. A deficient cholinergic system has been consistently implicated as one of the main factors associated with a heightened vulnerability to the aging process. In the present study we used mice lacking high affinity nicotinic receptors (β2-/-), which have been proposed as an animal model of accelerated/premature cognitive aging. Our aim was to identify behavioral signs that could serve as indicators or predictors of impending cognitive decline. We used test batteries in order to assess cognitive functions and additional tasks to investigate spontaneous behaviors, such as species-specific activities and exploration/locomotion in a novel environment. Our data confirm the hypothesis that β2-/- animals exhibit age-related cognitive impairments in spatial learning. In addition, they document age-related deficits in other areas, such as recognition memory, burrowing and nesting building, thereby extending the validity of this animal model for the study of pathological aging. Finally, our data reveal deficits in spontaneous behavior and habituation processes that precede the onset of cognitive decline and could therefore be useful as a non-invasive behavioral screen for identifying animals at risk. To our knowledge, this is the first study to perform an extensive behavioral assessment of an animal model of premature cognitive aging, and our results suggest that β2-nAChR dependent cognitive deterioration progressively evolves from initial subtle behavioral changes to global dementia due to the combined effect of the neuropathology and aging. PMID:27199738

  8. The C2 domains of granuphilin are high-affinity sensors for plasma membrane lipids.

    PubMed

    Lyakhova, Tatyana A; Knight, Jefferson D

    2014-09-01

    Membrane-targeting proteins are crucial components of many cell signaling pathways, including the secretion of insulin. Granuphilin, also known as synaptotagmin-like protein 4, functions in tethering secretory vesicles to the plasma membrane prior to exocytosis. Granuphilin docks to insulin secretory vesicles through interaction of its N-terminal domain with vesicular Rab proteins; however, the mechanisms of granuphilin plasma membrane targeting and release are less clear. Granuphilin contains two C2 domains, C2A and C2B, that interact with the plasma membrane lipid phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2]. The goal of this study was to determine membrane-binding mechanisms, affinities, and kinetics of both granuphilin C2 domains using fluorescence spectroscopic techniques. Results indicate that both C2A and C2B bind anionic lipids in a Ca(2+)-independent manner. The C2A domain binds liposomes containing a physiological mixture of lipids including 2% PI(4,5)P2 or PI(3,4,5)P3 with high affinity (apparent K(d, PIPx) of 2-5 nM), and binds nonspecifically with moderate affinity to anionic liposomes lacking phosphatidylinositol phosphate (PIPx) lipids. The C2B domain binds with sub-micromolar affinity to liposomes containing PI(4,5)P2 but does not have a measurable affinity for background anionic lipids. Both domains can be competed away from their target lipids by the soluble PIPx analog inositol-(1,2,3,4,5,6)-hexakisphosphate (IP6), which is a positive regulator of insulin secretion. Potential roles of these interactions in the docking and release of granuphilin from the plasma membrane are discussed.

  9. Early Signs of Pathological Cognitive Aging in Mice Lacking High-Affinity Nicotinic Receptors.

    PubMed

    Konsolaki, Eleni; Tsakanikas, Panagiotis; Polissidis, Alexia V; Stamatakis, Antonios; Skaliora, Irini

    2016-01-01

    In order to address pathological cognitive decline effectively, it is critical to adopt early preventive measures in individuals considered at risk. It is therefore essential to develop approaches that identify such individuals before the onset of irreversible dementia. A deficient cholinergic system has been consistently implicated as one of the main factors associated with a heightened vulnerability to the aging process. In the present study we used mice lacking high affinity nicotinic receptors (β2-/-), which have been proposed as an animal model of accelerated/premature cognitive aging. Our aim was to identify behavioral signs that could serve as indicators or predictors of impending cognitive decline. We used test batteries in order to assess cognitive functions and additional tasks to investigate spontaneous behaviors, such as species-specific activities and exploration/locomotion in a novel environment. Our data confirm the hypothesis that β2-/- animals exhibit age-related cognitive impairments in spatial learning. In addition, they document age-related deficits in other areas, such as recognition memory, burrowing and nesting building, thereby extending the validity of this animal model for the study of pathological aging. Finally, our data reveal deficits in spontaneous behavior and habituation processes that precede the onset of cognitive decline and could therefore be useful as a non-invasive behavioral screen for identifying animals at risk. To our knowledge, this is the first study to perform an extensive behavioral assessment of an animal model of premature cognitive aging, and our results suggest that β2-nAChR dependent cognitive deterioration progressively evolves from initial subtle behavioral changes to global dementia due to the combined effect of the neuropathology and aging.

  10. Impaired Presynaptic High-Affinity Choline Transporter Causes a Congenital Myasthenic Syndrome with Episodic Apnea.

    PubMed

    Bauché, Stéphanie; O'Regan, Seana; Azuma, Yoshiteru; Laffargue, Fanny; McMacken, Grace; Sternberg, Damien; Brochier, Guy; Buon, Céline; Bouzidi, Nassima; Topf, Ana; Lacène, Emmanuelle; Remerand, Ganaelle; Beaufrere, Anne-Marie; Pebrel-Richard, Céline; Thevenon, Julien; El Chehadeh-Djebbar, Salima; Faivre, Laurence; Duffourd, Yannis; Ricci, Federica; Mongini, Tiziana; Fiorillo, Chiara; Astrea, Guja; Burloiu, Carmen Magdalena; Butoianu, Niculina; Sandu, Carmen; Servais, Laurent; Bonne, Gisèle; Nelson, Isabelle; Desguerre, Isabelle; Nougues, Marie-Christine; Bœuf, Benoit; Romero, Norma; Laporte, Jocelyn; Boland, Anne; Lechner, Doris; Deleuze, Jean-François; Fontaine, Bertrand; Strochlic, Laure; Lochmuller, Hanns; Eymard, Bruno; Mayer, Michèle; Nicole, Sophie

    2016-09-01

    The neuromuscular junction (NMJ) is one of the best-studied cholinergic synapses. Inherited defects of peripheral neurotransmission result in congenital myasthenic syndromes (CMSs), a clinically and genetically heterogeneous group of rare diseases with fluctuating fatigable muscle weakness as the clinical hallmark. Whole-exome sequencing and Sanger sequencing in six unrelated families identified compound heterozygous and homozygous mutations in SLC5A7 encoding the presynaptic sodium-dependent high-affinity choline transporter 1 (CHT), which is known to be mutated in one dominant form of distal motor neuronopathy (DHMN7A). We identified 11 recessive mutations in SLC5A7 that were associated with a spectrum of severe muscle weakness ranging from a lethal antenatal form of arthrogryposis and severe hypotonia to a neonatal form of CMS with episodic apnea and a favorable prognosis when well managed at the clinical level. As expected given the critical role of CHT for multisystemic cholinergic neurotransmission, autonomic dysfunctions were reported in the antenatal form and cognitive impairment was noticed in half of the persons with the neonatal form. The missense mutations induced a near complete loss of function of CHT activity in cell models. At the human NMJ, a delay in synaptic maturation and an altered maintenance were observed in the antenatal and neonatal forms, respectively. Increased synaptic expression of butyrylcholinesterase was also observed, exposing the dysfunction of cholinergic metabolism when CHT is deficient in vivo. This work broadens the clinical spectrum of human diseases resulting from reduced CHT activity and highlights the complexity of cholinergic metabolism at the synapse. PMID:27569547

  11. Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124.

    PubMed

    Auld, Douglas S; Lovell, Scott; Thorne, Natasha; Lea, Wendy A; Maloney, David J; Shen, Min; Rai, Ganesha; Battaile, Kevin P; Thomas, Craig J; Simeonov, Anton; Hanzlik, Robert P; Inglese, James

    2010-03-16

    Firefly luciferase (FLuc), an ATP-dependent bioluminescent reporter enzyme, is broadly used in chemical biology and drug discovery assays. PTC124 (Ataluren; (3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid) discovered in an FLuc-based assay targeting nonsense codon suppression, is an unusually potent FLuc-inhibitor. Paradoxically, PTC124 and related analogs increase cellular FLuc activity levels by posttranslational stabilization. In this study, we show that FLuc inhibition and stabilization is the result of an inhibitory product formed during the FLuc-catalyzed reaction between its natural substrate, ATP, and PTC124. A 2.0 A cocrystal structure revealed the inhibitor to be the acyl-AMP mixed-anhydride adduct PTC124-AMP, which was subsequently synthesized and shown to be a high-affinity multisubstrate adduct inhibitor (MAI; K(D) = 120 pM) of FLuc. Biochemical assays, liquid chromatography/mass spectrometry, and near-attack conformer modeling demonstrate that formation of this novel MAI is absolutely dependent upon the precise positioning and reactivity of a key meta-carboxylate of PTC124 within the FLuc active site. We also demonstrate that the inhibitory activity of PTC124-AMP is relieved by free coenzyme A, a component present at high concentrations in luciferase detection reagents used for cell-based assays. This explains why PTC124 can appear to increase, instead of inhibit, FLuc activity in cell-based reporter gene assays. To our knowledge, this is an unusual example in which the "off-target" effect of a small molecule is mediated by an MAI mechanism.

  12. Targeting protein-protein interactions with trimeric ligands: high affinity inhibitors of the MAGUK protein family.

    PubMed

    Nissen, Klaus B; Haugaard-Kedström, Linda M; Wilbek, Theis S; Nielsen, Line S; Åberg, Emma; Kristensen, Anders S; Bach, Anders; Jemth, Per; Strømgaard, Kristian

    2015-01-01

    PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins.

  13. Water channel in the binding site of a high affinity anti-methotrexate antibody.

    PubMed

    Gayda, Susan; Longenecker, Kenton L; Manoj, Sharmila; Judge, Russell A; Saldana, Sylvia C; Ruan, Qiaoqiao; Swift, Kerry M; Tetin, Sergey Y

    2014-06-17

    In the present study, we report the structure of the free and drug-bound Fab fragment of a high affinity anti-methotrexate antibody and perform a thermodynamic analysis of the binding process. The anti-methotrexate Fab fragment features a remarkably rigid tunnel-like binding site that extends into a water channel serving as a specialized route to move solvent out and into the site upon ligand binding and dissociation. This new finding in antibody structure-function relationships directly relates to the fast association (1 × 10⁷ M⁻¹ s⁻¹) and slow dissociation (4 × 10⁻⁵ s⁻¹) rates determined for mAb ADD056, resulting in a very strong binding with a K(D) ~ 3.6 pM at 20 °C. As follows from the X-ray data analysis, the methotrexate-antibody complex is stabilized by an extended network of hydrogen bonds and stacking interactions. The analysis also shows structural involvement of the CDR H3 in formation of the water channel revealing another important role of this hypervariable region. This suggests a new direction in natural affinity maturation and opens a new possibility in antibody engineering. Methotrexate is a widely used therapeutic agent for many malignant diseases and inflammatory disorders. Unfortunately, it may also interfere with central aspects of metabolism and thereby cause inevitable side effects. Therefore, methotrexate therapy requires careful monitoring of drug blood levels, which is traditionally done by immunoassays. An understanding of the structure-function properties of antibodies selected for drug monitoring substantiates the performance and robustness of such tests. PMID:24832237

  14. Impaired Presynaptic High-Affinity Choline Transporter Causes a Congenital Myasthenic Syndrome with Episodic Apnea.

    PubMed

    Bauché, Stéphanie; O'Regan, Seana; Azuma, Yoshiteru; Laffargue, Fanny; McMacken, Grace; Sternberg, Damien; Brochier, Guy; Buon, Céline; Bouzidi, Nassima; Topf, Ana; Lacène, Emmanuelle; Remerand, Ganaelle; Beaufrere, Anne-Marie; Pebrel-Richard, Céline; Thevenon, Julien; El Chehadeh-Djebbar, Salima; Faivre, Laurence; Duffourd, Yannis; Ricci, Federica; Mongini, Tiziana; Fiorillo, Chiara; Astrea, Guja; Burloiu, Carmen Magdalena; Butoianu, Niculina; Sandu, Carmen; Servais, Laurent; Bonne, Gisèle; Nelson, Isabelle; Desguerre, Isabelle; Nougues, Marie-Christine; Bœuf, Benoit; Romero, Norma; Laporte, Jocelyn; Boland, Anne; Lechner, Doris; Deleuze, Jean-François; Fontaine, Bertrand; Strochlic, Laure; Lochmuller, Hanns; Eymard, Bruno; Mayer, Michèle; Nicole, Sophie

    2016-09-01

    The neuromuscular junction (NMJ) is one of the best-studied cholinergic synapses. Inherited defects of peripheral neurotransmission result in congenital myasthenic syndromes (CMSs), a clinically and genetically heterogeneous group of rare diseases with fluctuating fatigable muscle weakness as the clinical hallmark. Whole-exome sequencing and Sanger sequencing in six unrelated families identified compound heterozygous and homozygous mutations in SLC5A7 encoding the presynaptic sodium-dependent high-affinity choline transporter 1 (CHT), which is known to be mutated in one dominant form of distal motor neuronopathy (DHMN7A). We identified 11 recessive mutations in SLC5A7 that were associated with a spectrum of severe muscle weakness ranging from a lethal antenatal form of arthrogryposis and severe hypotonia to a neonatal form of CMS with episodic apnea and a favorable prognosis when well managed at the clinical level. As expected given the critical role of CHT for multisystemic cholinergic neurotransmission, autonomic dysfunctions were reported in the antenatal form and cognitive impairment was noticed in half of the persons with the neonatal form. The missense mutations induced a near complete loss of function of CHT activity in cell models. At the human NMJ, a delay in synaptic maturation and an altered maintenance were observed in the antenatal and neonatal forms, respectively. Increased synaptic expression of butyrylcholinesterase was also observed, exposing the dysfunction of cholinergic metabolism when CHT is deficient in vivo. This work broadens the clinical spectrum of human diseases resulting from reduced CHT activity and highlights the complexity of cholinergic metabolism at the synapse.

  15. Identification of a high affinity nucleocapsid protein binding element within the Moloney murine leukemia virus Psi-RNA packaging signal: implications for genome recognition.

    PubMed

    D'Souza, V; Melamed, J; Habib, D; Pullen, K; Wallace, K; Summers, M F

    2001-11-23

    Murine leukemia virus (MLV) is currently the most widely used gene delivery system in gene therapy trials. The simple retrovirus packages two copies of its RNA genome by a mechanism that involves interactions between the nucleocapsid (NC) domain of a virally-encoded Gag polyprotein and a segment of the RNA genome located just upstream of the Gag initiation codon, known as the Psi-site. Previous studies indicated that the MLV Psi-site contains three stem loops (SLB-SLD), and that stem loops SLC and SLD play prominent roles in packaging. We have developed a method for the preparation and purification of large quantities of recombinant Moloney MLV NC protein, and have studied its interactions with a series of oligoribonucleotides that contain one or more of the Psi-RNA stem loops. At RNA concentrations above approximately 0.3 mM, isolated stem loop SLB forms a duplex and stem loops SL-C and SL-D form kissing complexes, as expected from previous studies. However, neither the monomeric nor the dimeric forms of these isolated stem loops binds NC with significant affinity. Longer constructs containing two stem loops (SL-BC and SL-CD) also exhibit low affinities for NC. However, NC binds with high affinity and stoichiometrically to both the monomeric and dimeric forms of an RNA construct that contains all three stem loops (SL-BCD; K(d)=132(+/-55) nM). Titration of SL-BCD with NC also shifts monomer-dimer equilibrium toward the dimer. Mutagenesis experiments demonstrate that the conserved GACG tetraloops of stem loops C and D do not influence the monomer-dimer equilibrium of SL-BCD, that the tetraloop of stem loop B does not participate directly in NC binding, and that the tetraloops of stem loops C and D probably also do not bind to NC. These surprising results differ considerably from those observed for HIV-1, where NC binds to individual stem loops with high affinity via interactions with exposed residues of the tetraloops. The present results indicate that MLV NC binds

  16. Biochemical characterization of high-affinity 3H-opioid binding. Further evidence for Mu1 sites

    SciTech Connect

    Nishimura, S.L.; Recht, L.D.; Pasternak, G.W.

    1984-01-01

    In saturation studies with (/sup 3/H)dihydromorphine, unlabeled D-Ala2-D-Leu5-enkephalin (1 nM) inhibited the high-affinity binding component far more potently than the lower-affinity one. Similarly, morphine (1 nM) inhibited the higher-affinity binding of /sup 3/H-D-Ala2-D-Leu5-enkephalin to a greater extent than its lower-affinity binding component, consistent with a common high-affinity binding site for opiates and enkephalins. Treatment of tissue with either trypsin (1 microgram/ml) or N-ethylmaleimide (25 microM) effectively eliminated the high-affinity binding component of a series of /sup 3/H-opiates and opioid peptides. Competition studies following both treatments were consistent with a common high-affinity binding site. Both treatments also eliminated the ability of low morphine concentrations (less than 1 nM) to inhibit /sup 3/H-D-Ala2-D-Leu5-enkephalin binding and of low D-Ala2-D-Leu5-enkephalin concentrations (less than 1 nM) to inhibit (/sup 3/H)dihydromorphine binding. Protection experiments examining N-ethylmaleimide (25 microM) inhibition of (/sup 3/H)dihydromorphine binding showed significant protection (p less than 0.002) by both unlabeled D-Ala2-D-Leu5-enkephalin and morphine (both at 1 nM). When studied together, both naloxonazine and N-ethylmaleimide inhibited (/sup 3/H)dihydromorphine binding to a similar extent. Equally important, tissue previously treated with naloxonazine was far less sensitive to N-ethylmaleimide than was untreated control tissue, consistent with the possibility that both treatments affected the same site. Together, these results support the concept of a common high-affinity binding site for opiates and opioid peptides.

  17. Recording information on protein complexes in an information management system.

    PubMed

    Savitsky, Marc; Diprose, Jonathan M; Morris, Chris; Griffiths, Susanne L; Daniel, Edward; Lin, Bill; Daenke, Susan; Bishop, Benjamin; Siebold, Christian; Wilson, Keith S; Blake, Richard; Stuart, David I; Esnouf, Robert M

    2011-08-01

    The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein-protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described. PMID:21605682

  18. Visualizing active membrane protein complexes by electron cryotomography

    PubMed Central

    Gold, Vicki A.M.; Ieva, Raffaele; Walter, Andreas; Pfanner, Nikolaus; van der Laan, Martin; Kühlbrandt, Werner

    2014-01-01

    Unravelling the structural organization of membrane protein machines in their active state and native lipid environment is a major challenge in modern cell biology research. Here we develop the STAMP (Specifically TArgeted Membrane nanoParticle) technique as a strategy to localize protein complexes in situ by electron cryotomography (cryo-ET). STAMP selects active membrane protein complexes and marks them with quantum dots. Taking advantage of new electron detector technology that is currently revolutionizing cryotomography in terms of achievable resolution, this approach enables us to visualize the three-dimensional distribution and organization of protein import sites in mitochondria. We show that import sites cluster together in the vicinity of crista membranes, and we reveal unique details of the mitochondrial protein import machinery in action. STAMP can be used as a tool for site-specific labelling of a multitude of membrane proteins by cryo-ET in the future. PMID:24942077

  19. Immersion freezing of ice nucleation active protein complexes

    NASA Astrophysics Data System (ADS)

    Hartmann, S.; Augustin, S.; Clauss, T.; Wex, H.; Šantl-Temkiv, T.; Voigtländer, J.; Niedermeier, D.; Stratmann, F.

    2013-06-01

    Utilising the Leipzig Aerosol Cloud Interaction Simulator (LACIS), the immersion freezing behaviour of droplet ensembles containing monodisperse particles, generated from a Snomax™ solution/suspension, was investigated. Thereto ice fractions were measured in the temperature range between -5 °C to -38 °C. Snomax™ is an industrial product applied for artificial snow production and contains Pseudomonas syringae} bacteria which have long been used as model organism for atmospheric relevant ice nucleation active (INA) bacteria. The ice nucleation activity of such bacteria is controlled by INA protein complexes in their outer membrane. In our experiments, ice fractions increased steeply in the temperature range from about -6 °C to about -10 °C and then levelled off at ice fractions smaller than one. The plateau implies that not all examined droplets contained an INA protein complex. Assuming the INA protein complexes to be Poisson distributed over the investigated droplet populations, we developed the CHESS model (stoCHastic modEl of similar and poiSSon distributed ice nuclei) which allows for the calculation of ice fractions as function of temperature and time for a given nucleation rate. Matching calculated and measured ice fractions, we determined and parameterised the nucleation rate of INA protein complexes exhibiting class III ice nucleation behaviour. Utilising the CHESS model, together with the determined nucleation rate, we compared predictions from the model to experimental data from the literature and found good agreement. We found that (a) the heterogeneous ice nucleation rate expression quantifying the ice nucleation behaviour of the INA protein complex is capable of describing the ice nucleation behaviour observed in various experiments for both, Snomax™ and P. syringae bacteria, (b) the ice nucleation rate, and its temperature dependence, seem to be very similar regardless of whether the INA protein complexes inducing ice nucleation are attached

  20. Recording information on protein complexes in an information management system.

    PubMed

    Savitsky, Marc; Diprose, Jonathan M; Morris, Chris; Griffiths, Susanne L; Daniel, Edward; Lin, Bill; Daenke, Susan; Bishop, Benjamin; Siebold, Christian; Wilson, Keith S; Blake, Richard; Stuart, David I; Esnouf, Robert M

    2011-08-01

    The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein-protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described.

  1. Recording information on protein complexes in an information management system

    PubMed Central

    Savitsky, Marc; Diprose, Jonathan M.; Morris, Chris; Griffiths, Susanne L.; Daniel, Edward; Lin, Bill; Daenke, Susan; Bishop, Benjamin; Siebold, Christian; Wilson, Keith S.; Blake, Richard; Stuart, David I.; Esnouf, Robert M.

    2011-01-01

    The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein–protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described. PMID:21605682

  2. Coupling protein complex analysis to peptide based proteomics.

    PubMed

    Gao, Qiang; Madian, Ashraf G; Liu, Xiuping; Adamec, Jiri; Regnier, Fred E

    2010-12-01

    Proteolysis is a central component of most proteomics methods. Unfortunately much of the information relating to the structural diversity of proteins is lost during digestion. This paper describes a method in which the native proteome of yeast was subjected to preliminary fractionation by size exclusion chromatography (SEC) prior to trypsin digestion of SEC fractions and reversed phase chromatography-mass spectral analysis to identify tryptic peptides thus generated. Through this approach proteins associated with other proteins in high molecular mass complexes were recognized and identified. A focus of this work was on the identification of Hub proteins that associate with multiple interaction partners. A critical component of this strategy is to choose methods and conditions that maximize retention of native structure during the various stages of analysis prior to proteolysis, especially during cell lysis. Maximum survival of protein complexes during lysis was obtained with the French press and bead-beater methods of cell disruption at approximately pH 8 with 200 mM NaCl in the lysis buffer. Structure retention was favored by higher ionic strength, suggesting that hydrophobic effects are important in maintaining the structure of protein complexes. Recovery of protein complexes declined substantially with storage at any temperature, but storage at -20°C was best when low temperature storage was necessary. Slightly lower recovery was obtained with storage at -80°C while lowest recovery was achieved at 4°C. It was concluded that initial fractionation of native proteins in cell lysates by SEC prior to RPC-MS/MS of tryptic digests can be used to recognize and identify proteins in complexes along with their interaction partners in known protein complexes.

  3. A Diatom Light-Harvesting Pigment-Protein Complex 1

    PubMed Central

    Friedman, Alan L.; Alberte, Randall S.

    1984-01-01

    A light-harvesting pigment-protein complex was isolated from the diatom Phaeodactylum tricornutum using the zwitterionic detergent CHAPS (3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate). Detergent-solubilized membranes were fractionated by sucrose density gradient centrifugation into three components. The medium density fraction contained chlorophyll a, chlorophyll c, and fucoxanthin. This fraction was purified by DEAE-ion exchange chromatography, and contained chlorophyll a, chlorophyll c, and fucoxanthin in a molar ratio of 2.4:1.0:4.8. Fluorescence emission and excitation spectra of the isolated complex demonstrated that light energy absorbed by chlorophyll c and fucoxanthin was coupled to chlorophyll a fluorescence. Upon denaturation, the apoprotein yielded a polypeptide doublet at 17.5 to 18.0 kilodaltons which accounted for 30 to 40% of the toal membrane protein. These findings indicate that this pigment-protein complex is a major component of the diatom photosynthetic lammellae. The quantitative amino acid composition of the apoprotein was very similar to those reported for other membrane-bound pigment-protein complexes. Based on the protein to chlorophyll a ratio of 7700 grams protein per mole chlorophyll a for the complex, each apoprotein molecule contains, to the nearest integer, two chlorophyll a, one chlorophyll c, and five fucoxanthin molecules. Polyclonal antibodies raised against the 17.5 to 18.0 kilodaltons apoprotein showed a monospecific reaction with only the 17.5 to 18.0 protein zone from denatured P. tricornutum membranes as well as to the nondenatured pigment-protein complex. It appears that this complex is common to other diatom species. Images Fig. 2 Fig. 3 PMID:16663869

  4. Biclustering Protein Complex Interactions with a Biclique FindingAlgorithm

    SciTech Connect

    Ding, Chris; Zhang, Anne Ya; Holbrook, Stephen

    2006-12-01

    Biclustering has many applications in text mining, web clickstream mining, and bioinformatics. When data entries are binary, the tightest biclusters become bicliques. We propose a flexible and highly efficient algorithm to compute bicliques. We first generalize the Motzkin-Straus formalism for computing the maximal clique from L{sub 1} constraint to L{sub p} constraint, which enables us to provide a generalized Motzkin-Straus formalism for computing maximal-edge bicliques. By adjusting parameters, the algorithm can favor biclusters with more rows less columns, or vice verse, thus increasing the flexibility of the targeted biclusters. We then propose an algorithm to solve the generalized Motzkin-Straus optimization problem. The algorithm is provably convergent and has a computational complexity of O(|E|) where |E| is the number of edges. It relies on a matrix vector multiplication and runs efficiently on most current computer architectures. Using this algorithm, we bicluster the yeast protein complex interaction network. We find that biclustering protein complexes at the protein level does not clearly reflect the functional linkage among protein complexes in many cases, while biclustering at protein domain level can reveal many underlying linkages. We show several new biologically significant results.

  5. Ontology integration to identify protein complex in protein interaction networks

    PubMed Central

    2011-01-01

    Background Protein complexes can be identified from the protein interaction networks derived from experimental data sets. However, these analyses are challenging because of the presence of unreliable interactions and the complex connectivity of the network. The integration of protein-protein interactions with the data from other sources can be leveraged for improving the effectiveness of protein complexes detection algorithms. Methods We have developed novel semantic similarity method, which use Gene Ontology (GO) annotations to measure the reliability of protein-protein interactions. The protein interaction networks can be converted into a weighted graph representation by assigning the reliability values to each interaction as a weight. Following the approach of that of the previously proposed clustering algorithm IPCA which expands clusters starting from seeded vertices, we present a clustering algorithm OIIP based on the new weighted Protein-Protein interaction networks for identifying protein complexes. Results The algorithm OIIP is applied to the protein interaction network of Sacchromyces cerevisiae and identifies many well known complexes. Experimental results show that the algorithm OIIP has higher F-measure and accuracy compared to other competing approaches. PMID:22165991

  6. Synthesis and characterization of a high-affinity photoactivatable analogue of thyrotropin-releasing hormone.

    PubMed Central

    Brady, K D; Tashjian, A H

    1992-01-01

    An analogue of thyrotropin-releasing hormone (TRH, pGlu-His-ProNH2), i.e. pGlu-His-ProNH-(CH2)6-(4-azidosalicylamide) (TRH-ASA), has been synthesized and, in a radioiodinated form (TRH-IASA), characterized and used as a photoaffinity reagent to label the TRH receptor on rat pituitary GH4C1 cells. TRH-IASA bound to GH4C1 cells with high affinity (Kd = 8 nM), comparable with that of TRH binding. The binding of TRH-IASA was competitive with binding of TRH, two TRH analogues and a TRH receptor antagonist, chlordiazepoxide. TRH-IASA did not bind to or label GH12C1 cells, which lack functional TRH receptors. Labelling of GH4C1 cells with TRH-IASA followed by SDS/PAGE and autoradiography of membrane proteins demonstrated labelling of a single polypeptide which ran as a diffuse band between 71 and 91 kDa, centred at 76 kDa. No change in this labelling pattern was observed as a function of the length of time (between 5 min and 2 h) that GH4C1 cells were incubated with 3 nM-TRH-IASA. Using either a very short (5 s) photolysis interval or low TRH-IASA concentrations, only the 76 kDa band was labelled. Minor bands appeared only after extended photolysis and use of high TRH-IASA concentrations. We conclude that the TRH receptor from rat pituitary GH4C1 cells is a single peptide with an apparent molecular mass of 76 kDa. Details of the chemical synthesis of TRH-ASA are given in Supplementary Publication SUP 50167 (5 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1992) 281, 5. Images Fig. 3. Fig. 4. PMID:1310004

  7. High affinity group III mGluRs regulate mossy fiber input to CA3 interneurons

    PubMed Central

    Cosgrove, Kathleen E.; Meriney, Stephen D.; Barrionuevo, Germán

    2010-01-01

    Stratum lacunosum-moleculare interneurons (L-Mi) in hippocampal area CA3 target the apical dendrite of pyramidal cells providing feedforward inhibition. Here we report that selective activation of group III metabotropic glutamate receptors (mGluRs) 4/8 with L-(+)-2-amino-4-phosphnobytyric acid (L-AP4; 10 μM) decreased the probability of glutamate release from the mossy fiber (MF) terminals synapsing onto L-Mi. Consistent with this interpretation, application of L-AP4 in the presence of 3 mM strontium decreased the frequency of asynchronous MF EPSCs in L-Mi. Furthermore, the dose response curve showed that L-AP4 at 400 μM produced no further decrease in MF EPSC amplitude compared to 20 μM L-AP4, indicating the lack of mGluRs 7 at these MF terminals. We also found that one mechanism of mGluRs 4/8-mediated inhibition of release is linked to N-type voltage gated calcium channels at MF terminals. Application of the group III mGluR antagonist MSOP (100 μM) demonstrated that mGluRs 4/8 are neither tonically active nor activated by low and moderate frequencies of activity. However, trains of stimuli to the MF at 20 and 40Hz delivered during the application of MSOP revealed a relief of inhibition of transmitter release and an increase in the overall probability of action potential firing in the postsynaptic L-Mi. Interestingly, the time to first action potential was significantly shorter in the presence of MSOP, indicating that mGluR 4/8 activation delays L-Mi firing in response to MF activity. Taken together, our data demonstrate that the timing and probability of action potentials in L-Mi evoked by MF synaptic input is regulated by the activation of presynaptic high affinity group III mGluRs. PMID:20824730

  8. Induction of high-affinity GM-CSF receptors during all-trans retinoic acid treatment of acute promyelocytic leukemia.

    PubMed

    de Gentile, A; Toubert, M E; Dubois, C; Krawice, I; Schlageter, M H; Balitrand, N; Castaigne, S; Degos, L; Rain, J D; Najean, Y

    1994-10-01

    Differentiation of normal myeloid cells is accompanied by the increase of high-affinity GM-CSF receptors necessary for progenitor proliferation/differentiation and mature neutrophil function. All-trans retinoic acid (ATRA) induces terminal differentiation of acute promyelocytic leukemia cells (AML3 subtype). We report in this study that AML3 cells, like other AML subtypes, harbor high-affinity GM-CSF R (n = 138.3 +/- 69.3 sites/cell, Kd = 76.9 +/- 68.8 pM). In all cases, incubation with ATRA induces either an increase in the number of affinity of GM-CSF R (n = 212.7 +/- 116.2 sites/cell, Kd = 43.2 +/- 22.5 pM). The data presented show that modulation of GM-CSF receptors cells is correlated to the degree of ATRA-induced granulocytic differentiation but not to increased cell growth.

  9. Complementary DNA display selection of high-affinity peptides binding the vacuolating toxin (VacA) of Helicobacter pylori.

    PubMed

    Hayakawa, Yumiko; Matsuno, Mitsuhiro; Tanaka, Makoto; Wada, Akihiro; Kitamura, Koichiro; Takei, Osamu; Sasaki, Ryuzo; Mizukami, Tamio; Hasegawa, Makoto

    2015-09-01

    Artificial peptides designed for molecular recognition of a bacterial toxin have been developed. Vacuolating cytotoxin A protein (VacA) is a major virulence factor of Helicobacter pylori, a gram-negative microaerophilic bacterium inhabiting the upper gastrointestinal tract, particularly the stomach. This study attempted to identify specific peptide sequences with high affinity for VacA using systematic directed evolution in vitro, a cDNA display method. A surface plasmon resonance-based biosensor and fluorescence correlation spectroscopy to examine binding of peptides with VacA identified a peptide (GRVNQRL) with high affinity. Cyclization of the peptide by attaching cysteine residues to both termini improved its binding affinity to VacA, with a dissociation constant (Kd ) of 58 nm. This study describes a new strategy for the development of artificial functional peptides, which are promising materials in biochemical analyses and medical applications.

  10. Multipurpose ligand, DAKLI (Dynorphin A-analogue Kappa LIgand), with high affinity and selectivity for dynorphin (. kappa. opioid) binding sites

    SciTech Connect

    Goldstein, A.; Nestor, J.J. Jr.; Naidu, A.; Newman, S.R. )

    1988-10-01

    The authors describe a synthetic ligand, DALKI (Dynorphin A-analogue Kappa LIgand), related to the opioid peptide dynorphin A. A single reactive amino group at the extended carboxyl terminus permits various reporter groups to be attached, such as {sup 125}I-labeled Bolton-Hunter reagent, fluorescein isothiocyanate, or biotin. These derivatives have high affinity and selectivity for the dynorphin ({kappa} opioid) receptor. An incidental finding is that untreated guinea pig brain membranes have saturable avidin binding sites.

  11. Immunotherapy Expands and Maintains the Function of High-Affinity Tumor-Infiltrating CD8 T Cells In Situ.

    PubMed

    Moran, Amy E; Polesso, Fanny; Weinberg, Andrew D

    2016-09-15

    Cancer cells harbor high-affinity tumor-associated Ags capable of eliciting potent antitumor T cell responses, yet detecting these polyclonal T cells is challenging. Therefore, surrogate markers of T cell activation such as CD69, CD44, and programmed death-1 (PD-1) have been used. We report in this study that in mice, expression of activation markers including PD-1 is insufficient in the tumor microenvironment to identify tumor Ag-specific T cells. Using the Nur77GFP T cell affinity reporter mouse, we highlight that PD-1 expression can be induced independent of TCR ligation within the tumor. Given this, we characterized the utility of the Nur77GFP model system in elucidating mechanisms of action of immunotherapies independent of PD-1 expression. Coexpression of Nur77GFP and OX40 identifies a polyclonal population of high-affinity tumor-associated Ag-specific CD8(+) T cells, which produce more IFN-γ in situ than OX40 negative and doubles in quantity with anti-OX40 and anti-CTLA4 mAb therapy but not with anti-PD-1 or programmed death ligand-1. Moreover, expansion of these high-affinity CD8 T cells prolongs survival of tumor-bearing animals. Upon chronic stimulation in tumors and after adoptive cell therapy, CD8 TCR signaling and Nur77GFP induction is impaired, and tumors progress. However, this can be reversed and overall survival significantly enhanced after adoptive cell therapy with agonist OX40 immunotherapy. Therefore, we propose that OX40 agonist immunotherapy can maintain functional TCR signaling of chronically stimulated tumor-resident CD8 T cells, thereby increasing the frequency of cytotoxic, high-affinity, tumor-associated Ag-specific cells. PMID:27503208

  12. [3H]-RS-45041-190: a selective high-affinity radioligand for I2 imidazoline receptors.

    PubMed Central

    MacKinnon, A. C.; Redfern, W. S.; Brown, C. M.

    1995-01-01

    1. RS-45041-190 (4-chloro-2-(imidazolin-2-yl)isoindoline) is an I2 imidazoline receptor ligand with the highest affinity and selectivity so far described; [3H]-RS-45041-190 has a tritium atom attached to the 7-position on the isoindoline ring. 2. [3H]-RS-45041-190 binding to rat kidney membranes was saturable (Bmax = 223.1 +/- 18.4 fmol mg-1 protein) and of high affinity (Kd = 2.71 +/- 0.59 nM). Kinetic studies revealed that the binding was rapid and reversible, with [3H]-RS-45041-190 interacting with two sites or two affinity states. 3. Competition studies showed that 60-70% of [3H]-RS-45041-190 binding (1 nM) was specifically to imidazoline binding sites of the I2 subtype, characterized by high affinity for idazoxan (pIC50 7.85 +/- 0.03) and cirazoline (pIC50 8.16 +/- 0.05). The remaining 30-40% was displaced specifically by the monoamine oxidase A inhibitors, clorgyline and pargyline. 4. alpha 1- and alpha 2-adrenoceptor, I1 imidazoline, histamine, 5-hydroxytryptamine or dopamine receptor ligands had low affinity suggesting that [3H]-RS-45041-190 did not label receptors of these classes. 5. In autoradiography studies, [3H]-RS-45041-190 labelled discrete regions of rat brain corresponding to the distribution of I2 subtypes, notably the subfornical organ, arcuate nucleus, interpeduncular nucleus, medial habenular nucleus and lateral mammillary nucleus, and additional sites in the locus coeruleus, dorsal raphe and dorsomedial hypothalamic nucleus. 6. [3H]-RS-45041-190 therefore labels I2 receptors with high affinity, and an additional site which has high affinity for some monoamine oxidase inhibitors. Images Figure 6 Figure 7 Figure 8 PMID:8528552

  13. Sulfated Metabolites of Polychlorinated Biphenyls Are High-Affinity Ligands for the Thyroid Hormone Transport Protein Transthyretin

    PubMed Central

    Grimm, Fabian A.; Lehmler, Hans-Joachim; He, Xianran; Robertson, Larry W.

    2013-01-01

    Background: The displacement of l-thyroxine (T4) from binding sites on transthyretin (TTR) is considered a significant contributing mechanism in polychlorinated biphenyl (PCB)-induced thyroid disruption. Previous research has discovered hydroxylated PCB metabolites (OH-PCBs) as high-affinity ligands for TTR, but the binding potential of conjugated PCB metabolites such as PCB sulfates has not been explored. Objectives: We evaluated the binding of five lower-chlorinated PCB sulfates to human TTR and compared their binding characteristics to those determined for their OH-PCB precursors and for T4. Methods: We used fluorescence probe displacement studies and molecular docking simulations to characterize the binding of PCB sulfates to TTR. The stability of PCB sulfates and the reversibility of these interactions were characterized by HPLC analysis of PCB sulfates after their binding to TTR. The ability of OH-PCBs to serve as substrates for human cytosolic sulfotransferase 1A1 (hSULT1A1) was assessed by OH-PCB–dependent formation of adenosine-3´,5´-diphosphate, an end product of the sulfation reaction. Results: All five PCB sulfates were able to bind to the high-affinity binding site of TTR with equilibrium dissociation constants (Kd values) in the low nanomolar range (4.8–16.8 nM), similar to that observed for T4 (4.7 nM). Docking simulations provided corroborating evidence for these binding interactions and indicated multiple high-affinity modes of binding. All OH-PCB precursors for these sulfates were found to be substrates for hSULT1A1. Conclusions: Our findings show that PCB sulfates are high-affinity ligands for human TTR and therefore indicate, for the first time, a potential relevance for these metabolites in PCB-induced thyroid disruption. PMID:23584369

  14. Embracing proteins: structural themes in aptamer-protein complexes.

    PubMed

    Gelinas, Amy D; Davies, Douglas R; Janjic, Nebojsa

    2016-02-01

    Understanding the structural rules that govern specific, high-affinity binding characteristic of aptamer-protein interactions is important in view of the increasing use of aptamers across many applications. From the modest number of 16 aptamer-protein structures currently available, trends are emerging. The flexible phosphodiester backbone allows folding into precise three-dimensional structures using known nucleic acid motifs as scaffolds that orient specific functional groups for target recognition. Still, completely novel motifs essential for structure and function are found in modified aptamers with diversity-enhancing side chains. Aptamers and antibodies, two classes of macromolecules used as affinity reagents with entirely different backbones and composition, recognize protein epitopes of similar size and with comparably high shape complementarity. PMID:26919170

  15. Differences between high-affinity forskolin binding sites in dopamine-riche and other regions of rat brain

    SciTech Connect

    Poat, J.A.; Cripps, H.E.; Iversen, L.L.

    1988-05-01

    Forskolin labelled with (/sup 3/H) bound to high- and low-affinity sites in the rat brain. The high-affinity site was discretely located, with highest densities in the striatum, nucleus accumbens, olfactory tubercule, substantia nigra, hippocampus, and the molecular layers of the cerebellum. This site did not correlate well with the distribution of adenylate cyclase. The high-affinity striatal binding site may be associated with a stimulatory guanine nucleotide-binding protein. Thus, the number of sites was increased by the addition of Mg/sup 2 +/ and guanylyl imidodiphosphate. Cholera toxin stereotaxically injected into rat striatum increased the number of binding sites, and no further increase was noted following the subsequent addition of guanyl nucleotide. High-affinity forskolin binding sites in non-dopamine-rich brain areas (hippocampus and cerebullum) were modulated in a qualitatively different manner by guanyl nucleotides. In these areas the number of binding sites was significantly reduced by the addition of guanyl nucleotide. These results suggest that forskolin may have a potential role in identifying different functional/structural guanine nucleotide-binding proteins.

  16. High affinity IgM(+) memory B cells are generated through a germinal center-dependent pathway.

    PubMed

    Hara, Yasushi; Tashiro, Yasuyuki; Murakami, Akikazu; Nishimura, Miyuki; Shimizu, Takeyuki; Kubo, Masato; Burrows, Peter D; Azuma, Takachika

    2015-12-01

    During a T cell-dependent immune response, B cells undergo clonal expansion and selection and the induction of isotype switching and somatic hypermutation (SHM). Although somatically mutated IgM(+) memory B cells have been reported, it has not been established whether they are really high affinity B cells. We tracked (4-hydroxy-3-nitrophenyl) acetyl hapten-specific GC B cells from normal immunized mice based on affinity of their B cell receptor (BCR) and performed BCR sequence analysis. SHM was evident by day 7 postimmunization and increased with time, such that high affinity IgM(+) as well as IgG(+) memory B cells continued to be generated up to day 42. In contrast, class-switch recombination (CSR) was almost completed by day 7 and then the ratio of IgG1(+)/IgM(+) GC B cells remained unchanged. Together these findings suggest that IgM(+) B cells undergo SHM in the GC to generate high affinity IgM(+) memory cells and that this process continues even after CSR is accomplished.

  17. Tripartite ATP-independent Periplasmic (TRAP) Transporters Use an Arginine-mediated Selectivity Filter for High Affinity Substrate Binding*

    PubMed Central

    Fischer, Marcus; Hopkins, Adam P.; Severi, Emmanuele; Hawkhead, Judith; Bawdon, Daniel; Watts, Andrew G.; Hubbard, Roderick E.; Thomas, Gavin H.

    2015-01-01

    Tripartite ATP-independent periplasmic (TRAP) transporters are secondary transporters that have evolved an obligate dependence on a substrate-binding protein (SBP) to confer unidirectional transport. Different members of the DctP family of TRAP SBPs have binding sites that recognize a diverse range of organic acid ligands but appear to only share a common electrostatic interaction between a conserved arginine and a carboxylate group in the ligand. We investigated the significance of this interaction using the sialic acid-specific SBP, SiaP, from the Haemophilus influenzae virulence-related SiaPQM TRAP transporter. Using in vitro, in vivo, and structural methods applied to SiaP, we demonstrate that the coordination of the acidic ligand moiety of sialic acid by the conserved arginine (Arg-147) is essential for the function of the transporter as a high affinity scavenging system. However, at high substrate concentrations, the transporter can function in the absence of Arg-147 suggesting that this bi-molecular interaction is not involved in further stages of the transport cycle. As well as being required for high affinity binding, we also demonstrate that the Arg-147 is a strong selectivity filter for carboxylate-containing substrates in TRAP transporters by engineering the SBP to recognize a non-carboxylate-containing substrate, sialylamide, through water-mediated interactions. Together, these data provide biochemical and structural support that TRAP transporters function predominantly as high affinity transporters for carboxylate-containing substrates. PMID:26342690

  18. Current concepts. I. High affinity receptors for bombesin/GRP-like peptides on human small cell lung cancer

    SciTech Connect

    Moody, T.W.; Carney, D.N.; Cuttitta, F.; Quattrocchi, K.; Minna, J.D.

    1985-07-15

    The binding of a radiolabeled bombesin analogue to human small cell lung cancer (SCLC) cell lines was investigated. (/sup 125/I-Tyr/sup 4/)bombesin bound with high affinity (Kd = 0.5 nM) to a single class of sites (2000/cell) using SCLC line NCI-H446. Binding was reversible, saturable and specific. The pharmacology of binding was investigated, using NCI-H466 and SCLC line NCI-H345. Bombesin and structurally related peptides, such as gastrin releasing peptide (GRP), but not other peptides, such as substance P or vasopressin, inhibited high affinity (/sup 125/I-Tyr/sup 4/)BN binding activity. Finally, the putative receptor, a 78,000 dalton polypeptide, was identified by purifying radiolabeled cell lysates on bombesin or GRP affinity resins and then displaying the bound polypeptides on sodium dodecylsulfate polyacrylamide gels. Because SCLC both produces bombesin/GRP-like peptides and contains high affinity receptors for these peptides, they may function as important autocrine regulatory factors for human SCLC. 31 references, 6 figures, 2 tables.

  19. High-Affinity Glucose Transport in Aspergillus nidulans Is Mediated by the Products of Two Related but Differentially Expressed Genes

    PubMed Central

    Ventura, Luisa; González, Ramón; Ramón, Daniel; MacCabe, Andrew P.

    2014-01-01

    Independent systems of high and low affinity effect glucose uptake in the filamentous fungus Aspergillus nidulans. Low-affinity uptake is known to be mediated by the product of the mstE gene. In the current work two genes, mstA and mstC, have been identified that encode high-affinity glucose transporter proteins. These proteins' primary structures share over 90% similarity, indicating that the corresponding genes share a common origin. Whilst the function of the paralogous proteins is little changed, they differ notably in their patterns of expression. The mstC gene is expressed during the early phases of germination and is subject to CreA-mediated carbon catabolite repression whereas mstA is expressed as a culture tends toward carbon starvation. In addition, various pieces of genetic evidence strongly support allelism of mstC and the previously described locus sorA. Overall, our data define MstC/SorA as a high-affinity glucose transporter expressed in germinating conidia, and MstA as a high-affinity glucose transporter that operates in vegetative hyphae under conditions of carbon limitation. PMID:24751997

  20. High affinity and covalent-binding microtubule stabilizing agents show activity in chemotherapy-resistant acute myeloid leukemia cells

    PubMed Central

    Pera, Benet; Calvo-Vidal, M. Nieves; Ambati, Srikanth; Jordi, Michel; Kahn, Alissa; Díaz, J. Fernando; Fang, Weishuo; Altmann, Karl-Heinz; Cerchietti, Leandro; Moore, Malcolm A.S.

    2016-01-01

    Treatment failure in acute myeloid leukemia (AML) is frequently due to the persistence of a cell population resistant to chemotherapy through different mechanisms, in which drug efflux via ATP-binding cassette (ABC) proteins, specifically P-glycoprotein, is one of the most recognized. However, disappointing results from clinical trials employing inhibitors for these transporters have demonstrated the need to adopt different strategies. We hypothesized that microtubule targeting compounds presenting high affinity or covalent binding could overcome the effect of ABC transporters. We therefore evaluated the activity of the high-affinity paclitaxel analog CTX-40 as well as the covalent binder zampanolide (ZMP) in AML cells. Both molecules were active in chemosensitive as well as in chemoresistant cell lines overexpressing P-glycoprotein. Moreover, ZMP or CTX-40 in combination with daunorubicin showed synergistic killing without increased in vitro hematopoietic toxicity. In a primary AML sample, we further demonstrated that ZMP and CTX-40 are active in progenitor and differentiated leukemia cell populations. In sum, our data indicate that high affinity and covalent-binding anti-microtubule agents are active in AML cells otherwise chemotherapy resistant. PMID:26277539

  1. A pharmacological analysis of high-affinity sodium transport in barley (Hordeum vulgare L.): a 24Na+/42K+ study

    PubMed Central

    Schulze, Lasse M.; Britto, Dev T.; Li, Mingyuan; Kronzucker, Herbert J.

    2012-01-01

    Soil sodium, while toxic to most plants at high concentrations, can be beneficial at low concentrations, particularly when potassium is limiting. However, little is known about Na+ uptake in this ‘high-affinity’ range. New information is provided here with an insight into the transport characteristics, mechanism, and ecological significance of this phenomenon. High-affinity Na+ and K+ fluxes were investigated using the short-lived radiotracers 24Na and 42K, under an extensive range of measuring conditions (variations in external sodium, and in nutritional and pharmacological agents). This work was supported by electrophysiological, compartmental, and growth analyses. Na+ uptake was extremely sensitive to all treatments, displaying properties of high-affinity K+ transporters, K+ channels, animal Na+ channels, and non-selective cation channels. K+, NH4+NH4+, and Ca2+ suppressed Na+ transport biphasically, yielding IC50 values of 30, 10, and <5 μM, respectively. Reciprocal experiments showed that K+ influx is neither inhibited nor stimulated by Na+. Sodium efflux constituted 65% of influx, indicating a futile cycle. The thermodynamic feasibility of passive channel mediation is supported by compartmentation and electrophysiological data. Our study complements recent advances in the molecular biology of high-affinity Na+ transport by uncovering new physiological foundations for this transport phenomenon, while questioning its ecological relevance. PMID:22268152

  2. High-affinity glucose transport in Aspergillus nidulans is mediated by the products of two related but differentially expressed genes.

    PubMed

    Forment, Josep V; Flipphi, Michel; Ventura, Luisa; González, Ramón; Ramón, Daniel; Maccabe, Andrew P

    2014-01-01

    Independent systems of high and low affinity effect glucose uptake in the filamentous fungus Aspergillus nidulans. Low-affinity uptake is known to be mediated by the product of the mstE gene. In the current work two genes, mstA and mstC, have been identified that encode high-affinity glucose transporter proteins. These proteins' primary structures share over 90% similarity, indicating that the corresponding genes share a common origin. Whilst the function of the paralogous proteins is little changed, they differ notably in their patterns of expression. The mstC gene is expressed during the early phases of germination and is subject to CreA-mediated carbon catabolite repression whereas mstA is expressed as a culture tends toward carbon starvation. In addition, various pieces of genetic evidence strongly support allelism of mstC and the previously described locus sorA. Overall, our data define MstC/SorA as a high-affinity glucose transporter expressed in germinating conidia, and MstA as a high-affinity glucose transporter that operates in vegetative hyphae under conditions of carbon limitation. PMID:24751997

  3. Energy-dependent dissociation of ATP from high affinity catalytic sites of beef heart mitochondrial adenosine triphosphatase

    SciTech Connect

    Penefsky, H.S.

    1985-11-05

    Incubation of (gamma-TSP)ATP with a molar excess of the membrane-bound form of mitochondrial ATPase (F1) results in binding of the bulk of the radioactive nucleotide in high affinity catalytic sites (Ka = 10(12) M-1). Subsequent initiation of respiration by addition of succinate or NADH is accompanied by a profound decrease in the affinity for ATP. About one-third of the bound radioactive ATP appears to dissociate, that is, the (gamma-TSP)ATP becomes accessible to hexokinase. The NADH-stimulated dissociation of (gamma-TSP)ATP is energy-dependent since the stimulation is inhibited by uncouplers of oxidative phosphorylation and is prevented by respiratory chain inhibitors. The rate of the energy-dependent dissociation of ATP that occurs in the presence of NADH, ADP, and Pi is commensurate with the measured initial rate of ATP synthesis in NADH-supported oxidative phosphorylation catalyzed by the same submitochondrial particles. Thus, the rate of dissociation of ATP from the high affinity catalytic site of submitochondrial particles meets the criterion of kinetic competency under the conditions of oxidative phosphorylation. These experiments provide evidence in support of the argument that energy conserved during the oxidation of substrates by the respiratory chain can be utilized to reduce the very tight binding of product ATP in high affinity catalytic sites and to promote dissociation of the nucleotide.

  4. Chimeric Protein Complexes in Hybrid Species Generate Novel Phenotypes

    PubMed Central

    Piatkowska, Elzbieta M.; Naseeb, Samina; Knight, David; Delneri, Daniela

    2013-01-01

    Hybridization between species is an important mechanism for the origin of novel lineages and adaptation to new environments. Increased allelic variation and modification of the transcriptional network are the two recognized forces currently deemed to be responsible for the phenotypic properties seen in hybrids. However, since the majority of the biological functions in a cell are carried out by protein complexes, inter-specific protein assemblies therefore represent another important source of natural variation upon which evolutionary forces can act. Here we studied the composition of six protein complexes in two different Saccharomyces “sensu stricto” hybrids, to understand whether chimeric interactions can be freely formed in the cell in spite of species-specific co-evolutionary forces, and whether the different types of complexes cause a change in hybrid fitness. The protein assemblies were isolated from the hybrids via affinity chromatography and identified via mass spectrometry. We found evidence of spontaneous chimericity for four of the six protein assemblies tested and we showed that different types of complexes can cause a variety of phenotypes in selected environments. In the case of TRP2/TRP3 complex, the effect of such chimeric formation resulted in the fitness advantage of the hybrid in an environment lacking tryptophan, while only one type of parental combination of the MBF complex allowed the hybrid to grow under respiratory conditions. These phenotypes were dependent on both genetic and environmental backgrounds. This study provides empirical evidence that chimeric protein complexes can freely assemble in cells and reveals a new mechanism to generate phenotypic novelty and plasticity in hybrids to complement the genomic innovation resulting from gene duplication. The ability to exchange orthologous members has also important implications for the adaptation and subsequent genome evolution of the hybrids in terms of pattern of gene loss. PMID

  5. 3D complex: a structural classification of protein complexes.

    PubMed

    Levy, Emmanuel D; Pereira-Leal, Jose B; Chothia, Cyrus; Teichmann, Sarah A

    2006-11-17

    Most of the proteins in a cell assemble into complexes to carry out their function. It is therefore crucial to understand the physicochemical properties as well as the evolution of interactions between proteins. The Protein Data Bank represents an important source of information for such studies, because more than half of the structures are homo- or heteromeric protein complexes. Here we propose the first hierarchical classification of whole protein complexes of known 3-D structure, based on representing their fundamental structural features as a graph. This classification provides the first overview of all the complexes in the Protein Data Bank and allows nonredundant sets to be derived at different levels of detail. This reveals that between one-half and two-thirds of known structures are multimeric, depending on the level of redundancy accepted. We also analyse the structures in terms of the topological arrangement of their subunits and find that they form a small number of arrangements compared with all theoretically possible ones. This is because most complexes contain four subunits or less, and the large majority are homomeric. In addition, there is a strong tendency for symmetry in complexes, even for heteromeric complexes. Finally, through comparison of Biological Units in the Protein Data Bank with the Protein Quaternary Structure database, we identified many possible errors in quaternary structure assignments. Our classification, available as a database and Web server at http://www.3Dcomplex.org, will be a starting point for future work aimed at understanding the structure and evolution of protein complexes.

  6. Isolation of a novel lutein-protein complex from Chlorella vulgaris and its functional properties.

    PubMed

    Cai, Xixi; Huang, Qimin; Wang, Shaoyun

    2015-06-01

    A novel kind of lutein-protein complex (LPC) was extracted from heterotrophic Chlorella vulgaris through aqueous extraction. The purification procedure contained solubilization of thylakoid proteins by a zwitterionic detergent CHAPS, anion exchange chromatography and gel filtration chromatography. Both wavelength scanning and HPLC analysis confirmed that lutein was the major pigment of the protein-based complex, and the mass ratio of lutein and protein was determined to be 9.72 : 100. Besides showing lipid peroxidation inhibition activity in vitro, LPC exerted significant antioxidant effects against ABTS and DPPH radicals with IC50 of 2.90 and 97. 23 μg mL(-1), respectively. Meanwhile, in vivo antioxidant activity of the complex was evaluated using the mice hepatotoxicity model; LPC significantly suppressed the carbon tetrachloride-induced elevation of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, and decreased hepatic malondialdehyde (MDA) levels and the hepatosomatic index. Moreover, LPC could effectively restore the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) in the treated mice livers. Our findings further the progress in the research of natural protein-based lutein complexes, suggesting that LPC has the potential in hepatoprotection against chemical induced toxicity and in increasing the antioxidant capacity of the defense system in the human body.

  7. Protein Complex Production from the Drug Discovery Standpoint.

    PubMed

    Moarefi, Ismail

    2016-01-01

    Small molecule drug discovery critically depends on the availability of meaningful in vitro assays to guide medicinal chemistry programs that are aimed at optimizing drug potency and selectivity. As it becomes increasingly evident, most disease relevant drug targets do not act as a single protein. In the body, they are instead generally found in complex with protein cofactors that are highly relevant for their correct function and regulation. This review highlights selected examples of the increasing trend to use biologically relevant protein complexes for rational drug discovery to reduce costly late phase attritions due to lack of efficacy or toxicity.

  8. Detection of Streptavidin-Biotin Protein Complexes Using Three-Dimensional MOSFET in the Si Micro-Fluidic Channel

    NASA Astrophysics Data System (ADS)

    Han, Dae-Il; Kim, Dong-Sun; Park, Jee-Eun; Shin, Jang-Kyoo; Kong, Seong-Ho; Choi, Pyung; Lee, Jong-Hyun; Lim, Geunbae

    2005-07-01

    To detect the electrical reaction characteristics of streptavidin-biotin protein complexes, a three-dimensional (3-D) p-channel metal oxide semiconductor field effect transistor (PMOSFET)-type biosensor was fabricated in the convex corner of a micro-fluidic channel by tetramethyl ammonium hydroxide (TMAH) anisotropic etching. Au, which has a chemical affinity with thiol, was used as the gate metal for immobilizing a self-assembled monolayer (SAM). The SAM was used to immobilize streptavidin. The hydroxyl group of SAM was bound with the amine group of streptavidin. After that, streptavidin and biotin were bound by their high affinity (Ka˜ 1015 Mol-1). The measurements were performed in phosphate-buffered saline (PBS; pH 6.4, 20 mM) solution. Ag/AgCl was used as the reference electrode. The bindings of SAM, streptavidin and biotin caused a variation in the drain current of the 3-D MOSFET. To verify the interaction among the SAM, streptavidin and biotin, surface plasmon resonance (SPR) measurement was performed.

  9. Hamiltonian purification

    SciTech Connect

    Orsucci, Davide; Burgarth, Daniel; Facchi, Paolo; Pascazio, Saverio; Nakazato, Hiromichi; Yuasa, Kazuya; Giovannetti, Vittorio

    2015-12-15

    The problem of Hamiltonian purification introduced by Burgarth et al. [Nat. Commun. 5, 5173 (2014)] is formalized and discussed. Specifically, given a set of non-commuting Hamiltonians (h{sub 1}, …, h{sub m}) operating on a d-dimensional quantum system ℋ{sub d}, the problem consists in identifying a set of commuting Hamiltonians (H{sub 1}, …, H{sub m}) operating on a larger d{sub E}-dimensional system ℋ{sub d{sub E}} which embeds ℋ{sub d} as a proper subspace, such that h{sub j} = PH{sub j}P with P being the projection which allows one to recover ℋ{sub d} from ℋ{sub d{sub E}}. The notions of spanning-set purification and generator purification of an algebra are also introduced and optimal solutions for u(d) are provided.

  10. Hamiltonian purification

    NASA Astrophysics Data System (ADS)

    Orsucci, Davide; Burgarth, Daniel; Facchi, Paolo; Nakazato, Hiromichi; Pascazio, Saverio; Yuasa, Kazuya; Giovannetti, Vittorio

    2015-12-01

    The problem of Hamiltonian purification introduced by Burgarth et al. [Nat. Commun. 5, 5173 (2014)] is formalized and discussed. Specifically, given a set of non-commuting Hamiltonians {h1, …, hm} operating on a d-dimensional quantum system ℋd, the problem consists in identifying a set of commuting Hamiltonians {H1, …, Hm} operating on a larger dE-dimensional system ℋdE which embeds ℋd as a proper subspace, such that hj = PHjP with P being the projection which allows one to recover ℋd from ℋdE. The notions of spanning-set purification and generator purification of an algebra are also introduced and optimal solutions for 𝔲(d) are provided.

  11. Polymorphism in high-affinity calcium-binding proteins from crustacean sarcoplasm.

    PubMed

    Wnuk, W; Jauregui-Adell, J

    1983-03-01

    The sarcoplasmic calcium-binding proteins (SCP) from crayfish, lobster and shrimp myogen have been purified to homogeneity. These proteins exist as dimers and dissociate in the presence of sodium dodecyl sulfate or urea in subunits of 22000 molecular weight. During the last step of purification (DEAE-cellulose chromatography), SCP emerges in three peaks in the ratio of 14:1.5:1 for crayfish, of 7:2:1 for lobster and of 3:2:1 for shrimp. Gel electrophoresis and isoelectrofocusing experiments, under native and denaturing conditions, indicate that among the three SCP isotypes there are only two different polypeptide chains, alpha and beta, which appear in the form of three dimers: alpha 2, alpha beta and beta 2. The alpha and beta subunits differ slightly in polypeptide chain composition as found by amino acid analyses of the crayfish and lobster SCPs, and also by comparison of tryptic peptides for crayfish SCPs. The polymorphism observed in crustacean SCPs, which is increased by their ability to form dimers, contrasts with the situation prevailing among other invertebrate SCPs and vertebrate parvalbumins where only monomeric isotypes are found. Equilibrium binding studies show that all three SCP isotypes from both crayfish and lobster display the same metal-binding properties. They have in their dimeric form six Ca2+-binding sites: two calcium-specific sites, two Ca/Mg sites that interact with positive cooperativity and two Ca/Mg sites that interact with negative cooperativity. Interactions between the two subunits of SCP seem to result in cooperative binding of Ca2+, which in turn may control more efficiently Ca2+ fluxes in crustacean muscle. PMID:6832140

  12. Native Elution of Yeast Protein Complexes Obtained by Affinity Capture.

    PubMed

    LaCava, John; Fernandez-Martinez, Javier; Rout, Michael P

    2016-01-01

    This protocol describes two options for the native (nondenaturing) elution of protein complexes obtained by affinity capture. The first approach involves the elution of complexes purified through a tag that includes a human rhinovirus 3C protease (PreScission protease) cleavage site sequence between the protein of interest and the tag. Incubation with the protease cleaves immobilized complexes from the affinity medium. The second approach involves the release of protein A-tagged protein complexes using a competitive elution reagent called PEGylOx. The degree of purity of the native assemblies eluted is sample dependent and strongly influenced by the affinity capture. It should be noted that the efficiency of native elution is commonly lower than that of elution by a denaturing agent (e.g., SDS) and the release of the complex will be limited by the activity of the protease or the inhibition constant (Ki) of the competitive release agent. However, an advantage of native release is that some nonspecifically bound materials tend to stay adsorbed to the affinity medium, providing an eluted fraction of higher purity. Finally, keep in mind that the presence of the protease or elution peptide could potentially affect downstream applications; thus, their removal should be considered. PMID:27371597

  13. Transcriptional regulation of protein complexes within and across species.

    PubMed

    Tan, Kai; Shlomi, Tomer; Feizi, Hoda; Ideker, Trey; Sharan, Roded

    2007-01-23

    Yeast two-hybrid and coimmunoprecipitation experiments have defined large-scale protein-protein interaction networks for many model species. Separately, systematic chromatin immunoprecipitation experiments have enabled the assembly of large networks of transcriptional regulatory interactions. To investigate the functional interplay between these two interaction types, we combined both within a probabilistic framework that models the cell as a network of transcription factors regulating protein complexes. This framework identified 72 putative coregulated complexes in yeast and allowed the prediction of 120 previously uncharacterized transcriptional interactions. Several predictions were tested by new microarray profiles, yielding a confirmation rate (58%) comparable with that of direct immunoprecipitation experiments. Furthermore, we extended our framework to a cross-species setting, identifying 24 coregulated complexes that were conserved between yeast and fly. Analyses of these conserved complexes revealed different conservation levels of their regulators and provided suggestive evidence that protein-protein interaction networks may evolve more slowly than transcriptional interaction networks. Our results demonstrate how multiple molecular interaction types can be integrated toward a global wiring diagram of the cell, and they provide insights into the evolutionary dynamics of protein complex regulation.

  14. Solid-State NMR Spectroscopy of Protein Complexes

    PubMed Central

    Sun, Shangjin; Han, Yun; Paramasivam, Sivakumar; Yan, Si; Siglin, Amanda E.; Williams, John C.; Byeon, In-Ja L.; Ahn, Jinwoo; Gronenborn, Angela M.; Polenova, Tatyana

    2016-01-01

    Protein-protein interactions are vital for many biological processes. These interactions often result in the formation of protein assemblies that are large in size, insoluble and difficult to crystallize, and therefore are challenging to study by structure biology techniques, such as single crystal X-ray diffraction and solution NMR spectroscopy. Solid-state NMR (SSNMR) spectroscopy is emerging as a promising technique for studies of such protein assemblies because it is not limited by molecular size, solubility or lack of long-range order. In the past several years, we have applied magic angle spinning SSNMR based methods to study several protein complexes. In this chapter, we discuss the general solid-state NMR methodologies employed for structural and dynamics analyses of protein complexes with specific examples from our work on thioredoxin reassemblies, HIV-1 capsid protein assemblies and microtubule-associated protein assemblies. We present protocols for sample preparation and characterization, pulse sequences, SSNMR spectra collection and data analysis. PMID:22167681

  15. The role of Hsp90 in protein complex assembly.

    PubMed

    Makhnevych, Taras; Houry, Walid A

    2012-03-01

    Hsp90 is a ubiquitous and essential molecular chaperone that plays central roles in many signaling and other cellular pathways. The in vivo and in vitro activity of Hsp90 depends on its association with a wide variety of cochaperones and cofactors, which form large multi-protein complexes involved in folding client proteins. Based on our proteomic work mapping the molecular chaperone interaction networks in yeast, especially that of Hsp90, as well as, on experiments and results presented in the published literature, one major role of Hsp90 appears to be the promotion and maintenance of proper assembly of protein complexes. To highlight this role of Hsp90, the effect of the chaperone on the assembly of the following seven complexes is discussed in this review: snoRNP, RNA polymerase II, phosphatidylinositol-3 kinase-related protein kinase (PIKK), telomere complex, kinetochore, RNA induced silencing complexes (RISC), and 26S proteasome. For some complexes, it is observed that Hsp90 mediates complex assembly by stabilizing an unstable protein subunit and facilitating its incorporation into the complex; for other complexes, Hsp90 promotes change in the composition of that complex. In all cases, Hsp90 does not appear to be part of the final assembled complex. This article is part of a Special Issue entitled:Heat Shock Protein 90 (HSP90). PMID:21945180

  16. Inferring drug-disease associations based on known protein complexes.

    PubMed

    Yu, Liang; Huang, Jianbin; Ma, Zhixin; Zhang, Jing; Zou, Yapeng; Gao, Lin

    2015-01-01

    Inferring drug-disease associations is critical in unveiling disease mechanisms, as well as discovering novel functions of available drugs, or drug repositioning. Previous work is primarily based on drug-gene-disease relationship, which throws away many important information since genes execute their functions through interacting others. To overcome this issue, we propose a novel methodology that discover the drug-disease association based on protein complexes. Firstly, the integrated heterogeneous network consisting of drugs, protein complexes, and disease are constructed, where we assign weights to the drug-disease association by using probability. Then, from the tripartite network, we get the indirect weighted relationships between drugs and diseases. The larger the weight, the higher the reliability of the correlation. We apply our method to mental disorders and hypertension, and validate the result by using comparative toxicogenomics database. Our ranked results can be directly reinforced by existing biomedical literature, suggesting that our proposed method obtains higher specificity and sensitivity. The proposed method offers new insight into drug-disease discovery. Our method is publicly available at http://1.complexdrug.sinaapp.com/Drug_Complex_Disease/Data_Download.html.

  17. Molecular Signatures of Membrane Protein Complexes Underlying Muscular Dystrophy*

    PubMed Central

    Turk, Rolf; Hsiao, Jordy J.; Smits, Melinda M.; Ng, Brandon H.; Pospisil, Tyler C.; Jones, Kayla S.; Campbell, Kevin P.; Wright, Michael E.

    2016-01-01

    Mutations in genes encoding components of the sarcolemmal dystrophin-glycoprotein complex (DGC) are responsible for a large number of muscular dystrophies. As such, molecular dissection of the DGC is expected to both reveal pathological mechanisms, and provides a biological framework for validating new DGC components. Establishment of the molecular composition of plasma-membrane protein complexes has been hampered by a lack of suitable biochemical approaches. Here we present an analytical workflow based upon the principles of protein correlation profiling that has enabled us to model the molecular composition of the DGC in mouse skeletal muscle. We also report our analysis of protein complexes in mice harboring mutations in DGC components. Bioinformatic analyses suggested that cell-adhesion pathways were under the transcriptional control of NFκB in DGC mutant mice, which is a finding that is supported by previous studies that showed NFκB-regulated pathways underlie the pathophysiology of DGC-related muscular dystrophies. Moreover, the bioinformatic analyses suggested that inflammatory and compensatory mechanisms were activated in skeletal muscle of DGC mutant mice. Additionally, this proteomic study provides a molecular framework to refine our understanding of the DGC, identification of protein biomarkers of neuromuscular disease, and pharmacological interrogation of the DGC in adult skeletal muscle https://www.mda.org/disease/congenital-muscular-dystrophy/research. PMID:27099343

  18. Inferring drug-disease associations based on known protein complexes

    PubMed Central

    2015-01-01

    Inferring drug-disease associations is critical in unveiling disease mechanisms, as well as discovering novel functions of available drugs, or drug repositioning. Previous work is primarily based on drug-gene-disease relationship, which throws away many important information since genes execute their functions through interacting others. To overcome this issue, we propose a novel methodology that discover the drug-disease association based on protein complexes. Firstly, the integrated heterogeneous network consisting of drugs, protein complexes, and disease are constructed, where we assign weights to the drug-disease association by using probability. Then, from the tripartite network, we get the indirect weighted relationships between drugs and diseases. The larger the weight, the higher the reliability of the correlation. We apply our method to mental disorders and hypertension, and validate the result by using comparative toxicogenomics database. Our ranked results can be directly reinforced by existing biomedical literature, suggesting that our proposed method obtains higher specificity and sensitivity. The proposed method offers new insight into drug-disease discovery. Our method is publicly available at http://1.complexdrug.sinaapp.com/Drug_Complex_Disease/Data_Download.html. PMID:26044949

  19. A pp32-retinoblastoma protein complex modulates androgen receptor-mediated transcription and associates with components of the splicing machinery

    SciTech Connect

    Adegbola, Onikepe; Pasternack, Gary R. . E-mail: gpastern@jhmi.edu

    2005-08-26

    We have previously shown pp32 and the retinoblastoma protein interact. pp32 and the retinoblastoma protein are nuclear receptor transcriptional coregulators: the retinoblastoma protein is a coactivator for androgen receptor, the major regulator of prostate cancer growth, while pp32, which is highly expressed in prostate cancer, is a corepressor of the estrogen receptor. We now show pp32 increases androgen receptor-mediated transcription and the retinoblastoma protein modulates this activity. Using affinity purification and mass spectrometry, we identify members of the pp32-retinoblastoma protein complex as PSF and nonO/p54nrb, proteins implicated in coordinate regulation of nuclear receptor-mediated transcription and splicing. We show that the pp32-retinoblastoma protein complex is modulated during TPA-induced K562 differentiation. Present evidence suggests that nuclear receptors assemble multiprotein complexes to coordinately regulate transcription and mRNA processing. Our results suggest that pp32 and the retinoblastoma protein may be part of a multiprotein complex that coordinately regulates nuclear receptor-mediated transcription and mRNA processing.

  20. α4βδ GABA(A) receptors are high-affinity targets for γ-hydroxybutyric acid (GHB).

    PubMed

    Absalom, Nathan; Eghorn, Laura F; Villumsen, Inge S; Karim, Nasiara; Bay, Tina; Olsen, Jesper V; Knudsen, Gitte M; Bräuner-Osborne, Hans; Frølund, Bente; Clausen, Rasmus P; Chebib, Mary; Wellendorph, Petrine

    2012-08-14

    γ-Hydroxybutyric acid (GHB) binding to brain-specific high-affinity sites is well-established and proposed to explain both physiological and pharmacological actions. However, the mechanistic links between these lines of data are unknown. To identify molecular targets for specific GHB high-affinity binding, we undertook photolinking studies combined with proteomic analyses and identified several GABA(A) receptor subunits as possible candidates. A subsequent functional screening of various recombinant GABA(A) receptors in Xenopus laevis oocytes using the two-electrode voltage clamp technique showed GHB to be a partial agonist at αβδ- but not αβγ-receptors, proving that the δ-subunit is essential for potency and efficacy. GHB showed preference for α4 over α(1,2,6)-subunits and preferably activated α4β1δ (EC(50) = 140 nM) over α4β(2/3)δ (EC(50) = 8.41/1.03 mM). Introduction of a mutation, α4F71L, in α4β1(δ)-receptors completely abolished GHB but not GABA function, indicating nonidentical binding sites. Radioligand binding studies using the specific GHB radioligand [(3)H](E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene)acetic acid showed a 39% reduction (P = 0.0056) in the number of binding sites in α4 KO brain tissue compared with WT controls, corroborating the direct involvement of the α4-subunit in high-affinity GHB binding. Our data link specific GHB forebrain binding sites with α4-containing GABA(A) receptors and postulate a role for extrasynaptic α4δ-containing GABA(A) receptors in GHB pharmacology and physiology. This finding will aid in elucidating the molecular mechanisms behind the proposed function of GHB as a neurotransmitter and its unique therapeutic effects in narcolepsy and alcoholism.

  1. Expression cloning and functional characterization of the kidney cortex high-affinity proton-coupled peptide transporter.

    PubMed Central

    Boll, M; Herget, M; Wagener, M; Weber, W M; Markovich, D; Biber, J; Clauss, W; Murer, H; Daniel, H

    1996-01-01

    The presence of a proton-coupled electrogenic high-affinity peptide transporter in the apical membrane of tubular cells has been demonstrated by microperfusion studies and by use of brush border membrane vesicles. The transporter mediates tubular uptake of filtered di- and tripeptides and aminocephalosporin antibiotics. We have used expression cloning in Xenopus laevis oocytes for identification and characterization of the renal high-affinity peptide transporter. Injection of poly(A)+ RNA isolated from rabbit kidney cortex into oocytes resulted in expression of a pH-dependent transport activity for the aminocephalosporin antibiotic cefadroxil. After size fractionation of poly(A)+ RNA the transport activity was identified in the 3.0- to 5.0-kb fractions, which were used for construction of a cDNA library. The library was screened for expression of cefadroxil transport after injection of complementary RNA synthesized in vitro from different pools of clones. A single clone (rPepT2) was isolated that stimulated cefadroxil uptake into oocytes approximately 70-fold at a pH of 6.0. Kinetic analysis of cefadroxil uptake expressed by the transporter's complementary RNA showed a single saturable high-affinity transport system shared by dipeptides, tripeptides, and selected amino-beta-lactam antibiotics. Electrophysiological studies established that the transport activity is electrogenic and affected by membrane potential. Sequencing of the cDNA predicts a protein of 729 amino acids with 12 membrane-spanning domains. Although there is a significant amino acid sequence identity (47%) to the recently cloned peptide transporters from rabbit and human small intestine, the renal transporter shows distinct structural and functional differences. Images Fig. 7 PMID:8552623

  2. The ZnuABC high-affinity zinc uptake system and its regulator Zur in Escherichia coli.

    PubMed

    Patzer, S I; Hantke, K

    1998-06-01

    In Escherichia coli, lacZ operon fusions were isolated that were derepressed under iron repletion and repressed under iron depletion. Two fusions were localized in genes that formed an operon whose gene products had characteristics of a binding protein-dependent transport system. The growth defect of these mutants on TY medium containing 5mM EGTA was compensated for by the addition of Zn2+. In the presence of 0.5mM EGTA, only the parental strain was able to take up 65Zn2+. This high-affinity transport was energized by ATP. The genes were named znuACB (for zinc uptake; former name yebLMI) and localized at 42 min on the genetic map of E. coli. At high Zn2+ concentrations, the znu mutants took up more 65Zn2+ than the parental strain. The high-affinity 65Zn2+ uptake was repressed by growth in the presence of 10 microM Zn2+. A znuA-lacZ operon fusion was repressed by 5 microM Zn2+ and showed a more than 20-fold increase in beta-galactosidase activity when Zn2+ was bound to 1.5 microM TPEN [tetrakis-(2-pyridylmethyl) ethylenediamine]. To identify the Zn2+-dependent regulator, constitutive mutants were isolated and tested for complementation by a gene bank of E. coli. A complementing gene, yjbK of the E. coli genome, was identified and named zur (for zinc uptake regulation). The Zur protein showed 27% sequence identity with the iron regulator Fur. High-affinity 65Zn2+ transport of the constitutive zur mutant was 10-fold higher than that of the uninduced parental strain. An in vivo titration assay suggested that Zur binds to the bidirectional promoter region of znuA and znuCB. PMID:9680209

  3. Selection of a high-affinity and in vivo bioactive ssDNA aptamer against angiotensin II peptide.

    PubMed

    Heiat, Mohammad; Ranjbar, Reza; Latifi, Ali Mohammad; Rasaee, Mohammad Javad

    2016-08-01

    Unique features of aptamers have attracted interests for a broad range of applications. Aptamers are able to specifically bind to targets and inhibit their functions. This study, aimed to isolate the high affinity ssDNA aptamers against bio-regulator peptide angiotensin II (Ang II) and investigate their bioactivity in cellular and animal models. To isolate ssDNA aptamers, 12 rounds of affinity chromatography SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure were carried out. The SPR (surface plasmon resonance) and ELONA (enzyme linked oligonucleotide assay) analysis were used to determine the affinity and specificity of aptamers. The ability of selected aptamers to inhibit the proliferative effect of Ang II on human aortic vascular smooth muscle cells (HA-VSMCs) and their performance on Wistar rat urinary system and serum electrolyte levels were investigated. Two full-length aptamers (FLC112 and FLC125) with high affinity of respectively 7.52±2.44E-10 and 5.87±1.3E-9M were isolated against Ang II. The core regions of these aptamers (CRC112 and CRC125) also showed affinity of 5.33±1.15E-9 and 4.11±1.09E-9M. In vitro analysis revealed that FLC112 and FLC125 can inhibit the proliferative effect of Ang II on HA-VSMCs (P<0.05). They also significantly reduced the serum sodium level and increased the urine volume (P<0.05). The core regions of aptamers did not show high inhibitory potential against Ang II. It can be a spotlight that ssDNA aptamers have high potential for blocking Ang II. In conclusion, it appears that the researches focusing on high affinity and bioactive aptamers may lead to excellent results in blocking Ang II activity. PMID:27298205

  4. Insights from the Fungus Fusarium oxysporum Point to High Affinity Glucose Transporters as Targets for Enhancing Ethanol Production from Lignocellulose

    PubMed Central

    Ali, Shahin S.; Nugent, Brian; Mullins, Ewen; Doohan, Fiona M.

    2013-01-01

    Ethanol is the most-widely used biofuel in the world today. Lignocellulosic plant biomass derived from agricultural residue can be converted to ethanol via microbial bioprocessing. Fungi such as Fusarium oxysporum can simultaneously saccharify straw to sugars and ferment sugars to ethanol. But there are many bottlenecks that need to be overcome to increase the efficacy of microbial production of ethanol from straw, not least enhancement of the rate of fermentation of both hexose and pentose sugars. This research tested the hypothesis that the rate of sugar uptake by F. oxysporum would enhance the ethanol yields from lignocellulosic straw and that high affinity glucose transporters can enhance ethanol yields from this substrate. We characterized a novel hexose transporter (Hxt) from this fungus. The F. oxysporum Hxt represents a novel transporter with homology to yeast glucose signaling/transporter proteins Rgt2 and Snf3, but it lacks their C-terminal domain which is necessary for glucose signalling. Its expression level decreased with increasing glucose concentration in the medium and in a glucose uptake study the Km(glucose) was 0.9 mM, which indicated that the protein is a high affinity glucose transporter. Post-translational gene silencing or over expression of the Hxt in F. oxysporum directly affected the glucose and xylose transport capacity and ethanol yielded by F. oxysporum from straw, glucose and xylose. Thus we conclude that this Hxt has the capacity to transport both C5 and C6 sugars and to enhance ethanol yields from lignocellulosic material. This study has confirmed that high affinity glucose transporters are ideal candidates for improving ethanol yields from lignocellulose because their activity and level of expression is high in low glucose concentrations, which is very common during the process of consolidated processing. PMID:23382943

  5. Topography of the high-affinity lysine binding site of plasminogen as defined with a specific antibody probe

    SciTech Connect

    Miles, L.A.; Plow, E.F.

    1986-11-04

    An antibody population that reacted with the high-affinity lysine binding site of human plasminogen was elicited by immunizing rabbits with an elastase degradation product containing kringles 1-3 (EDP I). This antibody was immunopurified by affinity chromatography on plasminogen-Sepharose and elution with 0.2 M 6-aminohexanoic acid. The eluted antibodies bound (/sup 125/I)EDP I, (/sup 125/I)Glu-plasminogen, and (/sup 125/I)Lys-plasminogen in radioimmunoassays, and binding of each ligand was at least 99% inhibited by 0.2 M 6-aminohexanoic acid. The concentrations for 50% inhibition of (/sup 125/I)EDP I binding by tranexamic acid, 6-aminohexanoic acid, and lysine were 2.6, 46, and l730 ..mu..M, respectively. Similar values were obtained with plasminogen and suggested that an unoccupied high-affinity lysine binding site was required for antibody recognition. The antiserum reacted exclusively with plasminogen derivatives containing the EDP I region and did not react with those lacking an EDP I region, or with tissue plasminogen activator or prothrombin, which also contains kringles. By immunoblotting analyses, a chymotryptic degradation product of M/sub r/ 20,000 was derived from EDP I that retained reactivity with the antibody. ..cap alpha../sub 2/-Antiplasmin inhibited the binding of radiolabeled EDP I, Glu-plasminogen, or Lys-plasminogen by the antiserum, suggesting that the recognized site is involved in the noncovalent interaction of the inhibitor with plasminogen. The binding of (/sup 125/I)EDP I to fibrin was also inhibited by the antiserum. The observations provide independent evidence for the role of the high-affinity lysine binding site in the functional interactions of plasminogen with its primary substrate and inhibitor.

  6. Insights from the fungus Fusarium oxysporum point to high affinity glucose transporters as targets for enhancing ethanol production from lignocellulose.

    PubMed

    Ali, Shahin S; Nugent, Brian; Mullins, Ewen; Doohan, Fiona M

    2013-01-01

    Ethanol is the most-widely used biofuel in the world today. Lignocellulosic plant biomass derived from agricultural residue can be converted to ethanol via microbial bioprocessing. Fungi such as Fusarium oxysporum can simultaneously saccharify straw to sugars and ferment sugars to ethanol. But there are many bottlenecks that need to be overcome to increase the efficacy of microbial production of ethanol from straw, not least enhancement of the rate of fermentation of both hexose and pentose sugars. This research tested the hypothesis that the rate of sugar uptake by F. oxysporum would enhance the ethanol yields from lignocellulosic straw and that high affinity glucose transporters can enhance ethanol yields from this substrate. We characterized a novel hexose transporter (Hxt) from this fungus. The F. oxysporum Hxt represents a novel transporter with homology to yeast glucose signaling/transporter proteins Rgt2 and Snf3, but it lacks their C-terminal domain which is necessary for glucose signalling. Its expression level decreased with increasing glucose concentration in the medium and in a glucose uptake study the Km((glucose)) was 0.9 mM, which indicated that the protein is a high affinity glucose transporter. Post-translational gene silencing or over expression of the Hxt in F. oxysporum directly affected the glucose and xylose transport capacity and ethanol yielded by F. oxysporum from straw, glucose and xylose. Thus we conclude that this Hxt has the capacity to transport both C5 and C6 sugars and to enhance ethanol yields from lignocellulosic material. This study has confirmed that high affinity glucose transporters are ideal candidates for improving ethanol yields from lignocellulose because their activity and level of expression is high in low glucose concentrations, which is very common during the process of consolidated processing.

  7. (/sup 3/H)pirenzepine selectively identifies a high affinity population of muscarinic cholinergic receptors in the rat cerebral cortex

    SciTech Connect

    Watson, M.; Roeske, W.R.; Yamamura, H.I.

    1982-11-01

    The specific binding of (/sup 3/H)pirenzepine was investigated in homogenates of rat cerebral cortex, cerebellar cortex, and heart. Specific binding of (/sup 3/H)pirenzepine in the cerebral cortex as defined by displacement with atropine sulfate (1..mu..M) was of high affinity (K/sub d/ = 4-10 nM, receptor density = 1.06 pmoles/mg protein), stereoselective, and competitive with drugs specific for the muscarinic receptor. In contrast, few (/sup 3/H)pirenzepine binding sites were demonstrated in cerebellar and heart homogenates.

  8. Chloride homeostasis in Saccharomyces cerevisiae: high affinity influx, V-ATPase-dependent sequestration, and identification of a candidate Cl- sensor.

    PubMed

    Jennings, Michael L; Cui, Jian

    2008-04-01

    Chloride homeostasis in Saccharomyces cerevisiae has been characterized with the goal of identifying new Cl- transport and regulatory pathways. Steady-state cellular Cl- contents ( approximately 0.2 mEq/liter cell water) differ by less than threefold in yeast grown in media containing 0.003-5 mM Cl-. Therefore, yeast have a potent mechanism for maintaining constant cellular Cl- over a wide range of extracellular Cl-. The cell water:medium [Cl-] ratio is >20 in media containing 0.01 mM Cl- and results in part from sequestration of Cl- in organelles, as shown by the effect of deleting genes involved in vacuolar acidification. Organellar sequestration cannot account entirely for the Cl- accumulation, however, because the cell water:medium [Cl-] ratio in low Cl- medium is approximately 10 at extracellular pH 4.0 even in vma1 yeast, which lack the vacuolar H(+)-ATPase. Cellular Cl- accumulation is ATP dependent in both wild type and vma1 strains. The initial (36)Cl- influx is a saturable function of extracellular [(36)Cl-] with K(1/2) of 0.02 mM at pH 4.0 and >0.2 mM at pH 7, indicating the presence of a high affinity Cl- transporter in the plasma membrane. The transporter can exchange (36)Cl- for either Cl- or Br- far more rapidly than SO4=, phosphate, formate, HCO3-, or NO3-. High affinity Cl- influx is not affected by deletion of any of several genes for possible Cl- transporters. The high affinity Cl- transporter is activated over a period of approximately 45 min after shifting cells from high-Cl- to low-Cl- media. Deletion of ORF YHL008c (formate-nitrite transporter family) strongly reduces the rate of activation of the flux. Therefore, Yhl008cp may be part of a Cl(-)-sensing mechanism that activates the high affinity transporter in a low Cl- medium. This is the first example of a biological system that can regulate cellular Cl- at concentrations far below 1 mM. PMID:18378800

  9. Biomagnetic separation of Salmonella Typhimurium with high affine and specific ligand peptides isolated by phage display technique

    NASA Astrophysics Data System (ADS)

    Steingroewer, Juliane; Bley, Thomas; Bergemann, Christian; Boschke, Elke

    2007-04-01

    Analyses of food-borne pathogens are of great importance in order to minimize the health risk for customers. Thus, very sensitive and rapid detection methods are required. Current conventional culture techniques are very time consuming. Modern immunoassays and biochemical analysis also require pre-enrichment steps resulting in a turnaround time of at least 24 h. Biomagnetic separation (BMS) is a promising more rapid method. In this study we describe the isolation of high affine and specific peptides from a phage-peptide library, which combined with BMS allows the detection of Salmonella spp. with a similar sensitivity as that of immunomagnetic separation using antibodies.

  10. Negative Ions Enhance Survival of Membrane Protein Complexes

    NASA Astrophysics Data System (ADS)

    Liko, Idlir; Hopper, Jonathan T. S.; Allison, Timothy M.; Benesch, Justin L. P.; Robinson, Carol V.

    2016-06-01

    Membrane protein complexes are commonly introduced to the mass spectrometer solubilized in detergent micelles. The collisional activation used to remove the detergent, however, often causes protein unfolding and dissociation. As in the case for soluble proteins, electrospray in the positive ion mode is most commonly used for the study of membrane proteins. Here we show several distinct advantages of employing the negative ion mode. Negative polarity can yield lower average charge states for membrane proteins solubilized in saccharide detergents, with enhanced peak resolution and reduced adduct formation. Most importantly, we demonstrate that negative ion mode electrospray ionization (ESI) minimizes subunit dissociation in the gas phase, allowing access to biologically relevant oligomeric states. Together, these properties mean that intact membrane protein ions can be generated in a greater range of solubilizing detergents. The formation of negative ions, therefore, greatly expands the possibilities of using mass spectrometry on this intractable class of protein.

  11. Atomistic Simulation of Lignocellulosic Biomass and Associated Cellulosomal Protein Complexes

    SciTech Connect

    Petridis, Loukas; Crowley, Michael F; Smith, Jeremy C

    2010-01-01

    Computer simulations have been performed to obtain an atomic-level understanding of lignocellulose structure and the assembly of its associated cellulosomal protein complexes. First, a CHARMM molecular mechanics force field for lignin is derived and validated by performing a molecular dynamics simulation of a crystal of a lignin fragment molecule and comparing simulation-derived structural features with experimental results. Together with the existing force field for polysaccharides, this work provides the basis for full simulations of lignocellulose. Second, the underlying molecular mechanism governing the assembly of various cellulosomal modules is investigated by performing a novel free-energy calculation of the cohesin-dockerin dissociation. Our calculation indicates a free-energy barrier of ~17 kcal/mol and further reveals a stepwise dissociation pathway involving both the central -sheet interface and its adjacent solvent-exposed loop/turn regions clustered at both ends of the -barrel structure.

  12. Synthetic RNA-protein complex shaped like an equilateral triangle

    NASA Astrophysics Data System (ADS)

    Ohno, Hirohisa; Kobayashi, Tetsuhiro; Kabata, Rinko; Endo, Kei; Iwasa, Takuma; Yoshimura, Shige H.; Takeyasu, Kunio; Inoue, Tan; Saito, Hirohide

    2011-02-01

    Synthetic nanostructures consisting of biomacromolecules such as nucleic acids have been constructed using bottom-up approaches. In particular, Watson-Crick base pairing has been used to construct a variety of two- and three-dimensional DNA nanostructures. Here, we show that RNA and the ribosomal protein L7Ae can form a nanostructure shaped like an equilateral triangle that consists of three proteins bound to an RNA scaffold. The construction of the complex relies on the proteins binding to kink-turn (K-turn) motifs in the RNA, which allows the RNA to bend by ~60° at three positions to form a triangle. Functional RNA-protein complexes constructed with this approach could have applications in nanomedicine and synthetic biology.

  13. Integration of genomic datasets to predict protein complexes in yeast.

    PubMed

    Jansen, Ronald; Lan, Ning; Qian, Jiang; Gerstein, Mark

    2002-01-01

    The ultimate goal of functional genomics is to define the function of all the genes in the genome of an organism. A large body of information of the biological roles of genes has been accumulated and aggregated in the past decades of research, both from traditional experiments detailing the role of individual genes and proteins, and from newer experimental strategies that aim to characterize gene function on a genomic scale. It is clear that the goal of functional genomics can only be achieved by integrating information and data sources from the variety of these different experiments. Integration of different data is thus an important challenge for bioinformatics. The integration of different data sources often helps to uncover non-obvious relationships between genes, but there are also two further benefits. First, it is likely that whenever information from multiple independent sources agrees, it should be more valid and reliable. Secondly, by looking at the union of multiple sources, one can cover larger parts of the genome. This is obvious for integrating results from multiple single gene or protein experiments, but also necessary for many of the results from genome-wide experiments since they are often confined to certain (although sizable) subsets of the genome. In this paper, we explore an example of such a data integration procedure. We focus on the prediction of membership in protein complexes for individual genes. For this, we recruit six different data sources that include expression profiles, interaction data, essentiality and localization information. Each of these data sources individually contains some weakly predictive information with respect to protein complexes, but we show how this prediction can be improved by combining all of them. Supplementary information is available at http:// bioinfo.mbb.yale.edu/integrate/interactions/. PMID:12836664

  14. Supercharging Protein Complexes from Aqueous Solution Disrupts their Native Conformations

    NASA Astrophysics Data System (ADS)

    Sterling, Harry J.; Kintzer, Alexander F.; Feld, Geoffrey K.; Cassou, Catherine A.; Krantz, Bryan A.; Williams, Evan R.

    2012-02-01

    The effects of aqueous solution supercharging on the solution- and gas-phase structures of two protein complexes were investigated using traveling-wave ion mobility-mass spectrometry (TWIMS-MS). Low initial concentrations of m-nitrobenzyl alcohol ( m-NBA) in the electrospray ionization (ESI) solution can effectively increase the charge of concanavalin A dimers and tetramers, but at higher m-NBA concentrations, the increases in charge are accompanied by solution-phase dissociation of the dimers and up to a ~22% increase in the collision cross section (CCS) of the tetramers. With just 0.8% m-NBA added to the ESI solution of a ~630 kDa anthrax toxin octamer complex, the average charge is increased by only ~4% compared with the "native" complex, but it is sufficiently destabilized so that extensive gas-phase fragmentation occurs in the relatively high pressure regions of the TWIMS device. Anthrax toxin complexes exist in either a prechannel or a transmembrane channel state. With m-NBA, the prechannel state of the complex has the same CCS/charge ratio in the gas phase as the transmembrane channel state of the same complex formed without m-NBA, yet undergoes extensive dissociation, indicating that destabilization from supercharging occurs in the ESI droplet prior to ion formation and is not a result of Coulombic destabilization in the gas phase as a result of higher charging. These results demonstrate that the supercharging of large protein complexes is the result of conformational changes induced by the reagents in the ESI droplets, where enrichment of the supercharging reagent during droplet evaporation occurs.

  15. Identification of a high-affinity Ca sup 2+ pump associated with endocytotic vesicles in Dictyostelium discoideum

    SciTech Connect

    Milne, J.L.; Coukell, M.B. )

    1989-11-01

    In the cellular slime mold Dictyostelium discoideum, changes in free cytosolic Ca{sup 2+} are thought to regulate certain processes during cell aggregation and differentiation. To understand the mechanisms controlling free Ca{sup 2+} levels in this organism, the authors previously isolated and characterized an ATP/Mg{sup 2+}-dependent, high-affinity Ca{sup 2+} pump which appeared to be a component of inside-out plasma membrane vesicles. In this report, they demonstrate that a high-affinity Ca{sup 2+} pump, with properties virtually identical to the isolated pump, can be detected in filipin- or digitonin-permeabilized cells of Dictyostelium. Moreover, Ca{sup 2+}-pumping vesicles, which migrate on Percoll/KCl gradients like the vesicles identified earlier, can be isolated from the permeabilized cells. Results of additional experiments suggest that this intracellular Ca{sup 2+} transporter is associated with a high-capacity non-IP{sub 3}-releasable Ca{sup 2+} store which is generated by endocytosis. A possible role for this store in maintaining Ca{sup 2+} homeostasis in Dictyostelium is discussed.

  16. PtAAP11, a high affinity amino acid transporter specifically expressed in differentiating xylem cells of poplar.

    PubMed

    Couturier, Jérémy; de Faÿ, Elisabeth; Fitz, Michael; Wipf, Daniel; Blaudez, Damien; Chalot, Michel

    2010-06-01

    Amino acids are the currency of nitrogen exchange between source and sink tissues in plants and constitute a major source of the components used for cellular growth and differentiation. The characterization of a new amino acid transporter belonging to the amino acid permease (AAP) family, AAP11, expressed in the perennial species Populus trichocarpa is reported here. PtAAP11 expression analysis was performed by semi-quantitative RT-PCR and GUS activity after poplar transformation. PtAAP11 function was studied in detail by heterologous expression in yeast. The poplar genome contains 14 putative AAPs which is quite similar to other species analysed except Arabidopsis. PtAAP11 was mostly expressed in differentiating xylem cells in different organs. Functional characterization demonstrated that PtAAP11 was a high affinity amino acid transporter, more particularly for proline. Compared with other plant amino acid transporters, PtAAP11 represents a novel high-affinity system for proline. Thus, the functional characterization and expression studies suggest that PtAAP11 may play a major role in xylogenesis by providing proline required for xylem cell wall proteins. The present study provides important information highlighting the role of a specific amino acid transporter in xylogenesis in poplar.

  17. The Structure of a High-Affinity Kainate Receptor: GluK4 Ligand-Binding Domain Crystallized with Kainate.

    PubMed

    Kristensen, Ole; Kristensen, Lise Baadsgaard; Møllerud, Stine; Frydenvang, Karla; Pickering, Darryl S; Kastrup, Jette Sandholm

    2016-09-01

    Ionotropic glutamate receptors play a key role in fast neurotransmission in the CNS and have been linked to several neurological diseases and disorders. One subfamily is the kainate receptors, which are grouped into low-affinity (GluK1-3) and high-affinity (GluK4-5) receptors based on their affinity for kainate. Although structures of the ligand-binding domain (LBD) of all low-affinity kainate receptors have been reported, no structures of the high-affinity receptor subunits are available. Here, we present the X-ray structure of GluK4-LBD with kainate at 2.05 Å resolution, together with thermofluor and radiolabel binding affinity data. Whereas binding-site residues in GluK4 are most similar to the AMPA receptor subfamily, the domain closure and D1-D2 interlobe contacts induced by kainate are similar to the low-affinity kainate receptor GluK1. These observations provide a likely explanation for the high binding affinity of kainate at GluK4-LBD.

  18. Basement-membrane heparan sulphate with high affinity for antithrombin synthesized by normal and transformed mouse mammary epithelial cells.

    PubMed Central

    Pejler, G; David, G

    1987-01-01

    Basement-membrane proteoglycans, biosynthetically labelled with [35S]sulphate, were isolated from normal and transformed mouse mammary epithelial cells. Proteoglycans synthesized by normal cells contained mainly heparan sulphate and, in addition, small amounts of chondroitin sulphate chains, whereas transformed cells synthesized a relatively higher proportion of chondroitin sulphate. Polysaccharide chains from transformed cells were of lower average Mr and of lower anionic charge density compared with chains isolated from the untransformed counterparts, confirming results reported previously [David & Van den Berghe (1983) J. Biol. Chem. 258, 7338-7344]. A large proportion of the chains isolated from normal cells bound with high affinity to immobilized antithrombin, and the presence of 3-O-sulphated glucosamine residues, previously identified as unique markers for the antithrombin-binding region of heparin [Lindahl, Bäckström, Thunberg & Leder (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6551-6555], could be demonstrated. A significantly lower proportion of the chains derived from transformed cells bound with high affinity to antithrombin, and a corresponding decrease in the amount of incorporated 3-O-sulphate was observed. PMID:2963617

  19. Molecular cloning and functional characterization of a high-affinity zinc importer (DrZIP1) from zebrafish (Danio rerio).

    PubMed

    Qiu, Andong; Shayeghi, Majid; Hogstrand, Christer

    2005-06-15

    Zinc is a vital micronutrient to all organisms and a potential toxicant to aquatic animals. It is therefore of importance to understand the mechanism of zinc regulation. In the present study, we molecularly cloned and functionally characterized a zinc transporter of the SLC39A family [commonly referred to as the ZIP (Zrt- and Irt-related protein) family] from the gill of zebrafish (Danio rerio) (DrZIP1). DrZIP1 protein was found to localize at the plasma membrane and to function as a zinc uptake transporter when being expressed in either chinook salmon (Oncorhynchus tshawytscha) embryonic 214 cells or Xenopus laevis oocytes. In comparison with pufferfish transporter proteins (FrZIP2 and FrECaC) that are known to facilitate cellular zinc uptake, DrZIP1 appears to have high affinity to bind and transport zinc, suggesting that it maybe a high-affinity zinc uptake transporter (Km < 0.5 microM) in fish. Orthologues of DrZIP1 were also identified in both freshwater and seawater pufferfish (Tetraodon nigroviridis and Takifugu rubripes), indicating that these proteins may be functionally conserved among different fish species. DrZIP1 mRNA is expressed in all the tissues examined in the present study and thus DrZIP1 may be a constitutive zinc uptake transporter in many cell types of zebrafish.

  20. The Structure of the Amyloid-[beta] Peptide High-Affinity Copper II Binding Site in Alzheimer Disease

    SciTech Connect

    Streltsov, Victor A.; Titmuss, Stephen J.; Epa, V. Chandana; Barnham, Kevin J.; Masters, Colin L.; Varghese, Joseph N.

    2008-11-03

    Neurodegeneration observed in Alzheimer disease (AD) is believed to be related to the toxicity from reactive oxygen species (ROS) produced in the brain by the amyloid-{beta} (A{beta}) protein bound primarily to copper ions. The evidence for an oxidative stress role of A{beta}-Cu redox chemistry is still incomplete. Details of the copper binding site in A{beta} may be critical to the etiology of AD. Here we present the structure determined by combining x-ray absorption spectroscopy (XAS) and density functional theory analysis of A{beta} peptides complexed with Cu{sup 2+} in solution under a range of buffer conditions. Phosphate-buffered saline buffer salt (NaCl) concentration does not affect the high-affinity copper binding mode but alters the second coordination sphere. The XAS spectra for truncated and full-length A{beta}-Cu{sup 2+} peptides are similar. The novel distorted six-coordinated (3N3O) geometry around copper in the A{beta}-Cu{sup 2+} complexes include three histidines: glutamic, or/and aspartic acid, and axial water. The structure of the high-affinity Cu{sup 2+} binding site is consistent with the hypothesis that the redox activity of the metal ion bound to A{beta} can lead to the formation of dityrosine-linked dimers found in AD.

  1. Biphasic competition between opiates and enkephalins: does it indicate the existence of a common high affinity (mu-1) binding site

    SciTech Connect

    Sarne, Y.; Kenner, A.

    1987-08-03

    Displacement from brain membranes of labeled opiates by low concentrations of enkephalins and of labeled enkephalins by low concentrations of opiates has been previously explained by the existance of a common high affinity site termed mu-1. An alternative interpretation of the same results is that the trough seen in the low concentration zone of the displacement curves represents cross binding of mu and delta opioid ligands to delta and mu receptors, respectively. In three sets of experiments with brain membranes, the size of the trough is shown to be dependent on the labeled ligand used: The ratio between the size of troughs seen with (TH)D-Ala, D-Leu enkephalin and with (TH)morphine varies with experimental conditions (storage of membranes at 4C for 72h), with ratio of mu:delta receptors (e.g. in thalamus and cortex which are enriched in mu and delta sites, respectively) and with pretreatment of membranes with naloxonazine. These results cannot be explained by a common high affinity site, but rather by binding of (TH)D-Ala, D-Leu enkephalin to mu and of (TH)morphine to delta opioid receptors. 17 references, 3 figures.

  2. Bis-(1,2,3,4-tetrahydroisoquinolinium): a chiral scaffold for developing high-affinity ligands for SK channels.

    PubMed

    Liégeois, Jean-François; Wouters, Johan; Seutin, Vincent; Dilly, Sébastien

    2014-04-01

    N-Methyl-bis-(1,2,3,4-tetrahydroisoquinolinium) analogues derived from AG525 (1,1'-(propane-1,3-diyl)-bis-(6,7-dimethoxy-2- methyl-1,2,3,4-tetrahydroisoquinoline)) stereoisomers and tetrandrine, a rigid bis-(1,2,3,4-tetrahydroisoquinoline) analogue with an S,S configuration, were synthesized and tested for their affinity for small-conductance calcium-activated potassium channel (SK/KCa2) subtypes using radioligand binding assays. A significant increase in affinity was observed for the quaternized analogues over the parent 1,2,3,4-tetrahydroisoquinoline compounds. Interestingly, the impact of stereochemistry was not the same in the two groups of compounds. For quaternized analogues, affinities of S,S and R,R isomers for SK2 and SK3 channels were similar and in both cases higher than that of the meso derivative. Among the bis-tetrahydroisoquinoline compounds, the S,S isomers exhibited high affinity, while the R,R and meso isomers had similarly lower affinities. Furthermore, the SK2/SK3 selectivity ratio was slightly increased for quaternized analogues. Bis-(1,2,3,4-tetrahydroisoquinolinium) represents a new scaffold for the development of high-affinity ligands for SK channel subtypes. PMID:24829978

  3. Biphasic regulation of development of the high-affinity saxitoxin receptor by innervation in rat skeletal muscle

    SciTech Connect

    Sherman, S.J.; Catterall, W.A.

    1982-11-01

    Specific binding of /sup 3/H-saxitoxin (STX) was used to quantitate the density of voltage-sensitive sodium channels in developing rat skeletal muscle. In adult triceps surae, a single class of sites with a KD . 2.9 nM and a density of 21 fmol/mg wet wt was detected. The density of these high-affinity sites increased from 2.0 fmol/mg wet wt to the adult value in linear fashion during days 2-25 after birth. Denervation of the triceps surae at day 11 or 17 reduced final saxitoxin receptor site density to 10.4 or 9.2 fmol/mg wet wt, respectively, without changing KD. Denervation of the triceps surae at day 5 did not alter the subsequent development of saxitoxin receptor sites during days 5-9 and accelerated the increase of saxitoxin receptor sites during days 9-13. After day 13, saxitoxin receptor development abruptly ceased and the density of saxitoxin receptor sites declined to 11 fmol/wg wet wt. These results show that the regulation of high-affinity saxitoxin receptor site density by innervation is biphasic. During the first phase, which is independent of continuing innervation, the saxitoxin receptor density increases to 47-57% of the adult level. After day 11, the second phase of development, which is dependent on continuing innervation, gives rise to the adult density of saxitoxin receptors.

  4. Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

    PubMed Central

    Zlatic, Stephanie A.; Ryder, Pearl V.; Salazar, Gloria; Faundez, Victor

    2010-01-01

    The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 Å, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1. Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work. PMID:20216526

  5. Design of High-Affinity Stapled Peptides To Target the Repressor Activator Protein 1 (RAP1)/Telomeric Repeat-Binding Factor 2 (TRF2) Protein-Protein Interaction in the Shelterin Complex.

    PubMed

    Ran, Xu; Liu, Liu; Yang, Chao-Yie; Lu, Jianfeng; Chen, Yong; Lei, Ming; Wang, Shaomeng

    2016-01-14

    Shelterin, a six-protein complex, plays a fundamental role in protecting both the length and the stability of telomeres. Repressor activator protein 1 (RAP1) and telomeric repeat-binding factor 2 (TRF2) are two subunits in shelterin that interact with each other. Small-molecule inhibitors that block the RAP1/TRF2 protein-protein interaction can disrupt the structure of shelterin and may be employed as pharmacological tools to investigate the biology of shelterin. On the basis of the cocrystal structure of RAP1/TRF2 complex, we have developed first-in-class triazole-stapled peptides that block the protein-protein interaction between RAP1 and TRF2. Our most potent stapled peptide binds to RAP1 protein with a Ki value of 7 nM and is >100 times more potent than the corresponding wild-type TRF2 peptide. On the basis of our high-affinity peptides, we have developed and optimized a competitive, fluorescence polarization (FP) assay for accurate and rapid determination of the binding affinities of our designed compounds and this assay may also assist in the discovery of non-peptide, small-molecule inhibitors capable of blocking the RAP1/TRF2 protein-protein interaction.

  6. Affinity capture of biotinylated proteins at acidic conditions to facilitate hydrogen/deuterium exchange mass spectrometry analysis of multimeric protein complexes.

    PubMed

    Jensen, Pernille Foged; Jørgensen, Thomas J D; Koefoed, Klaus; Nygaard, Frank; Sen, Jette Wagtberg

    2013-08-01

    Characterization of conformational and dynamic changes associated with protein interactions can be done by hydrogen/deuterium exchange mass spectrometry (HDX-MS) by comparing the deuterium uptake in the bound and unbound state of the proteins. Investigation of local hydrogen/deuterium exchange in heteromultimeric protein complexes poses a challenge for the method due to the increased complexity of the mixture of peptides originating from all interaction partners in the complex. Previously, interference of peptides from one interaction partner has been removed by immobilizing the intact protein on beads prior to the HDX-MS experiment. However, when studying protein complexes of more than two proteins, immobilization can possibly introduce steric limitations to the interactions. Here, we present a method based on the high affinity biotin-streptavidin interaction that allows selective capture of biotinylated proteins even under the extreme conditions for hydrogen/deuterium exchange quenching i.e. pH 2.5 and 0 °C. This biotin-streptavidin capture strategy allows hydrogen/deuterium exchange to occur in proteins in solution and enables characterization of specific proteins in heteromultimeric protein complexes without interference of peptides originating from other interaction partners in the complex. The biotin-streptavidin strategy has been successfully implemented in a model system with two recombinant monoclonal antibodies that target nonoverlapping epitopes on the human epidermal growth factor receptor (EGFR). We present a workflow for biotinylation and characterization of recombinant antibodies and demonstrate affinity capture of biotinylated antibodies under hydrogen/deuterium exchange quench conditions by the biotin-streptavidin strategy.

  7. Polonium purification

    SciTech Connect

    Baker, J.D.

    1996-09-01

    Three processes for the purification of {sup 210}Po from irradiated bismuth targets are described. Safety equipment includes shielded hotcells for the initial separation from other activation products, gloveboxes for handling the volatile and highly toxic materials, and provisions for ventilation. All chemical separations must be performed under vacuum or in inerted systems. Two of the processes require large amounts of electricity; the third requires vessels made from exotic materials.

  8. Pool Purification

    NASA Technical Reports Server (NTRS)

    1988-01-01

    Caribbean Clear, Inc. used NASA's silver ion technology as a basis for its automatic pool purifier. System offers alternative approach to conventional purification chemicals. Caribbean Clear's principal markets are swimming pool owners who want to eliminate chlorine and bromine. Purifiers in Caribbean Clear System are same silver ions used in Apollo System to kill bacteria, plus copper ions to kill algae. They produce spa or pool water that exceeds EPA Standards for drinking water.

  9. Biodegradation of the chitin-protein complex in crustacean cuticle

    USGS Publications Warehouse

    Artur, Stankiewicz B.; Mastalerz, Maria; Hof, C.H.J.; Bierstedt, A.; Flannery, M.B.; Briggs, D.E.G.; Evershed, R.P.

    1998-01-01

    Arthropod cuticles consist predominantly of chitin cross-linked with proteins. While there is some experimental evidence that this chitin-protein complex may resist decay, the chemical changes that occur during degradation have not been investigated in detail. The stomatopod crustacean Neogonodactylus oerstedii was decayed in the laboratory under anoxic conditions. A combination of pyrolysis-gas chromatography/mass spectrometry and FTIR revealed extensive chemical changes after just 2 weeks that resulted in a cuticle composition dominated by chitin. Quantitative analysis of amino acids (by HPLC) and chitin showed that the major loss of proteins and chitin occurred between weeks 1 and 2. After 8 weeks tyrosine, tryptophan and valine are the most prominent amino acid moieties, showing their resistance to degradation. The presence of cyclic ketones in the pyrolysates indicates that mucopolysaccharides or other bound non-chitinous carbohydrates are also resistant to decay. There is no evidence of structural degradation of chitin prior to 8 weeks when FTIR revealed a reduction in chitin-specific bands. The chemical changes are paralleled by structural changes in the cuticle, which becomes an increasingly open structure consisting of loose chitinous fibres. The rapid rate of decay in the experiments suggests that where chitin and protein are preserved in fossil cuticles degradation must have been inhibited.Arthropod cuticles consist predominantly of chitin cross-linked with proteins. While there is some experimental evidence that this chitin-protein complex may resist decay, the chemical changes that occur during degradation have not been investigated in detail. The stomatopod crustacean Neogonodactylus oerstedii was decayed in the laboratory under anoxic conditions. A combination of pyrolysis-gas chromatography/mass spectrometry and FTIR revealed extensive chemical changes after just 2 weeks that resulted in a cuticle composition dominated by chitin. Quantitative

  10. Crystal Structure of the SH3 Domain of beta PIX in Complex with a High Affinity Peptide from PAK2

    SciTech Connect

    Hoelz,A.; Janz, J.; Lawrie, S.; Corwin, B.; Lee, A.; Sakmar, T.

    2006-01-01

    The p21-activated kinases (PAKs) are important effector proteins of the small GTPases Cdc42 and Rac and control cytoskeletal rearrangements and cell proliferation. The direct interaction of PAKs with guanine nucleotide exchange factors from the PIX/Cool family, which is responsible for the localization of PAK kinases to focal complexes in the cell, is mediated by a 24-residue peptide segment in PAKs and an N-terminal src homology 3 (SH3) domain in PIX/Cool. The SH3-binding segment of PAK contains the atypical consensus-binding motif PxxxPR, which is required for unusually high affinity binding. In order to understand the structural basis for the high affinity and specificity of the PIX-PAK interaction, we solved crystal structures for the N-terminal SH3 domain of {beta}PIX and for the complex of the atypical binding segment of PAK2 with the N-terminal SH3 domain of {beta}PIX at 0.92 Angstroms and 1.3 Angstroms resolution, respectively. The asymmetric unit of the crystal contains two SH3 domains and two peptide ligands. The bound peptide adopts a conformation that allows for intimate contacts with three grooves on the surface of the SH3 domain that lie between the n-Src and RT-loops. Most notably, the arginine residue of the PxxxPR motif forms a salt-bridge and is tightly coordinated by a number of residues in the SH3 domain. This arginine-specific interaction appears to be the key determinant for the high affinity binding of PAK peptides. Furthermore, C-terminal residues of the peptide engage in additional interactions with the surface of the RT-loop, which significantly increases binding specificity. Compared to a recent NMR structure of a similar complex, our crystal structure reveals an alternate binding mode. Finally, we compare our crystal structure with the recently published {beta}PIX/Cbl-b complex structure, and suggest the existence of a molecular switch.

  11. High-affinity consensus binding of target RNAs by the STAR/GSG proteins GLD-1, STAR-2 and Quaking

    PubMed Central

    2010-01-01

    Background STAR/GSG proteins regulate gene expression in metazoans by binding consensus sites in the 5' or 3' UTRs of target mRNA transcripts. Owing to the high degree of homology across the STAR domain, most STAR proteins recognize similar RNA consensus sequences. Previously, the consensus for a number of well-characterized STAR proteins was defined as a hexameric sequence, referred to as the SBE, for STAR protein binding element. C. elegans GLD-1 and mouse Quaking (Qk-1) are two representative STAR proteins that bind similar consensus hexamers, which differ only in the preferred nucleotide identities at certain positions. Earlier reports also identified partial consensus elements located upstream or downstream of a canonical consensus hexamer in target RNAs, although the relative contribution of these sequences to the overall binding energy remains less well understood. Additionally, a recently identified STAR protein called STAR-2 from C. elegans is thought to bind target RNA consensus sites similar to that of GLD-1 and Qk-1. Results Here, a combination of fluorescence-polarization and gel mobility shift assays was used to demonstrate that STAR-2 binds to a similar RNA consensus as GLD-1 and Qk-1. These assays were also used to further delineate the contributions of each hexamer consensus nucleotide to high-affinity binding by GLD-1, Qk-1 and STAR-2 in a variety of RNA contexts. In addition, the effects of inserting additional full or partial consensus elements upstream or downstream of a canonical hexamer in target RNAs were also measured to better define the sequence elements and RNA architecture recognized by different STAR proteins. Conclusions The results presented here indicate that a single hexameric consensus is sufficient for high-affinity RNA binding by STAR proteins, and that upstream or downstream partial consensus elements may alter binding affinities depending on the sequence and spacing. The general requirements determined for high-affinity RNA

  12. Assembly and solution structure of the core retromer protein complex.

    PubMed

    Norwood, Suzanne J; Shaw, Daniel J; Cowieson, Nathan P; Owen, David J; Teasdale, Rohan D; Collins, Brett M

    2011-01-01

    Retromer is a peripheral membrane protein complex that has pleiotropic roles in endosomal membrane trafficking. The core of retromer possesses three subunits, VPS35, VPS29 and VPS26, that play different roles in binding to cargo, regulatory proteins and complex stabilization. We have performed an investigation of the thermodynamics of core retromer assembly using isothermal titration calorimetry (ITC) demonstrating that VPS35 acts as the central subunit to which VPS29 and VPS26 bind independently. Furthermore, we confirm that the conserved PRLYL motif of the large VPS35 subunit is critical for direct VPS26 interaction. Heat capacity measurements of VPS29 and VPS26 binding to VPS35 indicate extensive binding interfaces and suggest conformational alterations in VPS29 or VPS35 upon complex formation. Solution studies of the retromer core using small-angle X-ray scattering allow us to propose a model whereby VPS35 forms an extended platform with VPS29 and VPS26 bound at distal ends, with the potential for forming dimeric assemblies. PMID:20875039

  13. DMS Footprinting of Structured RNAs and RNA-Protein Complexes

    PubMed Central

    Tijerina, Pilar; Mohr, Sabine; Russell, Rick

    2008-01-01

    We describe a protocol in which dimethyl sulfate (DMS) modification of the base-pairing faces of unpaired adenosine and cytidine nucleotides is used for structural analysis of RNAs and RNA-protein complexes (RNPs). The protocol is optimized for RNAs of small to moderate size (≤500 nucleotides). The RNA or RNP is first exposed to DMS under conditions that promote formation of the folded structure or complex, as well as ‘control’ conditions that do not allow folding or complex formation. The positions and extents of modification are then determined by primer extension, polyacrylamide gel electrophoresis (PAGE), and quantitative analysis. From changes in the extent of modification upon folding or protein binding (appearance of a ‘footprint’), it is possible to detect local changes in RNA secondary and tertiary structure, as well as the formation of RNA-protein contacts. This protocol takes 1.5–3 days to complete, depending on the type of analysis used. PMID:17948004

  14. Optimization of the electrostatic interactions in protein-protein complexes

    NASA Astrophysics Data System (ADS)

    Alexov, Emil; Brock, Kelly; Kundrotas, Petras

    2007-03-01

    Electrostatic energy is one of the driving forces of protein-protein association. Understanding the role of the energy components on the energetics of protein-protein association will help us in engineering protein-protein interactions and could lead to development of scoring functions that can rank alternative models and decoys. Here we investigate whether the components of the electrostatic energy of protein-protein complexes is optimized in respect to random distribution of the charged residues. We report a clear tendency that coulombic electrostatic interactions are optimized, while the reaction field energy is inversely optimized. It was found that the maximum of the coulombic energy Z-score is shifted 3 units away from the origin and the maximum of the reaction field energy by 2 units. Such a large shift of the Z-score of both coulombic and reaction field energies indicates that wild-type protein-protein interactions are in most cases optimized in terms of coulombic interactions while compromising reaction field energy. Based on these finding a scoring function was developed as a linear combination of the Z-score of the coulombic interactions minus Z-score of the reaction field energy. The scoring function was tested against the decoy sets and it was shown that in majority of the cases we can identify the wild-type complex among hundreds of decoys.

  15. Heterodimeric protein complex identification by naïve Bayes classifiers

    PubMed Central

    2013-01-01

    Background Protein complexes are basic cellular entities that carry out the functions of their components. It can be found that in databases of protein complexes of yeast like CYC2008, the major type of known protein complexes is heterodimeric complexes. Although a number of methods for trying to predict sets of proteins that form arbitrary types of protein complexes simultaneously have been proposed, it can be found that they often fail to predict heterodimeric complexes. Results In this paper, we have designed several features characterizing heterodimeric protein complexes based on genomic data sets, and proposed a supervised-learning method for the prediction of heterodimeric protein complexes. This method learns the parameters of the features, which are embedded in the naïve Bayes classifier. The log-likelihood ratio derived from the naïve Bayes classifier with the parameter values obtained by maximum likelihood estimation gives the score of a given pair of proteins to predict whether the pair is a heterodimeric complex or not. A five-fold cross-validation shows good performance on yeast. The trained classifiers also show higher predictability than various existing algorithms on yeast data sets with approximate and exact matching criteria. Conclusions Heterodimeric protein complex prediction is a rather harder problem than heteromeric protein complex prediction because heterodimeric protein complex is topologically simpler. However, it turns out that by designing features specialized for heterodimeric protein complexes, predictability of them can be improved. Thus, the design of more sophisticate features for heterodimeric protein complexes as well as the accumulation of more accurate and useful genome-wide data sets will lead to higher predictability of heterodimeric protein complexes. Our tool can be downloaded from http://imi.kyushu-u.ac.jp/~om/. PMID:24299017

  16. Omega-conotoxin GVIA binding to a high-affinity receptor in brain: characterization, calcium sensitivity, and solubilization

    SciTech Connect

    Wagner, J.A.; Snowman, A.M.; Biswas, A.; Olivera, B.M.; Snyder, S.H.

    1988-09-01

    We describe unique, high-affinity binding sites for omega(/sup 125/I)conotoxin GVIA in membranes from rat brain and rabbit sympathetic ganglia which appear to be primarily associated with N-type voltage-dependent calcium channels. The dissociation constant (KD) for the toxin in rat brain membranes is 60 pM. Physiologic extracellular concentrations of calcium inhibit toxin binding noncompetitively (IC50 = 0.2 mM). The regional distribution of the binding sites in rat brain differs markedly from that of dihydropyridine calcium antagonist receptors associated with L-type calcium channels. In detergent-solubilized brain membranes, toxin binding retains the same affinity, specificity, and ionic sensitivity as in particulate preparations.

  17. Peptide-based protein capture agents with high affinity, selectivity, and stability as antibody replacements in biodetection assays

    NASA Astrophysics Data System (ADS)

    Coppock, Matthew B.; Farrow, Blake; Warner, Candice; Finch, Amethist S.; Lai, Bert; Sarkes, Deborah A.; Heath, James R.; Stratis-Cullum, Dimitra

    2014-05-01

    Current biodetection assays that employ monoclonal antibodies as primary capture agents exhibit limited fieldability, shelf life, and performance due to batch-to-batch production variability and restricted thermal stability. In order to improve upon the detection of biological threats in fieldable assays and systems for the Army, we are investigating protein catalyzed capture (PCC) agents as drop-in replacements for the existing antibody technology through iterative in situ click chemistry. The PCC agent oligopeptides are developed against known protein epitopes and can be mass produced using robotic methods. In this work, a PCC agent under development will be discussed. The performance, including affinity, selectivity, and stability of the capture agent technology, is analyzed by immunoprecipitation, western blotting, and ELISA experiments. The oligopeptide demonstrates superb selectivity coupled with high affinity through multi-ligand design, and improved thermal, chemical, and biochemical stability due to non-natural amino acid PCC agent design.

  18. The Mycobacterium tuberculosis high-affinity iron importer, IrtA, contains an FAD-binding domain.

    PubMed

    Ryndak, Michelle B; Wang, Shuishu; Smith, Issar; Rodriguez, G Marcela

    2010-02-01

    Iron is an essential nutrient not freely available to microorganisms infecting mammals. To overcome iron deficiency, bacteria have evolved various strategies including the synthesis and secretion of high-affinity iron chelators known as siderophores. The siderophores produced and secreted by Mycobacterium tuberculosis, exomycobactins, compete for iron with host iron-binding proteins and, together with the iron-regulated ABC transporter IrtAB, are required for the survival of M. tuberculosis in iron deficient conditions and for normal replication in macrophages and in mice. This study further characterizes the role of IrtAB in M. tuberculosis iron acquisition. Our results demonstrate a role for IrtAB in iron import and show that the amino terminus domain of IrtA is a flavin-adenine dinucleotide-binding domain essential for iron acquisition. These results suggest a model in which the amino terminus of IrtA functions to couple iron transport and assimilation.

  19. Corticotropin-releasing factor antagonist blocks microwave-induced decreases in high-affinity choline uptake in the rat brain

    SciTech Connect

    Lai, H.; Carino, M.A.; Horita, A.; Guy, A.W. )

    1990-10-01

    Acute (45-min) irradiation with pulsed low-level microwaves (2450-MHz, 2 microseconds pulses at 500 pps, average power density of 1 mW/cm2, whole-body average specific absorption rate of 0.6 W/kg) decreased sodium-dependent high-affinity choline uptake (HACU) activity in the frontal cortex and hippocampus of the rat. These effects were blocked by pretreating the animals before exposure with intracerebroventricular injection of the specific corticotropin-releasing factor (CRF) receptor antagonist, alpha-helical-CRF9-41 (25 micrograms). Similar injection of the antagonist had no significant effect on HACU in the brain of the sham-exposed rats. These data suggest that low-level microwave irradiation activates CRF in the brain, which in turn causes the changes in central HACU.

  20. Human and rat mast cell high-affinity immunoglobulin E receptors: Characterization of putative. alpha. -chain gene products

    SciTech Connect

    Shimizu, Akira; Benfey, P.N.; Leder, P. ); Tepler, I. Brigham and Women's Hospital, Boston, MA ); Berenstein, E.H.; Siraganian, R.P. )

    1988-03-01

    The authors have cloned and determined the entire nucleotide sequence of cDNAs corresponding to the putative {alpha} subunits of the human and rat mast cell high-affinity IgE receptors. Both human and rat cDNAs encode an NH{sub 2}-terminal signal peptide, two immunoglobulin-like extracellular domains (encoded by discrete exons), a hydrophobic transmembrane region, and a positively charged cytoplasmic tail. The human and rat {alpha} subunits share an overall homology with one another and the immunoglobulin gene family, suggesting that they arose from a common ancestral gene and continue to share structural homology with their ligands. In addition, the rat gene is transcribed into at least three distinct forms, each of which yields a somewhat different coding sequence.

  1. Selectively Promiscuous Opioid Ligands: Discovery of High Affinity/Low Efficacy Opioid Ligands with Substantial Nociceptin Opioid Peptide Receptor Affinity

    PubMed Central

    2015-01-01

    Emerging clinical and preclinical evidence suggests that a compound displaying high affinity for μ, κ, and δ opioid (MOP, KOP, and DOP) receptors and antagonist activity at each, coupled with moderate affinity and efficacy at nociceptin opioid peptide (NOP) receptors will have utility as a relapse prevention agent for multiple types of drug abuse. Members of the orvinol family of opioid ligands have the desired affinity profile but have typically displayed substantial efficacy at MOP and or KOP receptors. In this study it is shown that a phenyl ring analogue (1d) of buprenorphine displays the desired profile in vitro with high, nonselective affinity for the MOP, KOP, and DOP receptors coupled with moderate affinity for NOP receptors. In vivo, 1d lacked any opioid agonist activity and was an antagonist of both the MOP receptor agonist morphine and the KOP receptor agonist ethylketocyclazocine, confirming the desired opioid receptor profile in vivo. PMID:24761755

  2. High-Affinity, Small-Molecule Peptidomimetic Inhibitors of MLL1/WDR5 Protein-Protein Interaction

    SciTech Connect

    Karatas, Hacer; Townsend, Elizabeth C; Cao, Fang; Chen, Yong; Bernard, Denzil; Liu, Liu; Lei, Ming; Dou, Yali; Wang, Shaomeng

    2013-02-12

    Mixed lineage leukemia 1 (MLL1) is a histone H3 lysine 4 (H3K4) methyltransferase, and targeting the MLL1 enzymatic activity has been proposed as a novel therapeutic strategy for the treatment of acute leukemia harboring MLL1 fusion proteins. The MLL1/WDR5 protein–protein interaction is essential for MLL1 enzymatic activity. In the present study, we designed a large number of peptidomimetics to target the MLL1/WDR5 interaction based upon -CO-ARA-NH–, the minimum binding motif derived from MLL1. Our study led to the design of high-affinity peptidomimetics, which bind to WDR5 with Ki < 1 nM and function as potent antagonists of MLL1 activity in a fully reconstituted in vitro H3K4 methyltransferase assay. Determination of co-crystal structures of two potent peptidomimetics in complex with WDR5 establishes their structural basis for high-affinity binding to WDR5. Evaluation of one such peptidomimetic, MM-102, in bone marrow cells transduced with MLL1-AF9 fusion construct shows that the compound effectively decreases the expression of HoxA9 and Meis-1, two critical MLL1 target genes in MLL1 fusion protein mediated leukemogenesis. MM-102 also specifically inhibits cell growth and induces apoptosis in leukemia cells harboring MLL1 fusion proteins. Our study provides the first proof-of-concept for the design of small-molecule inhibitors of the WDR5/MLL1 protein–protein interaction as a novel therapeutic approach for acute leukemia harboring MLL1 fusion proteins.

  3. Recombinant human nerve growth factor is biologically active and labels novel high-affinity binding sites in rat brain

    SciTech Connect

    Altar, C.A.; Burton, L.E.; Bennett, G.L.; Dugich-Djordjevic, M. )

    1991-01-01

    Iodinated recombinant human nerve growth factor (125I-rhNGF) stimulated neurite formation in PC12 cell cultures with a half-maximal potency of 35-49 pg/ml, compared with 39-52 pg/ml for rhNGF. In quantitative ligand autoradiography, the in vitro equilibrium binding of 125I-rhNGF to brain sections showed a 10-fold regional variation in density and was saturable, reversible, and specifically displaced by up to 74% with rhNGF or murine NGF (muNGF). At equilibrium, 125I-rhNGF bound to these sites with high affinity and low capacity (Bmax less than or equal to 13.2 fmol/mg of protein). Calculation of 125I-rhNGF binding affinity by kinetic methods gave average Kd values of 24 and 31 pM. Computer-generated maps revealed binding in brain regions not identified previously with 125I-muNGF, including hippocampus; dentate gyrus; amygdala; paraventricular thalamus; frontal, parietal, occipital, and cingulate cortices; nucleus accumbens; olfactory tubercle; subiculum; pineal gland; and medial geniculate nucleus. NGF binding sites were distributed in a 2-fold increasing medial-lateral gradient in the caudate-putamen and a 2-fold lateral-medial gradient in the nucleus accumbens. 125I-rhNGF binding sites were also found in most areas labeled by 125I-muNGF, including the interpedunucular nucleus, cerebellum, forebrain cholinergic nuclei, caudoventral caudate-putamen, and trigeminal nerve nucleus. 125I-rhNGF binding sites were absent from areas replete with low-affinity NGF binding sites, including circumventricular organs, myelinated fiber bundles, and choroid plexus. The present analysis provides an anatomical differentiation of high-affinity 125I-rhNGF binding sites and greatly expands the number of brain structures that may respond to endogenous NGF or exogenously administered rhNGF.

  4. High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization, Binding and Tumor Targeting

    PubMed Central

    Ullman, Christopher; Mathonet, Pascale; Oleksy, Arkadiusz; Diamandakis, Agata; Tomei, Licia; Demartis, Anna; Nardi, Chiara; Sambucini, Sonia; Missineo, Antonino; Alt, Karen; Hagemeyer, Christoph E.; Harris, Matt; Hedt, Amos; Weis, Roland; Gehlsen, Kurt R.

    2015-01-01

    Abdurins are a novel antibody-like scaffold derived from the engineering of a single isolated CH2 domain of human IgG. Previous studies established the prolonged serum half-life of Abdurins, the result of a retained FcRn binding motif. Here we present data on the construction of large, diverse, phage-display and cell-free DNA display libraries and the isolation of high affinity binders to the cancer target, membrane-bound ephrin receptor tyrosine kinase class A2 (EphA2). Antigen binding regions were created by designing combinatorial libraries into the structural loops and Abdurins were selected using phage display methods. Initial binders were reformatted into new maturation libraries and low nanomolar binders were isolated using cell-free DNA display, CIS display. Further characterization confirmed binding of the Abdurins to both human and murine EphA2 proteins and exclusively to cell lines that expressed EphA2, followed by rapid internalization. Two different EphA2 binders were labeled with 64Cu, using a bifunctional MeCOSar chelator, and administered to mice bearing tumors from transplanted human prostate cancer cells, followed by PET/CT imaging. The anti-EphA2 Abdurins localized in the tumors as early as 4 hours after injection and continued to accumulate up to 48 hours when the imaging was completed. These data demonstrate the ability to isolate high affinity binders from the engineered Abdurin scaffold, which retain a long serum half-life, and specifically target tumors in a xenograft model. PMID:26313909

  5. Role of the inhibitory guanine nucleotide regulatory protein in high affinity. cap alpha. /sub 2/ adrenergic agonist binding

    SciTech Connect

    Kim, M.H.

    1987-01-01

    The purpose of this study was to determine whether regulatory protein, N/sub i/ was required for high affinity agonist binding to the a/sub 2/ adrenergic receptor in human platelet membranes. Human platelet membranes treated under alkaline conditions (pH 11.5) exhibited a selective and complete loss of high affinity agonist binding as measured by the parital agonist (/sup 3/H)-p-aminoclonidine and full agonist (/sup 3/H)UK 14,304 in direct binding studies. The binding parameters for (/sup 3/H)UK 14,304 are as follows: for control platelet membranes, the K/sub d/ was 0.88 +/- 0.17 and nM and the B/sub max/ was 280 +/- 20 fmol/mg compared to 1.89 +/- 0.34 nM and 75 fmol/mg for pH 11.5 treated membranes. For (/sup 3/H)p-aminoclonidine, the data for pH 11.5 treated membranes is as follows: B/sub max/ = 100 +/- 20 fmol/mg, K/sub d/ = 3.4 +/- 0.1 nM, compared to control membranes: (best fit with a two site fit) K/sub d1/ = 0.7 nM, K/sub d2/ = 8 nM, B/sub max1/ = 76 fmol/mg, B/sub max2/ = 198 fmol/mg. The ..cap alpha../sub 2/ antagonists, (/sup 3/H)yohimbine, was used to assess the presence of the receptor.

  6. Synthesis and Characterization of [76Br]-Labeled High Affinity A3 Adenosine Receptor Ligands for Positron Emission Tomography

    PubMed Central

    Kiesewetter, Dale O.; Lang, Lixin; Ma, Ying; Bhattacharjee, Abesh Kumar; Gao, Zhan-Guo; Joshi, Bhalchandra V.; Melman, Artem; de Castro, Sonia; Jacobson, Kenneth A.

    2009-01-01

    Introduction Bromine-76 radiolabeled analogues of previously reported high affinity A3 adenosine receptor (A3AR) nucleoside ligands have been prepared as potential radiotracers for Positron Emission Tomography (PET). Methods The radiosyntheses were accomplished by oxidative radiobromination on the N6-benzyl moiety of trimethyltin precursors. Biodistribution studies of the kinetics of uptake were conducted in awake rats. Results We prepared an agonist ligand {[76Br](1′R,2′R,3′S,4′R,5′S)-4-{2-chloro-6-[(3-bromophenylmethyl)amino]purin-9-yl}-1-(methylaminocarbonyl)bicyclo[3.1.0]hexane-2,3-diol (MRS3581)} in 59% radiochemical yield (RCY) with a specific activity of 19.5 GBq/μmol and an antagonist ligand {[76Br](1R,2R,3S,4R,5S)-4-(6-(3-bromobenzylamino)-2-chloro-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol. (MRS5147)} in 65% RCY with a specific activity of 22 GBq/μmol). The resultant products exhibited the expected high affinity (Ki ~ 0.6 nM) and specific binding at the human A3AR in vitro. Biodistribution studies in the rat showed uptake in the organs of excretion and metabolism. The antagonist MRS5147 exhibited increasing uptake in testes, an organ that contains significant quantities of A3AR, over a 2 h time course, which suggests the presence of a specific A3AR retention mechanism. Conclusion We were able to compare uptake of the [76Br]labeled antagonist MRS5147 to [76Br]agonist MRS3581. The antagonist MRS5147 shows increasing uptake in the testes, an A3AR rich tissue, suggesting that this ligand may have promise as a molecular imaging agent. PMID:19181263

  7. Two perfectly conserved arginine residues are required for substrate binding in a high-affinity nitrate transporter.

    PubMed

    Unkles, Shiela E; Rouch, Duncan A; Wang, Ye; Siddiqi, M Yaeesh; Glass, Anthony D M; Kinghorn, James R

    2004-12-14

    This study represents the first attempt to investigate the molecular mechanisms by which nitrate, an anion of significant ecological, agricultural, and medical importance, is transported into cells by high-affinity nitrate transporters. Two charged residues, R87 and R368, located within hydrophobic transmembrane domains 2 and 8, respectively, are conserved in all 52 high-affinity nitrate transporters sequenced thus far. Site-directed replacements of either of R87 or R368 residues by lysine were found to be tolerated, but such residue changes increased the K(m) for nitrate influx from micromolar to millimolar values. Seven other amino acid substitutions of R87 or R368 all led to loss of function and lack of growth on nitrate. No evidence was obtained of R87 or R368 forming a salt-bridge with conserved acidic residues. Remarkably, the phenotype of loss-of-function mutant R87T was found to be alleviated by an alteration to lysine of N459, present in the second copy of the nitrate signature (transmembrane domain 11), suggesting a structural or functional interplay between residues R87 and N459 in the three-dimensional NrtA protein structure. Failure of the potential reciprocal second site suppressor N168K (in the first nitrate signature copy of transmembrane domain 5) to revert R368T was observed. Taken with recent structural studies of other major facilitator superfamily proteins, the results suggest that R87 and R368 are involved in substrate binding and probably located in a region of the protein close to N459. PMID:15576512

  8. Photoaffinity Labeling of High Affinity Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)-Binding Proteins in Sea Urchin Egg*♦

    PubMed Central

    Walseth, Timothy F.; Lin-Moshier, Yaping; Jain, Pooja; Ruas, Margarida; Parrington, John; Galione, Antony; Marchant, Jonathan S.; Slama, James T.

    2012-01-01

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is a messenger that regulates calcium release from intracellular acidic stores. Recent studies have identified two-pore channels (TPCs) as endolysosomal channels that are regulated by NAADP; however, the nature of the NAADP receptor binding site is unknown. To further study NAADP binding sites, we have synthesized and characterized [32P-5-azido]nicotinic acid adenine dinucleotide phosphate ([32P-5N3]NAADP) as a photoaffinity probe. Photolysis of sea urchin egg homogenates preincubated with [32P-5N3]NAADP resulted in specific labeling of 45-, 40-, and 30-kDa proteins, which was prevented by inclusion of nanomolar concentrations of unlabeled NAADP or 5N3-NAADP, but not by micromolar concentrations of structurally related nucleotides such as NAD, nicotinic acid adenine dinucleotide, nicotinamide mononucleotide, nicotinic acid, or nicotinamide. [32P-5N3]NAADP binding was saturable and displayed high affinity (Kd ∼10 nm) in both binding and photolabeling experiments. [32P-5N3]NAADP photolabeling was irreversible in a high K+ buffer, a hallmark feature of NAADP binding in the egg system. The proteins photolabeled by [32P-5N3]NAADP have molecular masses smaller than the sea urchin TPCs, and antibodies to TPCs do not detect any immunoreactivity that comigrates with either the 45-kDa or the 40-kDa photolabeled proteins. Interestingly, antibodies to TPC1 and TPC3 were able to immunoprecipitate a small fraction of the 45- and 40-kDa photolabeled proteins, suggesting that these proteins associate with TPCs. These data suggest that high affinity NAADP binding sites are distinct from TPCs. PMID:22117077

  9. High-Affinity Inhibitors of Human NAD+-Dependent 15-Hydroxyprostaglandin Dehydrogenase: Mechanisms of Inhibition and Structure-Activity Relationships

    PubMed Central

    Niesen, Frank H.; Schultz, Lena; Jadhav, Ajit; Bhatia, Chitra; Guo, Kunde; Maloney, David J.; Pilka, Ewa S.; Wang, Minghua; Oppermann, Udo; Heightman, Tom D.; Simeonov, Anton

    2010-01-01

    Background 15-hydroxyprostaglandin dehydrogenase (15-PGDH, EC 1.1.1.141) is the key enzyme for the inactivation of prostaglandins, regulating processes such as inflammation or proliferation. The anabolic pathways of prostaglandins, especially with respect to regulation of the cyclooxygenase (COX) enzymes have been studied in detail; however, little is known about downstream events including functional interaction of prostaglandin-processing and -metabolizing enzymes. High-affinity probes for 15-PGDH will, therefore, represent important tools for further studies. Principal Findings To identify novel high-affinity inhibitors of 15-PGDH we performed a quantitative high-throughput screen (qHTS) by testing >160 thousand compounds in a concentration-response format and identified compounds that act as noncompetitive inhibitors as well as a competitive inhibitor, with nanomolar affinity. Both types of inhibitors caused strong thermal stabilization of the enzyme, with cofactor dependencies correlating with their mechanism of action. We solved the structure of human 15-PGDH and explored the binding modes of the inhibitors to the enzyme in silico. We found binding modes that are consistent with the observed mechanisms of action. Conclusions Low cross-reactivity in screens of over 320 targets, including three other human dehydrogenases/reductases, suggest selectivity of the present inhibitors for 15-PGDH. The high potencies and different mechanisms of action of these chemotypes make them a useful set of complementary chemical probes for functional studies of prostaglandin-signaling pathways. Enhanced version This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S2. PMID:21072165

  10. High-affinity lead binding proteins in rat kidney cytosol mediate cell-free nuclear translocation of lead

    SciTech Connect

    Mistry, P.; Lucier, G.W.; Fowler, B.A.

    1985-02-01

    The PbII binding characteristics of the previously reported PbII binding proteins of rat kidney cytosol were investigated further. Saturation and Scatchard analysis of /sup 203/Pb binding in whole cytosol and in 40% saturated ammonium sulfate precipitated fractions disclosed a class of relatively high-affinity sites with an apparent Kd of approximately 50 nM and binding capacities of approximately 41 and 9 pmol/mg of protein, respectively. Two /sup 203/Pb binding proteins with approximate molecular masses of 63K and 11.5K daltons and a high molecular weight component (greater than 200K) were isolated by Sepharose-6B column chromatography. The time course of association of /sup 203/Pb with cytosol and the 63K protein showed maximum binding at 18 hr which was stable up to 25 hr at 4 degrees C. The approximate half-time dissociation rate (T 1/2) of specifically bound /sup 203/Pb to the 63K protein was 100 min at 4 degrees C whereas the 11.5K protein showed little dissociation of specifically bound ligand at this temperature. Saturation analysis of the three isolated proteins disclosed low capacity, high-affinity sites with similar apparent Kd values to the cytosol assay. Sucrose density gradient analysis of kidney cytosol showed approximate sedimentation coefficients of 2S, 4.6S and 7S for the 11.5K, 63K and the high molecular weight proteins, respectively. Competitive binding studies with cytosol demonstrated displacement of /sup 203/Pb by PbII, CdII and ZnII ions but not CaII ions.

  11. High-affinity α4β2 nicotinic receptors mediate the impairing effects of acute nicotine on contextual fear extinction.

    PubMed

    Kutlu, Munir Gunes; Holliday, Erica; Gould, Thomas J

    2016-02-01

    Previously, studies from our lab have shown that while acute nicotine administered prior to training and testing enhances contextual fear conditioning, acute nicotine injections prior to extinction sessions impair extinction of contextual fear. Although there is also strong evidence showing that the acute nicotine's enhancing effects on contextual fear conditioning require high-affinity α4β2 nicotinic acetylcholine receptors (nAChRs), it is unknown which nAChR subtypes are involved in the acute nicotine-induced impairment of contextual fear extinction. In this study, we investigated the effects of acute nicotine administration on contextual fear extinction in knock-out (KO) mice lacking α4, β2 or α7 subtypes of nAChRs and their wild-type (WT) littermates. Both KO and WT mice were first trained and tested for contextual fear conditioning and received a daily contextual extinction session for 4 days. Subjects received intraperitoneal injections of nicotine (0.18 mg/kg) or saline 2-4 min prior to each extinction session. Our results showed that the mice that lack α4 and β2 subtypes of nAChRs showed normal contextual fear extinction but not the acute nicotine-induced impairment while the mice that lack the α7 subtype showed both normal contextual extinction and nicotine-induced impairment of contextual extinction. In addition, control experiments showed that acute nicotine-induced impairment of contextual fear extinction persisted when nicotine administration was ceased and repeated acute nicotine administrations alone did not induce freezing behavior in the absence of context-shock learning. These results clearly demonstrate that high-affinity α4β2 nAChRs are necessary for the effects of acute nicotine on contextual fear extinction.

  12. Histones have high affinity for the glomerular basement membrane. Relevance for immune complex formation in lupus nephritis

    SciTech Connect

    Schmiedeke, T.M.; Stoeckl, F.W.W.; Weber, R.; Sugisaki, Y.; Batsford, S.R.; Vogt, A.

    1989-06-01

    An effort has been made to integrate insights on charge-based interactions in immune complex glomerulonephritis with nuclear antigen involvement in lupus nephritis. Attention was focussed on the histones, a group of highly cationic nuclear constituents, which could be expected to bind to fixed anionic sites present in the glomerular basement membrane (GBM). We demonstrated that all histone subfractions, prepared according to Johns, have a high affinity for GBM and the basement membrane of peritubular capillaries. Tissue uptake of /sup 125/I-labeled histones was measured by injecting 200 micrograms of each fraction into the left kidney via the aorta and measuring organ uptake after 15 min. In glomeruli isolated from the left kidneys, the following quantities of histones were found: f1, 13 micrograms; f2a (f2al + f2a2), 17 micrograms; f2b, 17 micrograms; and f3, 32 micrograms. Kinetic studies of glomerular binding showed that f1 disappeared much more rapidly than f2a. The high affinity of histones (pI between 10.5 and 11.0; mol wt 10,000-22,000) for the GBM correlates well with their ability to form aggregates (mol wt greater than 100,000) for comparison lysozyme (pI 11, mol wt 14,000), which does not aggregate spontaneously bound poorly (0.4 micrograms in isolated glomeruli). The quantity of histones and lysozyme found in the isolated glomeruli paralleled their in vitro affinity for a Heparin-Sepharose column (gradient elution studies). This gel matrix contains the sulfated, highly anionic polysaccharide heparin, which is similar to the negatively charged heparan sulfate present in the GBM. Lysozyme eluted with 0.15 M NaCl, f1 with 1 M NaCl, and f2a, f2b, and f3 could not be fully desorbed even with 2 M NaCl; 6 M guanidine-HCl was necessary.

  13. High-affinity α4β2 nicotinic receptors mediate the impairing effects of acute nicotine on contextual fear extinction.

    PubMed

    Kutlu, Munir Gunes; Holliday, Erica; Gould, Thomas J

    2016-02-01

    Previously, studies from our lab have shown that while acute nicotine administered prior to training and testing enhances contextual fear conditioning, acute nicotine injections prior to extinction sessions impair extinction of contextual fear. Although there is also strong evidence showing that the acute nicotine's enhancing effects on contextual fear conditioning require high-affinity α4β2 nicotinic acetylcholine receptors (nAChRs), it is unknown which nAChR subtypes are involved in the acute nicotine-induced impairment of contextual fear extinction. In this study, we investigated the effects of acute nicotine administration on contextual fear extinction in knock-out (KO) mice lacking α4, β2 or α7 subtypes of nAChRs and their wild-type (WT) littermates. Both KO and WT mice were first trained and tested for contextual fear conditioning and received a daily contextual extinction session for 4 days. Subjects received intraperitoneal injections of nicotine (0.18 mg/kg) or saline 2-4 min prior to each extinction session. Our results showed that the mice that lack α4 and β2 subtypes of nAChRs showed normal contextual fear extinction but not the acute nicotine-induced impairment while the mice that lack the α7 subtype showed both normal contextual extinction and nicotine-induced impairment of contextual extinction. In addition, control experiments showed that acute nicotine-induced impairment of contextual fear extinction persisted when nicotine administration was ceased and repeated acute nicotine administrations alone did not induce freezing behavior in the absence of context-shock learning. These results clearly demonstrate that high-affinity α4β2 nAChRs are necessary for the effects of acute nicotine on contextual fear extinction. PMID:26688111

  14. Substrate specificity and mapping of residues critical for transport in the high-affinity glutathione transporter Hgt1p.

    PubMed

    Zulkifli, Mohammad; Yadav, Shambhu; Thakur, Anil; Singla, Shiffalli; Sharma, Monika; Bachhawat, Anand Kumar

    2016-08-01

    The high-affinity glutathione transporter Hgt1p of Saccharomyces cerevisiae belongs to a relatively new and structurally uncharacterized oligopeptide transporter (OPT) family. To understand the structural features required for interaction with Hgt1p, a quantitative investigation of substrate specificity of Hgt1p was carried out. Hgt1p showed a higher affinity for reduced glutathione (GSH), whereas it transported oxidized glutathione (GSSG) and other glutathione conjugates with lower affinity. To identify the residues of Hgt1p critical for substrate binding and translocation, all amino acid residues of the 13 predicted transmembrane domains (TMDs) have been subjected to mutagenesis. Functional evaluation of these 269 mutants by growth and biochemical assay followed by kinetic analysis of the severely defective mutants including previous mutagenic studies on this transporter have led to the identification of N124 (TMD1), V185 (TMD3), Q222, G225 and Y226 (TMD4), P292 (TMD5), Y374 (TMD6), L429 (TMD7) and F523 and Q526 (TMD9) as critical for substrate binding with at least 3-fold increase in Km upon mutagenesis to alanine. In addition residues Y226 and Y374 appeared to be important for differential substrate specificity. An ab initio model of Hgt1p was built and refined using these mutagenic data that yielded a helical arrangement that includes TMD3, TMD4, TMD5, TMD6, TMD7, TMD9 and TMD13 as pore-lining helices with the functionally important residues in a channel-facing orientation. Taken together the results of this study provides the first mechanistic insights into glutathione transport by a eukaryotic high-affinity glutathione transporter. PMID:27252386

  15. PURIFICATION PROCESS

    DOEpatents

    Wibbles, H.L.; Miller, E.I.

    1958-01-14

    This patent deals with the separation of uranium from molybdenum compounds, and in particular with their separation from ether solutions containing the molybdenum in the form of acids, such as silicomolybdic and phosphomolybdic acids. After the nitric acid leach of pitchblende, the molybdenum values present in the ore are found in the leach solution in the form of complex acids. The uranium bearing solution may be purified of this molybdenum content by comtacting it with activated charcoal. The purification is improved when the acidity of the solution is low ad agitation is also beneficial. The molybdenum may subsequently be recovered from the charcosl ad the charcoal reused.

  16. Water Purification

    NASA Technical Reports Server (NTRS)

    1992-01-01

    Silver ionization water purification technology was originally developed for Apollo spacecraft. It was later used to cleanse swimming pools and has now been applied to industrial cooling towers and process coolers. Sensible Technologies, Inc. has added two other technologies to the system, which occupies only six square feet. It is manufactured in three capacities, and larger models are custom built on request. The system eliminates scale, corrosion, algae, bacteria and debris, and because of the NASA technology, viruses and waterborne bacteria are also destroyed. Applications include a General Motors cooling tower, amusement parks, ice manufacture and a closed-loop process cooling system.

  17. Affinity Chromatography Purification of Cytochrome c Binding Enzymes

    NASA Astrophysics Data System (ADS)

    Azzi, Angelo; Bill, Kurt; Broger, Clemens

    1982-04-01

    An efficient affinity chromatography procedure for the isolation of mitochondrial cytochrome c oxidase and reductase is described. Saccharomyces cerevisiae cytochrome c was used as a ligand, bound to a thiol-Sepharose 4B gel through cysteine-107. In this way, the site of interaction of cytochrome c with cytochrome oxidase and reductase remained unmodified and available for binding to a number of partner enzymes. The procedure is adequate for the purification of all those proteins having in common the property of binding with high affinity to cytochrome c--e.g., cytochrome c oxidase, reductase, and peroxidase, sulfite oxidase, and reaction centers of photosynthetic bacteria.

  18. Protein complexes and functional modules in molecular networks

    NASA Astrophysics Data System (ADS)

    Spirin, Victor; Mirny, Leonid A.

    2003-10-01

    Proteins, nucleic acids, and small molecules form a dense network of molecular interactions in a cell. Molecules are nodes of this network, and the interactions between them are edges. The architecture of molecular networks can reveal important principles of cellular organization and function, similarly to the way that protein structure tells us about the function and organization of a protein. Computational analysis of molecular networks has been primarily concerned with node degree [Wagner, A. & Fell, D. A. (2001) Proc. R. Soc. London Ser. B 268, 1803-1810; Jeong, H., Tombor, B., Albert, R., Oltvai, Z. N. & Barabasi, A. L. (2000) Nature 407, 651-654] or degree correlation [Maslov, S. & Sneppen, K. (2002) Science 296, 910-913], and hence focused on single/two-body properties of these networks. Here, by analyzing the multibody structure of the network of protein-protein interactions, we discovered molecular modules that are densely connected within themselves but sparsely connected with the rest of the network. Comparison with experimental data and functional annotation of genes showed two types of modules: (i) protein complexes (splicing machinery, transcription factors, etc.) and (ii) dynamic functional units (signaling cascades, cell-cycle regulation, etc.). Discovered modules are highly statistically significant, as is evident from comparison with random graphs, and are robust to noise in the data. Our results provide strong support for the network modularity principle introduced by Hartwell et al. [Hartwell, L. H., Hopfield, J. J., Leibler, S. & Murray, A. W. (1999) Nature 402, C47-C52], suggesting that found modules constitute the "building blocks" of molecular networks.

  19. Protein purification using PDZ affinity chromatography.

    PubMed

    Walkup, Ward G; Kennedy, Mary B

    2015-01-01

    PDZ domains function in nature as protein-binding domains within scaffold and membrane-associated proteins. They comprise approximately 90 residues and undergo specific, high-affinity interactions with complementary C-terminal peptide sequences, other PDZ domains, and/or phospholipids. We have previously shown that the specific, strong interactions of PDZ domains with their ligands make them well suited for use in affinity chromatography. This unit provides protocols for the PDZ affinity chromatography procedure that are applicable for the purification of proteins that contain PDZ domains or PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We detail the preparation of affinity resins composed of PDZ domains or PDZ domain peptide ligands coupled to solid supports. These resins can be used to purify proteins containing endogenous or genetically introduced PDZ domains or ligands, eluting the proteins with free PDZ domain peptide ligands. PMID:25829303

  20. Identifying functions of protein complexes based on topology similarity with random forest.

    PubMed

    Li, Zhan-Chao; Lai, Yan-Hua; Chen, Li-Li; Xie, Yun; Dai, Zong; Zou, Xiao-Yong

    2014-03-01

    Elucidating the functions of protein complexes is critical for understanding disease mechanisms, diagnosis and therapy. In this study, based on the concept that protein complexes with similar topology may have similar functions, we firstly model protein complexes as weighted graphs with nodes representing the proteins and edges indicating interaction between proteins. Secondly, we use topology features derived from the graphs to characterize protein complexes based on the graph theory. Finally, we construct a predictor by using random forest and topology features to identify the functions of protein complexes. Effectiveness of the current method is evaluated by identifying the functions of mammalian protein complexes. And then the predictor is also utilized to identify the functions of protein complexes retrieved from human protein-protein interaction networks. We identify some protein complexes with significant roles in the occurrence of tumors, vesicles and retinoblastoma. It is anticipated that the current research has an important impact on pathogenesis and the pharmaceutical industry. The source code of Matlab and the dataset are freely available on request from the authors. PMID:24389559

  1. Identifying dynamic protein complexes based on gene expression profiles and PPI networks.

    PubMed

    Li, Min; Chen, Weijie; Wang, Jianxin; Wu, Fang-Xiang; Pan, Yi

    2014-01-01

    Identification of protein complexes from protein-protein interaction networks has become a key problem for understanding cellular life in postgenomic era. Many computational methods have been proposed for identifying protein complexes. Up to now, the existing computational methods are mostly applied on static PPI networks. However, proteins and their interactions are dynamic in reality. Identifying dynamic protein complexes is more meaningful and challenging. In this paper, a novel algorithm, named DPC, is proposed to identify dynamic protein complexes by integrating PPI data and gene expression profiles. According to Core-Attachment assumption, these proteins which are always active in the molecular cycle are regarded as core proteins. The protein-complex cores are identified from these always active proteins by detecting dense subgraphs. Final protein complexes are extended from the protein-complex cores by adding attachments based on a topological character of "closeness" and dynamic meaning. The protein complexes produced by our algorithm DPC contain two parts: static core expressed in all the molecular cycle and dynamic attachments short-lived. The proposed algorithm DPC was applied on the data of Saccharomyces cerevisiae and the experimental results show that DPC outperforms CMC, MCL, SPICi, HC-PIN, COACH, and Core-Attachment based on the validation of matching with known complexes and hF-measures. PMID:24963481

  2. Docking and scoring protein complexes: CAPRI 3rd Edition.

    PubMed

    Lensink, Marc F; Méndez, Raúl; Wodak, Shoshana J

    2007-12-01

    The performance of methods for predicting protein-protein interactions at the atomic scale is assessed by evaluating blind predictions performed during 2005-2007 as part of Rounds 6-12 of the community-wide experiment on Critical Assessment of PRedicted Interactions (CAPRI). These Rounds also included a new scoring experiment, where a larger set of models contributed by the predictors was made available to groups developing scoring functions. These groups scored the uploaded set and submitted their own best models for assessment. The structures of nine protein complexes including one homodimer were used as targets. These targets represent biologically relevant interactions involved in gene expression, signal transduction, RNA, or protein processing and membrane maintenance. For all the targets except one, predictions started from the experimentally determined structures of the free (unbound) components or from models derived by homology, making it mandatory for docking methods to model the conformational changes that often accompany association. In total, 63 groups and eight automatic servers, a substantial increase from previous years, submitted docking predictions, of which 1994 were evaluated here. Fifteen groups submitted 305 models for five targets in the scoring experiment. Assessment of the predictions reveals that 31 different groups produced models of acceptable and medium accuracy-but only one high accuracy submission-for all the targets, except the homodimer. In the latter, none of the docking procedures reproduced the large conformational adjustment required for correct assembly, underscoring yet again that handling protein flexibility remains a major challenge. In the scoring experiment, a large fraction of the groups attained the set goal of singling out the correct association modes from incorrect solutions in the limited ensembles of contributed models. But in general they seemed unable to identify the best models, indicating that current scoring

  3. Multiple mode of binding of phencyclidines: high affinity association between phencyclidine receptors in rat brain and a monovalent ion-sensitive polypeptide

    SciTech Connect

    Haring, R.; Kloog, Y.; Harshak-Felixbrodt, N.A.; Sokolovsky, M.

    1987-01-30

    Two populations of phencyclidine (PCP) binding sites are shown to exist in the rat brain: a high-affinity monovalent ion-sensitive site (Kd of 10-14 nM for (/sup 3/H)TCP, (/sup 3/H)N-(1-(2-thienyl)cyclohexyl)piperidine), which exists in both the frontal cortex and the hippocampus, and a lower affinity site (Kd of 80-130 nM for (/sup 3/H)TCP) which is found in the hippocampus but not in the frontal cortex. The nature of the interactions between the ion-binding sites and the high affinity PCP receptors depend on both ligand structure (PCP or TCP) and the ion involved (K or Na). The high-affinity sites are associated with an Mr 90,000 polypeptide whose labeling by (/sup 3/H)azido phencyclidine is selectively inhibited by monovalent ions.

  4. Positive allosteric modulation of the GHB high-affinity binding site by the GABAA receptor modulator monastrol and the flavonoid catechin.

    PubMed

    Eghorn, Laura F; Hoestgaard-Jensen, Kirsten; Kongstad, Kenneth T; Bay, Tina; Higgins, David; Frølund, Bente; Wellendorph, Petrine

    2014-10-01

    γ-Hydroxybutyric acid (GHB) is a metabolite of γ-aminobutyric acid (GABA) and a proposed neurotransmitter in the mammalian brain. We recently identified α4βδ GABAA receptors as possible high-affinity GHB targets. GABAA receptors are highly sensitive to allosteric modulation. Thus to investigate whether GHB high-affinity binding sites are also sensitive to allosteric modulation, we screened both known GABAA receptor ligands and a library of natural compounds in the rat cortical membrane GHB specific high-affinity [3H]NCS-382 binding assay. Two hits were identified: Monastrol, a positive allosteric modulator of GABA function at δ-containing GABAA receptors, and the naturally occurring flavonoid catechin. These compounds increased [3H]NCS-382 binding to 185-272% in high micromolar concentrations. Monastrol and (+)-catechin significantly reduced [3H]NCS-382 dissociation rates and induced conformational changes in the binding site, demonstrating a positive allosteric modulation of radioligand binding. Surprisingly, binding of [3H]GHB and the GHB high-affinity site-specific radioligands [125I]BnOPh-GHB and [3H]HOCPCA was either decreased or only weakly increased, indicating that the observed modulation was critically probe-dependent. Both monastrol and (+)-catechin were agonists at recombinant α4β3δ receptors expressed in Xenopus laevis oocytes. When monastrol and GHB were co-applied no changes were seen compared to the individual responses. In summary, we have identified the compounds monastrol and catechin as the first allosteric modulators of GHB high-affinity binding sites. Despite their relatively weak affinity, these compounds may aid in further characterization of the GHB high-affinity sites that are likely to represent certain GABAA receptors.

  5. A high affinity kidney targeting by chitobionic acid-conjugated polysorbitol gene transporter alleviates unilateral ureteral obstruction in rats.

    PubMed

    Islam, Mohammad Ariful; Kim, Sanghwa; Firdous, Jannatul; Lee, Ah-Young; Hong, Seong-Ho; Seo, Min Kyeong; Park, Tae-Eun; Yun, Cheol-Heui; Choi, Yun-Jaie; Chae, Chanhee; Cho, Chong-Su; Cho, Myung-Haing

    2016-09-01

    Aside from kidney transplantation - a procedure which is exceedingly dependent on donor-match and availability leading to excessive costs - there are currently no permanent treatments available which reverse kidney injury and failure. However, kidney-specific targeted gene therapy has outstanding potential to treat kidney-related dysfunction. Herein we report a novel kidney-specific targeted gene delivery system developed through the conjugation of chitobionic acid (CBA) to a polysorbitol gene transporter (PSGT) synthesized from sorbitol diacrylate and low molecular weight polyethylenimine (PEI) carrying hepatocyte growth factor (HGF) gene to alleviate unilateral ureteral obstruction (UUO) in rats. CBA-PSGT performed exceptionally well for targeted delivery of HGF to kidney tissues compared to its non-targeted counterparts (P < 0.001) after systemic tail-vein injection and significantly reduced the UUO symptoms, returning the UUO rats to a normal health status. The kidney-targeted CBA-PSGT-delivered HGF also strikingly reduced various pathologic and molecular markers in vivo such as the level of collagens (type I and II), blood urea nitrogen (BUN), creatinine, and the expressions of ICAM-1, TIMP-1 and α-SMA which play a critical role in obstructive kidney functions. Therefore, CBA-PSGT should be further investigated because of its potential to alleviate UUO and kidney-related diseases using high affinity kidney targeting. PMID:27318934

  6. Rational design, synthesis and biological evaluation of modular fluorogenic substrates with high affinity and selectivity for PTP1B.

    PubMed

    Sanchini, Silvano; Perruccio, Francesca; Piizzi, Grazia

    2014-05-01

    Protein-tyrosine phosphatase 1B (PTP1B) is a key regulatory enzyme in several signal transduction pathways, and its upregulation has been associated with type-2 diabetes, obesity and cancer. Selective determination of the functional significance of PTP1B remains a major challenge because the activity of this crucial enzyme is currently evaluated through the use of fluorescent probes that lack selectivity and are limited to biochemical assays. Here we describe the rational design, synthesis and biological evaluation of new modular PTP1B fluorogenic substrates. The self-immolative 4-hydroxybenzyl alcohol has been used as a key component for the design of phosphotyrosine mimics linked to a latent chromophore, which is released through an enzyme-initiated domino reaction. Preliminary biological investigations showed that, by optimising the stereoelectronic properties and the binding interactions at the enzyme active site, it is possible to achieve substrates with high affinity and promising selectivity. Due to their modular nature, the synthesised fluorogenic probes represent versatile tools; customisation of the different subunits could widen the scope of these probes to a broader range of in vitro assays. Finally, these studies elucidate the critical role played by Asp181 in the PTP1B-catalysed dephosphorylation mechanism: disruption of the native conformation of this key amino acid residue on the WDP loop yields fluorogenic inhibitors, rather than substrates. For this reason, our studies also represent a step forward for the development of improved PTP1B noncovalent inhibitors. PMID:24719298

  7. Synthesis of tetravalent LacNAc-glycoclusters as high-affinity cross-linker against Erythrina cristagalli agglutinin.

    PubMed

    Ogata, Makoto; Chuma, Yasushi; Yasumoto, Yoshinori; Onoda, Takashi; Umemura, Myco; Usui, Taichi; Park, Enoch Y

    2016-01-01

    Four kinds of tetravalent double-headed glycoclusters [(LacNAc)4-DHGs] were designed with linkers of varying lengths consisting of alkanedioic carboxyamido groups (C6, C12, C18 and C24) between two bi-antennary LacNAc-glycosides. These glycoclusters served as high-affinity cross-linking ligands for the LacNAc-binding lectin Erythrina cristagalli agglutinin (ECA). The binding activity and cross-linking between each ligand and ECA were characterized by a hemagglutination inhibition (HI) assay, isothermal titration calorimetry (ITC), a quantitative precipitation assay and dynamic light scattering (DLS). For the precipitation assay and DLS measurement, the synthesized (LacNAc)4-DHGs were found to be capable of binding and precipitating the ECA as multivalent ligands. ITC analysis indicated the binding of (LacNAc)4-DHGs was driven by a favorable enthalpy change. Furthermore, the entropy penalty from binding (LacNAc)4-DHGs clearly decreased in a spacer length-dependent manner. The binding affinities of flexible (LacNAc)4-DHGs (C18 and C24) with long spacers were found to be more favorable than those of the clusters having short spacers (C6 and C12). These results were supported by molecular dynamics simulations with explicit water molecules for the tetravalent glycoclusters with ECA. We concluded that the subtle modification in the epitope-presenting scaffolds exerts the significant effect in the recognition efficiency involved in the LacNAc moieties by ECA.

  8. Processivity and Subcellular Localization of Glycogen Synthase Depend on a Non-catalytic High Affinity Glycogen-binding Site*

    PubMed Central

    Díaz, Adelaida; Martínez-Pons, Carlos; Fita, Ignacio; Ferrer, Juan C.; Guinovart, Joan J.

    2011-01-01

    Glycogen synthase, a central enzyme in glucose metabolism, catalyzes the successive addition of α-1,4-linked glucose residues to the non-reducing end of a growing glycogen molecule. A non-catalytic glycogen-binding site, identified by x-ray crystallography on the surface of the glycogen synthase from the archaeon Pyrococcus abyssi, has been found to be functionally conserved in the eukaryotic enzymes. The disruption of this binding site in both the archaeal and the human muscle glycogen synthases has a large impact when glycogen is the acceptor substrate. Instead, the catalytic efficiency remains essentially unchanged when small oligosaccharides are used as substrates. Mutants of the human muscle enzyme with reduced affinity for glycogen also show an altered intracellular distribution and a marked decrease in their capacity to drive glycogen accumulation in vivo. The presence of a high affinity glycogen-binding site away from the active center explains not only the long-recognized strong binding of glycogen synthase to glycogen but also the processivity and the intracellular localization of the enzyme. These observations demonstrate that the glycogen-binding site is a critical regulatory element responsible for the in vivo catalytic efficiency of GS. PMID:21464127

  9. High-affinity binding of short peptides to major histocompatibility complex class II molecules by anchor combinations.

    PubMed Central

    Hammer, J; Belunis, C; Bolin, D; Papadopoulos, J; Walsky, R; Higelin, J; Danho, W; Sinigaglia, F; Nagy, Z A

    1994-01-01

    We have previously identified four anchor positions in HLA-DRB1*0101-binding peptides, and three anchors involved in peptide binding to DRB1*0401 and DRB1*1101 molecules, by screening of an M13 peptide display library (approximately 20 million independent nonapeptides) for DR-binding activity. In this study, high stringency screening of the M13 library for DRB1*0401 binding has resulted in identification of three further anchor positions. Taken together, a peptide-binding motif has been obtained, in which six of seven positions show enrichment of certain residues. We have demonstrated an additive effect of anchors in two different ways: (i) the addition of more anchors is shown to compensate for progressive truncation of designer peptides; (ii) the incorporation of an increasing number of anchors into 6- or 7-residue-long designer peptides is shown to result in a gradual increase of binding affinity to the level of 13-residue-long high-affinity epitopes. The anchor at relative position 1 seems to be obligatory, in that its substitution abrogates binding completely, whereas the elimination of other anchors results only in partial loss of binding affinity. The spacing between anchors is critical, since their effect is lost by shifting them one position toward the N or C terminus. The information born out of this study has been successfully used to identify DR-binding sequences from natural proteins. PMID:8183931

  10. Elongated fibrillar structure of a streptococcal adhesin assembled by the high-affinity association of [alpha]- and PPII-helices

    SciTech Connect

    Larson, Matthew R.; Rajashankar, Kanagalaghatta R.; Patel, Manisha H.; Robinette, Rebekah A.; Crowley, Paula J.; Michalek, Suzanne; Brady, L. Jeannine; Deivanayagam, Champion

    2010-08-18

    Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein adhesin that interacts with salivary components within the salivary pellicle. AgI/II contributes to virulence and has been studied as an immunological and structural target, but a fundamental understanding of its underlying architecture has been lacking. Here we report a high-resolution (1.8 {angstrom}) crystal structure of the A{sub 3}VP{sub 1} fragment of S. mutans AgI/II that demonstrates a unique fibrillar form (155 {angstrom}) through the interaction of two noncontiguous regions in the primary sequence. The A{sub 3} repeat of the alanine-rich domain adopts an extended {alpha}-helix that intertwines with the P{sub 1} repeat polyproline type II (PPII) helix to form a highly extended stalk-like structure heretofore unseen in prokaryotic or eukaryotic protein structures. Velocity sedimentation studies indicate that full-length AgI/II that contains three A/P repeats extends over 50 nanometers in length. Isothermal titration calorimetry revealed that the high-affinity association between the A{sub 3} and P{sub 1} helices is enthalpically driven. Two distinct binding sites on AgI/II to the host receptor salivary agglutinin (SAG) were identified by surface plasmon resonance (SPR). The current crystal structure reveals that AgI/II family proteins are extended fibrillar structures with the number of alanine- and proline-rich repeats determining their length.

  11. ZK91587: a novel synthetic antimineralocorticoid displays high affinity for corticosterone (type I) receptors in the rat hippocampus

    SciTech Connect

    Sutanto, W.; de Kloet, E.R.

    1988-01-01

    In vitro cytosol binding assays have shown the properties of binding of a novel steroid, ZK91587 (15..beta.., 16..beta..b-methylene-mexrenone) in the brain of rats. Scatchard and Woolf analyses of the binding data reveal the binding of (/sup 3/H) ZK91587 to the total hippocampal coritcosteroid receptor sites with high affinity, and low capacity. When 100-fold excess RU28362 was included simultaneously with (/sup 3/H) ZK91587, the labelled steroid binds with the same affinity and capacity. Relative binding affinities (RBA) of various steroids for the Type I or Type II corticosteroid receptor in these animals are: Type I: ZK91587 = corticosterone (B) > cortisol (F); Type II: B > F >>> ZK91587. In the binding kinetic study, ZK91587 has a high association rate of binding in the rat. The steroid dissociates following a one slope pattern, indicating, the present data demonstrate that in the rat hippocampus, ZK91587 binds specifically to the Type I (corticosterone-preferring/mineralocorticoid-like receptor.

  12. Combination of isothermal titration calorimetry and time-resolved luminescence for high affinity antibody-ligand interaction thermodynamics and kinetics.

    PubMed

    Aweda, Tolulope A; Meares, Claude F

    2012-02-01

    For experiments using synthetic ligands as probes for biological experiments, it is useful to determine the specificity and affinity of the ligands for their receptors. As ligands with higher affinities are developed (K(A)>10(8)M(-1); K(D)<10(-8)M), a new challenge arises: to measure these values accurately. Isothermal titration calorimetry measures heat produced or consumed during ligand binding, and also provides the equilibrium binding constant. However, as normally practiced, its range is limited. Displacement titration, where a competing weaker ligand is used to lower the apparent affinity of the stronger ligand, can be used to determine the binding affinity as well as the complete thermodynamic data for ligand-antibody complexes with very high affinity. These equilibrium data have been combined with kinetic measurements to yield the rate constants as well. We describe this methodology, using as an example antibody 2D12.5, which captures yttrium S-2-(4-aminobenzyl)-1, 4, 7, 10-tetraazacyclododecanetetraacetate.

  13. The high-affinity receptor for IgG, FcγRI, of humans and non-human primates.

    PubMed

    Chenoweth, Alicia M; Trist, Halina M; Tan, Peck-Szee; Wines, Bruce D; Hogarth, P Mark

    2015-11-01

    Non-human primate (NHP) models, especially involving macaques, are considered important models of human immunity and have been essential in preclinical testing for vaccines and therapeutics. Despite this, much less characterization of macaque Fc receptors has occurred compared to humans or mice. Much of the characterization of macaque Fc receptors so far has focused on the low-affinity Fc receptors, particularly FcγRIIIa. From these studies, it is clear that there are distinct differences between the human and macaque low-affinity receptors and their interaction with human IgG. Relatively little work has been performed on the high-affinity IgG receptor, FcγRI, especially in NHPs. This review will focus on what is currently known of how FcγRI interacts with IgG, from mutation studies and recent crystallographic studies of human FcγRI, and how amino acid sequence differences in the macaque FcγRI may affect this interaction. Additionally, this review will look at the functional consequences of differences in the amino acid sequences between humans and macaques.

  14. Genetically encoded photocrosslinkers locate the high-affinity binding site of antidepressant drugs in the human serotonin transporter.

    PubMed

    Rannversson, Hafsteinn; Andersen, Jacob; Sørensen, Lena; Bang-Andersen, Benny; Park, Minyoung; Huber, Thomas; Sakmar, Thomas P; Strømgaard, Kristian

    2016-01-01

    Despite the well-established role of the human serotonin transporter (hSERT) in the treatment of depression, the molecular details of antidepressant drug binding are still not fully understood. Here we utilize amber codon suppression in a membrane-bound transporter protein to encode photocrosslinking unnatural amino acids (UAAs) into 75 different positions in hSERT. UAAs are incorporated with high specificity, and functionally active transporters have similar transport properties and pharmacological profiles compared with wild-type transporters. We employ ultraviolet-induced crosslinking with p-azido-L-phenylalanine (azF) at selected positions in hSERT to map the binding site of imipramine, a prototypical tricyclic antidepressant, and vortioxetine, a novel multimodal antidepressant. We find that the two antidepressants crosslink with azF incorporated at different positions within the central substrate-binding site of hSERT, while no crosslinking is observed at the vestibular-binding site. Taken together, our data provide direct evidence for defining the high-affinity antidepressant binding site in hSERT. PMID:27089947

  15. The CBL-Interacting Protein Kinase CIPK23 Regulates HAK5-Mediated High-Affinity K+ Uptake in Arabidopsis Roots.

    PubMed

    Ragel, Paula; Ródenas, Reyes; García-Martín, Elena; Andrés, Zaida; Villalta, Irene; Nieves-Cordones, Manuel; Rivero, Rosa M; Martínez, Vicente; Pardo, Jose M; Quintero, Francisco J; Rubio, Francisco

    2015-12-01

    Plant growth and development requires efficient acquisition of essential elements. Potassium (K(+)) is an important macronutrient present in the soil solution at a wide range of concentrations. Regulation of the K(+) uptake systems in the roots is essential to secure K(+) supply. It has been shown in Arabidopsis (Arabidopsis thaliana) that when the external K(+) concentration is very low (<10 µm), K(+) nutrition depends exclusively on the high-affinity K(+) transporter5 (HAK5). Low-K(+)-induced transcriptional activation of the gene encoding HAK5 has been previously reported. Here, we show the posttranscriptional regulation of HAK5 transport activity by phosphorylation. Expression in a heterologous system showed that the Ca(2+) sensors calcineurin B-like (CBL1), CBL8, CBL9, and CBL10, together with CBL-interacting protein kinase23 (CIPK23), activated HAK5 in vivo. This activation produced an increase in the affinity and the Vmax of K(+) transport. In vitro experiments show that the N terminus of HAK5 is phosphorylated by CIPK23. This supports the idea that phosphorylation of HAK5 induces a conformational change that increases its affinity for K(+). Experiments of K(+) (Rb(+)) uptake and growth measurements in low-K(+) medium with Arabidopsis single mutants hak5, akt1, and cipk23, double mutants hak5 akt1, hak5 cipk23, and akt1 cipk23, and the triple mutant hak5 akt1 cipk23 confirmed the regulatory role of CIPK23 in planta.

  16. High affinity RGD-binding sites at the plasma membrane of Arabidopsis thaliana links the cell wall.

    PubMed

    Canut, H; Carrasco, A; Galaud, J P; Cassan, C; Bouyssou, H; Vita, N; Ferrara, P; Pont-Lezica, R

    1998-10-01

    The heptapeptide Tyr-Gly-Arg-Gly-Asp-Ser-Pro containing the sequence Arg-Gly-Asp (RGD--the essential structure recognised by animal cells in substrate adhesion molecules) was tested on epidermal cells of onion and cultured cells of Arabidopsis upon plasmolysis. Dramatic changes were observed on both types of cells following treatment: on onion cells, Hechtian strands linking the cell wall to the membrane were lost, while Arabidopsis cells changed from concave to convex plasmolysis. A control heptapeptide Tyr-Gly-Asp-Gly-Arg-Ser-Pro had no effect on the shape of plasmolysed cells. Protoplasts isolated from Arabidopsis cells agglutinate in the presence of ProNectinF, a genetically engineered protein of 72 kDa containing 13 RGD sequences: several protoplasts may adhere to a single molecule of ProNectinF. The addition of the RGD-heptapeptide disrupted the adhesion between the protoplasts. Purified plasma membrane from Arabidopsis cells exhibits specific binding sites for the iodinated RGD-heptapeptide. The binding is saturable, reversible, and two types of high affinity sites (Kd1 approximately 1 nM, and Kd2 approximately 40 nM) can be discerned. Competitive inhibition by several structurally related peptides and proteins noted the specific requirement for the RGD sequence. Thus, the RGD-binding activity of Arabidopsis fulfils the adhesion features of integrins, i.e. peptide specificity, subcellular location, and involvement in plasma membrane-cell wall attachments.

  17. Peptides in headlock – a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy

    PubMed Central

    Braun, Michael B.; Traenkle, Bjoern; Koch, Philipp A.; Emele, Felix; Weiss, Frederik; Poetz, Oliver; Stehle, Thilo; Rothbauer, Ulrich

    2016-01-01

    Nanobodies are highly valuable tools for numerous bioanalytical and biotechnical applications. Here, we report the characterization of a nanobody that binds a short peptide epitope with extraordinary affinity. Structural analysis reveals an unusual binding mode where the extended peptide becomes part of a β-sheet structure in the nanobody. This interaction relies on sequence-independent backbone interactions augmented by a small number of specificity-determining side chain contacts. Once bound, the peptide is fastened by two nanobody side chains that clamp it in a headlock fashion. Exploiting this unusual binding mode, we generated a novel nanobody-derived capture and detection system. Matrix-coupled nanobody enables the fast and efficient isolation of epitope-tagged proteins from prokaryotic and eukaryotic expression systems. Additionally, the fluorescently labeled nanobody visualizes subcellular structures in different cellular compartments. The high-affinity-binding and modifiable peptide tag of this system renders it a versatile and robust tool to combine biochemical analysis with microscopic studies. PMID:26791954

  18. Regulation of the high-affinity copper transporter (hCtr1) expression by cisplatin and heavy metals.

    PubMed

    Liang, Zheng Dong; Long, Yan; Chen, Helen H W; Savaraj, Niramol; Kuo, Macus Tien

    2014-01-01

    Platinum-based antitumor agents have been the mainstay in cancer chemotherapy for many human malignancies. Drug resistance is an important obstacle to achieving the maximal therapeutic efficacy of these drugs. Understanding how platinum drugs enter cells is of great importance in improving therapeutic efficacy. It has been demonstrated that human high-affinity copper transporter 1 (hCtr1) is involved in transporting cisplatin into cells to elicit cytotoxic effects, although other mechanisms may exist. In this communication, we demonstrate that cisplatin transcriptionally induces the expression of hCtr1 in time- and concentration-dependent manners. Cisplatin functions as a competitor for hCtr1-mediated copper transport, resulting in reduced cellular copper levels and leading to upregulated expression of Sp1, which is a positive regulator for hCtr1 expression. Thus, regulation of hCtr1 expression by cisplatin is an integral part of the copper homeostasis regulation system. We also demonstrate that Ag(I) and Zn(II), which are known to suppress hCtr1-mediated copper transport, can also induce hCtr1/Sp1 expression. In contrast, Cd(II), another inhibitor of copper transport, downregulates hCtr1 expression by suppressing Sp1 expression. Collectively, our results demonstrate diverse mechanisms of regulating copper metabolism by these heavy metals.

  19. Positive modulation of synaptic and extrasynaptic GABAA receptors by an antagonist of the high affinity benzodiazepine binding site.

    PubMed

    Middendorp, Simon J; Maldifassi, Maria C; Baur, Roland; Sigel, Erwin

    2015-08-01

    GABAA receptors are the major inhibitory neurotransmitter receptors in the brain and are the target for many clinically important drugs such as the benzodiazepines. Benzodiazepines act at the high-affinity binding site at the α+/γ- subunit interface. Previously, an additional low affinity binding site for diazepam located in the transmembrane (TM) domain has been described. The compound SJM-3 was recently identified in a prospective screening of ligands for the benzodiazepine binding site and investigated for its site of action. We determined the binding properties of SJM-3 at GABAA receptors recombinantly expressed in HEK-cells using radioactive ligand binding assays. Impact on function was assessed in Xenopus laevis oocytes with electrophysiological experiments using the two-electrode voltage clamp method. SJM-3 was shown to act as an antagonist at the α+/γ- site. At the same time it strongly potentiated GABA currents via the binding site for diazepam in the transmembrane domain. Mutation of a residue in M2 of the α subunit strongly reduced receptor modulation by SJM-3 and a homologous mutation in the β subunit abolished potentiation. SJM-3 acts as a more efficient modulator than diazepam at the site in the trans-membrane domain. In contrast to low concentrations of benzodiazepines, SJM-3 modulates both synaptic and extrasynaptic receptors. A detailed exploration of the membrane site may provide the basis for the design and identification of subtype-selective modulatory drugs.

  20. Genetically encoded photocrosslinkers locate the high-affinity binding site of antidepressant drugs in the human serotonin transporter

    PubMed Central

    Rannversson, Hafsteinn; Andersen, Jacob; Sørensen, Lena; Bang-Andersen, Benny; Park, Minyoung; Huber, Thomas; Sakmar, Thomas P.; Strømgaard, Kristian

    2016-01-01

    Despite the well-established role of the human serotonin transporter (hSERT) in the treatment of depression, the molecular details of antidepressant drug binding are still not fully understood. Here we utilize amber codon suppression in a membrane-bound transporter protein to encode photocrosslinking unnatural amino acids (UAAs) into 75 different positions in hSERT. UAAs are incorporated with high specificity, and functionally active transporters have similar transport properties and pharmacological profiles compared with wild-type transporters. We employ ultraviolet-induced crosslinking with p-azido-L-phenylalanine (azF) at selected positions in hSERT to map the binding site of imipramine, a prototypical tricyclic antidepressant, and vortioxetine, a novel multimodal antidepressant. We find that the two antidepressants crosslink with azF incorporated at different positions within the central substrate-binding site of hSERT, while no crosslinking is observed at the vestibular-binding site. Taken together, our data provide direct evidence for defining the high-affinity antidepressant binding site in hSERT. PMID:27089947

  1. Shark Attack: high affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation.

    PubMed

    Zielonka, Stefan; Weber, Niklas; Becker, Stefan; Doerner, Achim; Christmann, Andreas; Christmann, Christine; Uth, Christina; Fritz, Janine; Schäfer, Elena; Steinmann, Björn; Empting, Martin; Ockelmann, Pia; Lierz, Michael; Kolmar, Harald

    2014-12-10

    A novel method for stepwise in vitro affinity maturation of antigen-specific shark vNAR domains is described that exclusively relies on semi-synthetic repertoires derived from non-immunized sharks. Target-specific molecules were selected from a CDR3-randomized bamboo shark (Chiloscyllium plagiosum) vNAR library using yeast surface display as platform technology. Various antigen-binding vNAR domains were easily isolated by screening against several therapeutically relevant antigens, including the epithelial cell adhesion molecule (EpCAM), the Ephrin type-A receptor 2 (EphA2), and the human serine protease HTRA1. Affinity maturation was demonstrated for EpCAM and HTRA1 by diversifying CDR1 of target-enriched populations which allowed for the rapid selection of nanomolar binders. EpCAM-specific vNAR molecules were produced as soluble proteins and more extensively characterized via thermal shift assays and biolayer interferometry. Essentially, we demonstrate that high-affinity binders can be generated in vitro without largely compromising the desirable high thermostability of the vNAR scaffold.

  2. Whole-Virion Influenza Vaccine Recalls an Early Burst of High-Affinity Memory B Cell Response through TLR Signaling.

    PubMed

    Onodera, Taishi; Hosono, Akira; Odagiri, Takato; Tashiro, Masato; Kaminogawa, Shuichi; Okuno, Yoshinobu; Kurosaki, Tomohiro; Ato, Manabu; Kobayashi, Kazuo; Takahashi, Yoshimasa

    2016-05-15

    Inactivated influenza vaccines have two formulations, whole- and split-virion types; however, how differential formulations impact their booster effects remain unknown. In this study, we demonstrate that whole-virion vaccines recall two waves of Ab responses, early T cell-independent (TI) and late T cell-dependent responses, whereas split-virion vaccines elicit the late T cell-dependent response only. Notably, higher-affinity Abs with improved neutralizing activity are provided from the early TI response, which emphasizes the important contribution of the formulation-dependent response in the protective immunity. Moreover, we show that the early TI response completely requires B cell-intrinsic TLR7 signaling, which can be delivered through viral RNAs within whole-virion vaccine. Thus, our results indicate that TLR agonists in whole-virion type improve recall Ab responses by directly targeting memory B cells, a finding with important implications for vaccine strategies aimed at the prompt recall of high-affinity neutralizing Abs. PMID:27053762

  3. Synthesis of tetravalent LacNAc-glycoclusters as high-affinity cross-linker against Erythrina cristagalli agglutinin.

    PubMed

    Ogata, Makoto; Chuma, Yasushi; Yasumoto, Yoshinori; Onoda, Takashi; Umemura, Myco; Usui, Taichi; Park, Enoch Y

    2016-01-01

    Four kinds of tetravalent double-headed glycoclusters [(LacNAc)4-DHGs] were designed with linkers of varying lengths consisting of alkanedioic carboxyamido groups (C6, C12, C18 and C24) between two bi-antennary LacNAc-glycosides. These glycoclusters served as high-affinity cross-linking ligands for the LacNAc-binding lectin Erythrina cristagalli agglutinin (ECA). The binding activity and cross-linking between each ligand and ECA were characterized by a hemagglutination inhibition (HI) assay, isothermal titration calorimetry (ITC), a quantitative precipitation assay and dynamic light scattering (DLS). For the precipitation assay and DLS measurement, the synthesized (LacNAc)4-DHGs were found to be capable of binding and precipitating the ECA as multivalent ligands. ITC analysis indicated the binding of (LacNAc)4-DHGs was driven by a favorable enthalpy change. Furthermore, the entropy penalty from binding (LacNAc)4-DHGs clearly decreased in a spacer length-dependent manner. The binding affinities of flexible (LacNAc)4-DHGs (C18 and C24) with long spacers were found to be more favorable than those of the clusters having short spacers (C6 and C12). These results were supported by molecular dynamics simulations with explicit water molecules for the tetravalent glycoclusters with ECA. We concluded that the subtle modification in the epitope-presenting scaffolds exerts the significant effect in the recognition efficiency involved in the LacNAc moieties by ECA. PMID:26672510

  4. The CBL-Interacting Protein Kinase CIPK23 Regulates HAK5-Mediated High-Affinity K+ Uptake in Arabidopsis Roots.

    PubMed

    Ragel, Paula; Ródenas, Reyes; García-Martín, Elena; Andrés, Zaida; Villalta, Irene; Nieves-Cordones, Manuel; Rivero, Rosa M; Martínez, Vicente; Pardo, Jose M; Quintero, Francisco J; Rubio, Francisco

    2015-12-01

    Plant growth and development requires efficient acquisition of essential elements. Potassium (K(+)) is an important macronutrient present in the soil solution at a wide range of concentrations. Regulation of the K(+) uptake systems in the roots is essential to secure K(+) supply. It has been shown in Arabidopsis (Arabidopsis thaliana) that when the external K(+) concentration is very low (<10 µm), K(+) nutrition depends exclusively on the high-affinity K(+) transporter5 (HAK5). Low-K(+)-induced transcriptional activation of the gene encoding HAK5 has been previously reported. Here, we show the posttranscriptional regulation of HAK5 transport activity by phosphorylation. Expression in a heterologous system showed that the Ca(2+) sensors calcineurin B-like (CBL1), CBL8, CBL9, and CBL10, together with CBL-interacting protein kinase23 (CIPK23), activated HAK5 in vivo. This activation produced an increase in the affinity and the Vmax of K(+) transport. In vitro experiments show that the N terminus of HAK5 is phosphorylated by CIPK23. This supports the idea that phosphorylation of HAK5 induces a conformational change that increases its affinity for K(+). Experiments of K(+) (Rb(+)) uptake and growth measurements in low-K(+) medium with Arabidopsis single mutants hak5, akt1, and cipk23, double mutants hak5 akt1, hak5 cipk23, and akt1 cipk23, and the triple mutant hak5 akt1 cipk23 confirmed the regulatory role of CIPK23 in planta. PMID:26474642

  5. Ectomycorrhiza-mediated repression of the high-affinity ammonium importer gene AmAMT2 in Amanita muscaria.

    PubMed

    Willmann, Anita; Weiss, Michael; Nehls, Uwe

    2007-02-01

    A main function of ectomycorrhizas, a symbiosis between certain soil fungi and fine roots of woody plants, is the exchange of plant-derived carbohydrates for fungus-derived nutrients. As it is required in large amounts, nitrogen is of special interest. A gene (AmAMT2) coding for a putative fungal ammonium importer was identified in an EST project of functional Amanita muscaria/poplar ectomycorrhizas. Heterologous expression of the entire AmAMT2 coding region in yeast revealed the corresponding protein to be a high-affinity ammonium importer. In axenically grown Amanita hyphae AmAMT2 expression was strongly repressed by nitrogen, independent of whether the offered nitrogen source was transported by AmAMT2 or not. In functional ectomycorrhizas the AmAMT2 transcript level was further decreased in both hyphal networks (sheath and Hartig net), while extraradical hyphae revealed strong gene expression. Together our data suggest that (1) AmAMT2 expression is regulated by the endogenous nitrogen content of hyphae and (2) fungal hyphae in ectomycorrhizas are well supported with nitrogen even when the extraradical mycelium is nitrogen limited. As a consequence of AmAMT2 repression in mycorrhizas, ammonium can be suggested as a potential nitrogen source delivered by fungal hyphae in symbiosis.

  6. The high-affinity poplar ammonium importer PttAMT1.2 and its role in ectomycorrhizal symbiosis.

    PubMed

    Selle, Anita; Willmann, Martin; Grunze, Nina; Gessler, Arthur; Weiss, Michael; Nehls, Uwe

    2005-12-01

    One way to elucidate whether ammonium could act as a nitrogen (N) source delivered by the fungus in ectomycorrhizal symbiosis is to investigate plant ammonium importers. Expression analysis of a high-affinity ammonium importer from Populus tremulax tremuloides (PttAMT1.2) and of known members of the AMT1 gene family from Populus trichocarpa was performed. In addition, PttAMT1.2 function was studied in detail by heterologous expression in yeast. PttAMT1.2 expression proved to be root-specific, affected by N nutrition, and strongly increased in a N-independent manner upon ectomycorrhiza formation. The corresponding protein had a K(M) value for ammonium of c. 52 microm. From the seven members of the AMT1 gene family, one gene was exclusively expressed in roots while four genes were detectable in all poplar organs but with varying degrees of expression. Ectomycorrhiza formation resulted in a strong upregulation of three of these genes. Our results indicate an increased ammonium uptake capacity of mycorrhized poplar roots and suggest, together with the expression of putative ammonium exporter genes in the ectomycorrhizal fungus Amanita muscaria, that ammonium could be a major N source delivered from the fungus towards the plant in symbiosis.

  7. New High Affinity Monoclonal Antibodies Recognize Non-Overlapping Epitopes On Mesothelin For Monitoring And Treating Mesothelioma

    PubMed Central

    Zhang, Yi-Fan; Phung, Yen; Gao, Wei; Kawa, Seiji; Hassan, Raffit; Pastan, Ira; Ho, Mitchell

    2015-01-01

    Mesothelin is an emerging cell surface target in mesothelioma and other solid tumors. Most antibody drug candidates recognize highly immunogenic Region I (296–390) on mesothelin. Here, we report a group of high-affinity non-Region I rabbit monoclonal antibodies. These antibodies do not compete for mesothelin binding with the immunotoxin SS1P that binds Region I of mesothelin. One pair of antibodies (YP218 and YP223) is suitable to detect soluble mesothelin in a sandwich ELISA with high sensitivity. The new assay can also be used to measure serum mesothelin concentration in mesothelioma patients, indicating its potential use for monitoring patients treated with current antibody therapies targeting Region I. The antibodies are highly specific and sensitive in immunostaining of mesothelioma. To explore their use in tumor therapy, we have generated the immunotoxins based on the Fv of these antibodies. One immunotoxin (YP218 Fv-PE38) exhibits potent anti-tumor cytotoxicity towards primary mesothelioma cell lines in vitro and an NCI-H226 xenograft tumor in mice. Furthermore, we have engineered a humanized YP218 Fv that retains full binding affinity for mesothelin-expressing cancer cells. In conclusion, with their unique binding properties, these antibodies may be promising candidates for monitoring and treating mesothelioma and other mesothelin-expressing cancers. PMID:25996440

  8. ZrFsy1, a High-Affinity Fructose/H+ Symporter from Fructophilic Yeast Zygosaccharomyces rouxii

    PubMed Central

    Leandro, Maria José; Sychrová, Hana; Prista, Catarina; Loureiro-Dias, Maria C.

    2013-01-01

    Zygosaccharomyces rouxii is a fructophilic yeast than can grow at very high sugar concentrations. We have identified an ORF encoding a putative fructose/H+ symporter in the Z. rouxii CBS 732 genome database. Heterologous expression of this ORF in a S. cerevisiae strain lacking its own hexose transporters (hxt-null) and subsequent kinetic characterization of its sugar transport activity showed it is a high-affinity low-capacity fructose/H+ symporter, with Km 0.45±0.07 mM and Vmax 0.57±0.02 mmol h−1 (gdw) −1. We named it ZrFsy1. This protein also weakly transports xylitol and sorbose, but not glucose or other hexoses. The expression of ZrFSY1 in Z. rouxii is higher when the cells are cultivated at extremely low fructose concentrations (<0.2%) and on non-fermentable carbon sources such as mannitol and xylitol, where the cells have a prolonged lag phase, longer duplication times and change their microscopic morphology. A clear phenotype was determined for the first time for the deletion of a fructose/H+ symporter in the genome where it occurs naturally. The effect of the deletion of ZrFSY1 in Z. rouxii cells is only evident when the cells are cultivated at very low fructose concentrations, when the ZrFsy1 fructose symporter is the main active fructose transporter system. PMID:23844167

  9. Surfactant protein A binds TGF-β1 with high affinity and stimulates the TGF-β pathway.

    PubMed

    Willems, Coen H M P; Zimmermann, Luc J I; Kloosterboer, Nico; Kramer, Boris W; van Iwaarden, J Freek

    2014-02-01

    We were able to demonstrate reversible, specific and high-affinity binding of radioactively-labelled TGF-β1 ((125)I-TGF-β1) to immobilized surfactant protein A (SP-A), with an apparent dissociation constant of 53 picomolar at ∼21. Addition of a 200-fold molar excess of the latency associated peptide (LAP) prevented and dissociated the binding of (125)I-TGF-β1 to SP-A, whereas latent TGF-β1 had no effect. Using a bioassay for TGF-β1 activity--a luciferase reporter assay--we were able to show that SP-A in the presence of TGF-β1 stimulated the TGF-β1 pathway, whereas SP-A alone had no effect. Studies with structural analogues of the distinct SP-A tail domain and head domain indicated that stimulatory activity of SP-A resided in the head domain. No activation of latent TGF-β1 by SP-A was observed. In addition, we observed that SP-A inhibited TGF-β1 inactivation by LAP. These results indicate that SP-A may have a regulatory role in the TGF-β1-mediated processes in the lung. PMID:23685990

  10. High-affinity FRβ-specific CAR T cells eradicate AML and normal myeloid lineage without HSC toxicity.

    PubMed

    Lynn, R C; Feng, Y; Schutsky, K; Poussin, M; Kalota, A; Dimitrov, D S; Powell, D J

    2016-06-01

    Acute myeloid leukemia (AML) is an aggressive malignancy, and development of new treatments to prolong remissions is warranted. Chimeric antigen receptor (CAR) T-cell therapies appear promising but on-target, off-tumor recognition of antigen in healthy tissues remains a concern. Here we isolated a high-affinity (HA) folate receptor beta (FRβ)-specific single-chain variable fragment (2.48 nm KD) for optimization of FRβ-redirected CAR T-cell therapy for AML. T cells stably expressing the HA-FRβ CAR exhibited greatly enhanced antitumor activity against FRβ(+) AML in vitro and in vivo compared with a low-affinity FRβ CAR (54.3 nm KD). Using the HA-FRβ immunoglobulin G, FRβ expression was detectable in myeloid-lineage hematopoietic cells; however, expression in CD34(+) hematopoietic stem cells (HSCs) was nearly undetectable. Accordingly, HA-FRβ CAR T cells lysed mature CD14(+) monocytes, while HSC colony formation was unaffected. Because of the potential for elimination of mature myeloid lineage, mRNA CAR electroporation for transient CAR expression was evaluated. mRNA-electroporated HA-FRβ CAR T cells retained effective antitumor activity in vitro and in vivo. Together, our results highlight the importance of antibody affinity in target protein detection and CAR development and suggest that transient delivery of potent HA-FRβ CAR T cells is highly effective against AML and reduces the risk for long-term myeloid toxicity.

  11. Reconstitution of interactions between tyrosine kinases and the high affinity IgE receptor which are controlled by receptor clustering.

    PubMed Central

    Scharenberg, A M; Lin, S; Cuenod, B; Yamamura, H; Kinet, J P

    1995-01-01

    High affinity IgE receptor (Fc epsilon RI) signaling after contact with antigen occurs in response to receptor clustering. This paper describes methodology, based on vaccinia virus driven protein expression, for probing signaling pathways and its application to Fc epsilon RI interactions with the lyn and syk tyrosine kinases. Reconstitution of the complete tetrameric Fc epsilon RI receptor, lyn and syk in a non-hematopoietic 'null' cell line is sufficient to reconstruct clustering-controlled receptor tyrosine phosphorylation and activation of syk, without apparent requirement for hematopoietic specific phosphatases. The src family kinase lyn phosphorylates Fc epsilon RI in response to receptor clustering, resulting in syk binding to the phosphorylated Fc epsilon RI. Lyn also participates in the tyrosine phosphorylation and activation of syk in a manner which is dependent on phosphorylated Fc epsilon RI. Using overexpression of active and dominant negative syk proteins in a mast cell line which naturally expresses Fc epsilon RI, we corroborate syk's role downstream of receptor phosphorylation, and demonstrate that syk SH2 domains protect receptor ITAMs from ongoing dephosphorylation. Based on these results, we propose that receptor clustering controls lyn-mediated Fc epsilon RI tyrosine phosphorylation by shifting a balance between phosphorylation and dephosphorylation towards accumulation of tyrosine phosphorylated Fc epsilon RI. Fc epsilon RI tyrosine phosphorylation functions to bring syk into a microenvironment where it becomes tyrosine phosphorylated and activated, thereby allowing clustering to indirectly control syk activity. Images PMID:7628439

  12. Whole-Virion Influenza Vaccine Recalls an Early Burst of High-Affinity Memory B Cell Response through TLR Signaling.

    PubMed

    Onodera, Taishi; Hosono, Akira; Odagiri, Takato; Tashiro, Masato; Kaminogawa, Shuichi; Okuno, Yoshinobu; Kurosaki, Tomohiro; Ato, Manabu; Kobayashi, Kazuo; Takahashi, Yoshimasa

    2016-05-15

    Inactivated influenza vaccines have two formulations, whole- and split-virion types; however, how differential formulations impact their booster effects remain unknown. In this study, we demonstrate that whole-virion vaccines recall two waves of Ab responses, early T cell-independent (TI) and late T cell-dependent responses, whereas split-virion vaccines elicit the late T cell-dependent response only. Notably, higher-affinity Abs with improved neutralizing activity are provided from the early TI response, which emphasizes the important contribution of the formulation-dependent response in the protective immunity. Moreover, we show that the early TI response completely requires B cell-intrinsic TLR7 signaling, which can be delivered through viral RNAs within whole-virion vaccine. Thus, our results indicate that TLR agonists in whole-virion type improve recall Ab responses by directly targeting memory B cells, a finding with important implications for vaccine strategies aimed at the prompt recall of high-affinity neutralizing Abs.

  13. High affinity RGD-binding sites at the plasma membrane of Arabidopsis thaliana links the cell wall.

    PubMed

    Canut, H; Carrasco, A; Galaud, J P; Cassan, C; Bouyssou, H; Vita, N; Ferrara, P; Pont-Lezica, R

    1998-10-01

    The heptapeptide Tyr-Gly-Arg-Gly-Asp-Ser-Pro containing the sequence Arg-Gly-Asp (RGD--the essential structure recognised by animal cells in substrate adhesion molecules) was tested on epidermal cells of onion and cultured cells of Arabidopsis upon plasmolysis. Dramatic changes were observed on both types of cells following treatment: on onion cells, Hechtian strands linking the cell wall to the membrane were lost, while Arabidopsis cells changed from concave to convex plasmolysis. A control heptapeptide Tyr-Gly-Asp-Gly-Arg-Ser-Pro had no effect on the shape of plasmolysed cells. Protoplasts isolated from Arabidopsis cells agglutinate in the presence of ProNectinF, a genetically engineered protein of 72 kDa containing 13 RGD sequences: several protoplasts may adhere to a single molecule of ProNectinF. The addition of the RGD-heptapeptide disrupted the adhesion between the protoplasts. Purified plasma membrane from Arabidopsis cells exhibits specific binding sites for the iodinated RGD-heptapeptide. The binding is saturable, reversible, and two types of high affinity sites (Kd1 approximately 1 nM, and Kd2 approximately 40 nM) can be discerned. Competitive inhibition by several structurally related peptides and proteins noted the specific requirement for the RGD sequence. Thus, the RGD-binding activity of Arabidopsis fulfils the adhesion features of integrins, i.e. peptide specificity, subcellular location, and involvement in plasma membrane-cell wall attachments. PMID:9807828

  14. Constitutive expression of high-affinity sulfate transporter (HAST) gene in Indian mustard showed enhanced sulfur uptake and assimilation.

    PubMed

    Abdin, M Z; Akmal, M; Ram, M; Nafis, T; Alam, P; Nadeem, M; Khan, M A; Ahmad, A

    2011-07-01

    Lycopersicon esculantum sulfate transporter gene (LeST 1.1) encodes a high-affinity sulfate transporter (HAST) located in root epidermis. In this study, the LeST 1.1 gene was constitutively expressed in Indian mustard (Brassica juncea cv. Pusa Jai Kisan). Transgenic as well as untransformed plants were grown in sulfur-insufficient (25 and 50 μM) and sulfur-sufficient (1,000 μM) conditions for 30 days. Two-fold increase was noticed in the sulfate uptake rate of transgenic plants grown in both sulfur-insufficient and -sufficient conditions as compared to untransformed plants. The transgenic B. juncea plants were able to accumulate higher biomass and showed improved sulfur status even in sulfur-insufficient conditions when compared with untransformed plants. Chlorophyll content, ATP sulfurylase activity and protein content were also higher in transgenic plants than untranformed plants under sulfur-insufficient conditions. Our results, thus, clearly indicate that constitutive expression of LeST 1.1 gene in B. juncea had led to enhanced capacity of sulfur uptake and assimilation even in sulfur-insufficient conditions. This approach can also be used in other crops to enhance their sulfate uptake and assimilation potential under S-insufficient conditions. PMID:20938698

  15. A high-affinity receptor for urokinase plasminogen activator on human keratinocytes: characterization and potential modulation during migration.

    PubMed Central

    McNeill, H; Jensen, P J

    1990-01-01

    Low passage cultures of normal human keratinocytes produce several components of the plasminogen activator/plasmin proteolytic cascade, including urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), and two specific inhibitors. Studies here presented demonstrate that these cells also contain a high-affinity (Kd = 3 x 10(-10) M) plasma membrane-binding site for uPA. High molecular weight uPA, either as the single-chain precursor or two-chain activated form, bound to the receptor; however, low molecular weight (33 kD) uPA, tPA, or epidermal growth factor did not compete for binding, demonstrating specificity. Acid treatment, which removed endogenous uPA from the receptor, was required to detect maximal binding (45,000 sites per cell). To investigate the possibility that the uPA receptor on keratinocytes may be involved in epithelial migration during wound repair, cultures were wounded and allowed to migrate into the wounded site. Binding sites for uPA were localized by autoradiographic analysis of 125I-uPA binding as well as by immunocytochemical studies using anti-uPA IgG. With both techniques uPA binding sites were detected selectively on the plasma membrane of cells at the leading edge of the migrating epithelial sheet. This localization pattern suggests that uPA receptor expression on keratinocytes may be coupled to cell migration during cutaneous wounding. Images PMID:1965151

  16. 4-Methylumbelliferyl-beta-N-Acetylglucosaminide Hydrolysis by a High-Affinity Enzyme, a Putative Marker of Protozoan Bacterivory.

    PubMed

    Vrba, J; Simek, K; Nedoma, J; Hartman, P

    1993-09-01

    Hydrolysis of an artificial fluorogenic substrate, 4-methylumbelliferyl-beta-N-acetylglucosaminide, has been studied in a monoculture predator-prey system with either a flagellate (Bodo saltans) or a ciliate (Cyclidium sp.) fed upon pure bacterial culture (Aeromonas hydrophila or Alcaligenes xylosoxidans). Aeromonas hydrophila produced a low-affinity beta-N-acetylglucosaminidase-like enzyme (K(m), >100 mumol liter) but Alcaligenes xylosoxidans did not. Inoculation of both bacterial strains with bacterivorous protozoa induced the occurrence of another, high-affinity, beta-N-acetylglucosaminidase-like enzyme (K(m), <0.5 mumol liter). The latter enzyme showed significant, close correlations with total grazing rates of both B. saltans (r = 0.96) and Cyclidium sp. (r = 0.89) estimated by using uptake of fluorescently labelled bacteria. Further significant correlations between several protozoan parameters and kinetic parameters of this enzyme suggest its likely protozoan origin. If both types of enzyme occurred together, they could be satisfactorily distinguished by using kinetic data analysis. Hence, measurements of beta-N-acetylglucosaminidase-like activities might be promising to use to improve estimations of protozoan bacterivory.

  17. 4-Methylumbelliferyl-β-N-Acetylglucosaminide Hydrolysis by a High-Affinity Enzyme, a Putative Marker of Protozoan Bacterivory

    PubMed Central

    Vrba, Jaroslav; Šimek, Karel; Nedoma, Jiří; Hartman, Petr

    1993-01-01

    Hydrolysis of an artificial fluorogenic substrate, 4-methylumbelliferyl-β-N-acetylglucosaminide, has been studied in a monoculture predator-prey system with either a flagellate (Bodo saltans) or a ciliate (Cyclidium sp.) fed upon pure bacterial culture (Aeromonas hydrophila or Alcaligenes xylosoxidans). Aeromonas hydrophila produced a low-affinity β-N-acetylglucosaminidase-like enzyme (Km, ≫100 μmol liter-1) but Alcaligenes xylosoxidans did not. Inoculation of both bacterial strains with bacterivorous protozoa induced the occurrence of another, high-affinity, β-N-acetylglucosaminidase-like enzyme (Km, <0.5 μmol liter-1). The latter enzyme showed significant, close correlations with total grazing rates of both B. saltans (r2 = 0.96) and Cyclidium sp. (r2 = 0.89) estimated by using uptake of fluorescently labelled bacteria. Further significant correlations between several protozoan parameters and kinetic parameters of this enzyme suggest its likely protozoan origin. If both types of enzyme occurred together, they could be satisfactorily distinguished by using kinetic data analysis. Hence, measurements of β-N-acetylglucosaminidase-like activities might be promising to use to improve estimations of protozoan bacterivory. PMID:16349049

  18. Characterization of a common high-affinity receptor for reovirus serotypes 1 and 3 on endothelial cells

    SciTech Connect

    Verdin, E.M.; King, G.L.; Maratos-Flier, E.

    1989-03-01

    During viremia, viruses may be cleared from the blood stream and taken up by specific organs. The uptake of virus from the blood stream is dependent on the association of viral particles with endothelial cells that line the luminal surfaces of large and small blood vessels. To understand the nature of this interaction, the authors have studied the binding of reovirus serotypes 1 and 3 to these cells in vitro. Both serotypes of reovirus productively infected endothelial cells. By using (/sup 35/S)methionine-biolabeled reovirus as a tracer ligand, they found that both viruses rapidly bind to endothelial cells and that equilibrium is reached after 4 h. The binding of the radiolabeled viruses was saturable and mediated by a homogeneous population of cellular receptors with very high affinity for the virus ligands. Exposure of labeled virus to monoclonal antibodies directed against the viral hemagglutinin inhibited binding, demonstrating that the attachment of reovirus to endothelial cells is mediated by the hemagglutinin for both serotypes. By using a novel ligand-blotting assay, the binding of both viruses to a 54,000-dalton protein could be demonstrated. By using cell fractionation after homogenization, they demonstrated that this 54-kilodalton protein is a membrane protein, in agreement with its proposed role as a cell surface receptor for reovirus serotypes 1 and 3.

  19. Identification and properties of very high affinity brain membrane-binding sites for a neurotoxic phospholipase from the taipan venom

    SciTech Connect

    Lambeau, G.; Barhanin, J.; Schweitz, H.; Qar, J.; Lazdunski, M. )

    1989-07-05

    Four new monochain phospholipases were purified from the Oxyuranus scutellatus (taipan) venom. Three of them were highly toxic when injected into mice brain. One of these neurotoxic phospholipases, OS2, was iodinated and used in binding experiments to demonstrate the presence of two families of specific binding sites in rat brain synaptic membranes. The affinities were exceptionally high, Kd1 = 1.5 +/- 0.5 pM and Kd2 = 45 +/- 10 pM, and the maximal binding capacities were Bmax 1 = 1 +/- 0.4 and Bmax 2 = 3 +/- 0.5 pmol/mg of protein. Both binding sites were sensitive to proteolysis and demonstrated to be located on proteins of Mr 85,000-88,000 and 36,000-51,000 by cross-linking and photoaffinity labeling techniques. The binding of {sup 125}I-OS2 to synaptic membranes was dependent on Ca2+ ions and enhanced by Zn2+ ions which inhibit phospholipase activity. Competition experiments have shown that, except for beta-bungarotoxin, a number of known toxic snake or bee phospholipases have very high affinities for the newly identified binding sites. A good correlation (r = 0.80) was observed between toxicity and affinity but not between phospholipase activity and affinity.

  20. Development of indazolylpyrimidine derivatives as high-affine EphB4 receptor ligands and potential PET radiotracers.

    PubMed

    Ebert, Kristin; Wiemer, Jens; Caballero, Julio; Köckerling, Martin; Steinbach, Jörg; Pietzsch, Jens; Mamat, Constantin

    2015-09-01

    Due to their essential role in the pathogenesis of cancer, members of the Eph (erythropoietin-producing hepatoma cell line-A2) receptor tyrosine kinase family represent promising candidates for molecular imaging. Thus, the development and preparation of novel radiotracers for the noninvasive imaging of the EphB4 receptor via positron emission tomography (PET) is described. First in silico investigations with the indazolylpyrimidine lead compound which is known to be highly affine to EphB4 were executed to identify favorable labeling positions for an introduction of fluorine-18 to retain the affinity. Based on this, reference compounds as well as precursors were developed and labeled with carbon-11 and fluorine-18, respectively. For this purpose, a protecting group strategy essentially had to be generated to prevent unwanted methylation and to enable the introduction of fluorine-18. Further, a convenient radiolabeling strategy using [(11)C]methyl iodide was established which afforded the isotopically labeled radiotracer in 30-35% RCY (d.c.) which is identical with the original inhibitor molecule. A spiro ammonium precursor was prepared for radiolabeling with fluorine-18. Unfortunately, the labeling did not lead to the desired (18)F-radiotracer under the chosen conditions. PMID:26189032

  1. Demonstration of high-affinity folate binding activity associated with the brush border membranes of rat kidney.

    PubMed Central

    Selhub, J; Rosenberg, I H

    1978-01-01

    Folate binding activity of high affinity was identified in the particulate fractions of rat kidney homogenates. This binding activity cofractionated with alkaline phosphatase and maltase, two brush border membranes markers. With an enriched preparation of brush border membranes, freed of endogenous folate by acid treatment, the binding of [3H]olate was found to be saturable (Kb = 4.2 X 10(-11)M) and rapid. Binding was optimal at pH 6.4-7.7. At neutral pH, competition for binding with [3H]folic acid required 1.45 equivalents of pteroylheptaglutamate, 6.25 equivalents of N5-methyltetrathydrofolate, 29 equivalents of methotrexate, and 125 equivalents of N5-formyltetrahydrofolate. At alkaline pH, N5-methyltetrahydrofolate was as effective a competitor as folic acid. In view of reports that renal tubular reabsorption of folate includes an initial tight binding step, the binding activity associated with the brush border membranes may participate in this process. PMID:28521

  2. The M2 selective antagonist AF-DX 116 shows high affinity for muscarine receptors in bovine tracheal membranes.

    PubMed

    Roffel, A F; in't Hout, W G; de Zeeuw, R A; Zaagsma, J

    1987-05-01

    We have characterized the muscarine receptors in bovine tracheal and left ventricular membranes using 3H-dexetimide/pirenzepine and 3H-dexetimide/AF-DX 116 competition studies. Pirenzepine exhibited low (M2) affinity binding to both preparations; Kd was 590 nM in left ventricle and 463 nM in trachea. AF-DX 116 exhibited high (M2) affinity binding to left ventricle (Kd = 95.6 nM); in tracheal membranes it bound with high (M2) affinity (Kd = 40.7 nM) to 74% of the receptors and with low (M3) affinity (Kd = 2.26 microM) to 26% of the receptors. It is concluded that bovine tracheal muscle membranes contain a heterogeneous population of muscarine binding sites, the majority having M2 (heart) subtype characteristics and being located on the smooth muscle membranes; a minority having M3 (exocrine gland) subtype characteristics and presumed to be located in submucosal glands. This is the first report of high affinity binding of AF-DX 116 to non-cardiac peripheral muscarine receptors. PMID:3614390

  3. High affinity ( sup 3 H)glibenclamide binding sites in rat neuronal and cardiac tissue: Localization and developmental characteristics

    SciTech Connect

    Miller, J.A.; Velayo, N.L.; Dage, R.C.; Rampe, D. )

    1991-01-01

    We examined the binding of the antidiabetic sulfonylurea (3H) glibenclamide to rat brain and heart membranes. High affinity binding was observed in adult rat forebrain (Kd = 137.3 pM, maximal binding site density = 91.8 fmol/mg of protein) and ventricle (Kd = 77.1 pM, maximal binding site density = 65.1 fmol/mg of protein). Binding site density increased approximately 250% in forebrain membranes during postnatal development but was constant in ventricular membranes. Quantitative autoradiography was used to examine the regional distribution of (3H) glibenclamide binding sites in sections from rat brain, spinal cord and heart. The greatest density of binding in adult brain was found in the substantia nigra and globus pallidus, whereas the other areas displayed heterogenous binding. In agreement with the membrane binding studies, 1-day-old rat brain had significantly fewer (3H)glibenclamide binding sites than adult brain. Additionally, the pattern of distribution of these sites was qualitatively different from that of the adult. In adult rat spinal cord, moderate binding densities were observed in spinal cord gray and displayed a rostral to caudal gradient. In adult rat heart, moderate binding densities were observed and the sites were distributed homogeneously. In conclusion, significant development of (3H)glibenclamide binding sites was seen in the brain but not the heart during postnatal maturation. Furthermore, a heterogeneous distribution of binding sites was observed in both the brain and spinal cord of adult rats.

  4. Identification of an outer membrane protein of Fusobacterium necrophorum subsp. necrophorum that binds with high affinity to bovine endothelial cells.

    PubMed

    Kumar, Amit; Menon, Sailesh; Nagaraja, T G; Narayanan, Sanjeev

    2015-03-23

    Fusobacterium necrophorum, a Gram-negative anaerobe, is the primary etiologic agent of liver abscesses in cattle. There are two subspecies; subsp. necrophorum and subsp. funduliforme, which differ in morphological, biochemical, molecular characteristics, and virulence. The subsp. necrophorum, which is more virulent, occurs more frequently in liver abscesses than the subsp. funduliforme. Bacterial adhesion to the host cell surface is critical to the pathogenesis of several bacterial infections, and in F. necrophorum, outer membrane proteins (OMP) have been shown to mediate adhesion to bovine endothelial cells. The objective of this study was to identify potential adhesins that are involved in adhesion of F. necrophorum subsp. necrophorum to the host cells. An OMP of 42.4 kDa, which binds with high affinity to the bovine endothelial cells and is recognized by the sera from cattle with liver abscesses, was identified. N-terminal sequencing of the protein showed 96% homology to the FomA protein of F. nucleatum. The PCR analysis showed that this fomA gene was present in several strains of subsp. necrophorum, subsp. funduliforme of bovine and subsp. funduliforme of human origin. The purified native and recombinantly expressed protein when preincubated with the endothelial cells, prevented the attachment of subsp. necrophorum significantly. In addition, the polyclonal antibody produced against the protein prevented the binding of subsp. necrophorum to bovine endothelial cells.

  5. Qualitative and Quantitative Protein Complex Prediction Through Proteome-Wide Simulations.

    PubMed

    Rizzetto, Simone; Priami, Corrado; Csikász-Nagy, Attila

    2015-10-01

    Despite recent progress in proteomics most protein complexes are still unknown. Identification of these complexes will help us understand cellular regulatory mechanisms and support development of new drugs. Therefore it is really important to establish detailed information about the composition and the abundance of protein complexes but existing algorithms can only give qualitative predictions. Herein, we propose a new approach based on stochastic simulations of protein complex formation that integrates multi-source data--such as protein abundances, domain-domain interactions and functional annotations--to predict alternative forms of protein complexes together with their abundances. This method, called SiComPre (Simulation based Complex Prediction), achieves better qualitative prediction of yeast and human protein complexes than existing methods and is the first to predict protein complex abundances. Furthermore, we show that SiComPre can be used to predict complexome changes upon drug treatment with the example of bortezomib. SiComPre is the first method to produce quantitative predictions on the abundance of molecular complexes while performing the best qualitative predictions. With new data on tissue specific protein complexes becoming available SiComPre will be able to predict qualitative and quantitative differences in the complexome in various tissue types and under various conditions.

  6. A larger number of L chains (Tac) enhance the association rate of interleukin 2 to the high affinity site of the interleukin 2 receptor

    PubMed Central

    1988-01-01

    The IL-2-R is composed of at least two proteins, that is, a 55-kD protein (p55, the L chain, or Tac) and a 75-kD protein (p75, the H chain, or converter). The high affinity binding of IL-2 results in the formation of the ternary complex consisting of IL-2, and the L and H chains. To distinguish the affinity conversion model and the binary complex model we have carried out kinetic studies on the IL-2 binding to the high affinity IL-2-R on T lymphocytes expressing various numbers of L chains and a relatively constant number of H chains. We found that expression of a larger number of L chains accelerated the association of IL-2 to the high affinity receptor. The results are not compatible with the binary complex model that assumes a fixed number of high affinity sites determined by the numbers of a limiting chain. Instead, the results are consistent with the prediction of the affinity conversion model that assumes association of IL-2 to the L chain as the first step of the ternary complex formation and they indicate that the possible role of excess L chains is to accelerate the formation of the ternary complex. The reaction rate constants calculated from the affinity conversion model were reasonably constant. PMID:3263463

  7. Cross-linking of epidermal growth factor receptors in intact cells: detection of initial stages of receptor clustering and determination of molecular weight of high-affinity receptors

    SciTech Connect

    Fanger, B.O.; Austin, K.S.; Earp, H.S.; Cidlowski, J.A.

    1986-10-21

    A method was developed to label epidermal growth factor (EGF) receptors with /sup 125/I-EGF in whole cells using chemical cross-linking reagents. Polyacrylamide gel electrophoresis resolved an M/sub r/ approx. 180,000 EGF-receptor complex and larger M/sub r/ greater than or equal to 360,000 aggregates. The formation of the larger complexes was timed and temperature dependent and appeared to represent the initial events of EGF receptor clustering. Alteration of the ratio of /sup 125/I-EGF-labeled high- and low- affinity complexes by competition with unlabeled EGF or by induction of additional high-affinity sites with dexamethasone suggested that both sites were represented by the M/sub r/ approx. 180,000 /sup 125/I-EGF-receptor complexes. Digestion of cells before cross-linking detected a small population of trypsin-resistant M/sub r/ approx. 180,000 receptors, which could represent previously described cryptic and/or high-affinity receptors. Few of the M/sub r/ approx. 360,000 receptors were trypsin resistant. Glucocorticoid induction of high-affinity EGF receptors failed to induce detectable changes in the microclustering of EGF receptors but did result in a 50% increase in EGF-induced receptor phosphorylation in HeLa S/sub 3/ cell membranes at 4/sup 0/C. Thus, glucocorticoids increase high-affinity EGF binding sites, EGF-induced receptor phosphorylation, and cell growth.

  8. Identifying high-affinity aptamer ligands with defined cross-reactivity using high-throughput guided systematic evolution of ligands by exponential enrichment

    PubMed Central

    Levay, Agata; Brenneman, Randall; Hoinka, Jan; Sant, David; Cardone, Marco; Trinchieri, Giorgio; Przytycka, Teresa M.; Berezhnoy, Alexey

    2015-01-01

    Oligonucleotide aptamers represent a novel platform for creating ligands with desired specificity, and they offer many potentially significant advantages over monoclonal antibodies in terms of feasibility, cost, and clinical applicability. However, the isolation of high-affinity aptamer ligands from random oligonucleotide pools has been challenging. Although high-throughput sequencing (HTS) promises to significantly facilitate systematic evolution of ligands by exponential enrichment (SELEX) analysis, the enormous datasets generated in the process pose new challenges for identifying those rare, high-affinity aptamers present in a given pool. We show that emulsion PCR preserves library diversity, preventing the loss of rare high-affinity aptamers that are difficult to amplify. We also demonstrate the importance of using reference targets to eliminate binding candidates with reduced specificity. Using a combination of bioinformatics and functional analyses, we show that the rate of amplification is more predictive than prevalence with respect to binding affinity and that the mutational landscape within a cluster of related aptamers can guide the identification of high-affinity aptamer ligands. Finally, we demonstrate the power of this selection process for identifying cross-species aptamers that can bind human receptors and cross-react with their murine orthologs. PMID:26007661

  9. Identifying high-affinity aptamer ligands with defined cross-reactivity using high-throughput guided systematic evolution of ligands by exponential enrichment.

    PubMed

    Levay, Agata; Brenneman, Randall; Hoinka, Jan; Sant, David; Cardone, Marco; Trinchieri, Giorgio; Przytycka, Teresa M; Berezhnoy, Alexey

    2015-07-13

    Oligonucleotide aptamers represent a novel platform for creating ligands with desired specificity, and they offer many potentially significant advantages over monoclonal antibodies in terms of feasibility, cost, and clinical applicability. However, the isolation of high-affinity aptamer ligands from random oligonucleotide pools has been challenging. Although high-throughput sequencing (HTS) promises to significantly facilitate systematic evolution of ligands by exponential enrichment (SELEX) analysis, the enormous datasets generated in the process pose new challenges for identifying those rare, high-affinity aptamers present in a given pool. We show that emulsion PCR preserves library diversity, preventing the loss of rare high-affinity aptamers that are difficult to amplify. We also demonstrate the importance of using reference targets to eliminate binding candidates with reduced specificity. Using a combination of bioinformatics and functional analyses, we show that the rate of amplification is more predictive than prevalence with respect to binding affinity and that the mutational landscape within a cluster of related aptamers can guide the identification of high-affinity aptamer ligands. Finally, we demonstrate the power of this selection process for identifying cross-species aptamers that can bind human receptors and cross-react with their murine orthologs.

  10. Two plant bacteria, S. meliloti and Ca. Liberibacter asiaticus, share functional znuABC homologues that encode for a high affinity Zinc uptake system

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Znu system, encoded for by znuABC, can be found in multiple genera of bacteria and has been shown to be responsible for the import of zinc under low zinc conditions. Although this high-affinity uptake system is known to be important for both growth and/or pathogenesis in bacteria, it has not bee...

  11. [125I]AT-1012, a New High Affinity Radioligand for the α3β4 Nicotinic Acetylcholine Receptors

    PubMed Central

    Wu, Jinhua; Perry, David C.; Bupp, James E.; Jiang, Faming; Polgar, Willma E.; Toll, Lawrence; Zaveri, Nurulain T.

    2013-01-01

    Recent genetic and pharmacological studies have implicated the α3, β4 and a5 subunits of the nicotinic acetylcholine receptor (nAChR) in dependence to nicotine and other abused drugs and nicotine withdrawal. The α3β4* nAChR subtype has been shown to co-assemble with the α5 or β3 nAChR subunits, and is found mainly in the autonomic ganglia and select brain regions. It has been difficult to study the α3β4 nAChR because there have been no selective nonpeptidic ligands available to independently examine its pharmacology. We recently reported the synthesis of a [125I]-radiolabeled analog of a high affinity, selective small-molecule α3β4 nAChR ligand, AT-1012. We report here the vitro characterization of this radioligand in receptor binding and in vitro autoradiographic studies targeting the α3β4* nAChR. Binding of [125I]AT-1012 was characterized at the rat α3β4- and α4β2 nAChR transfected into HEK cells as well as at the human α3β4α5 nAChR in HEK cells. Binding affinity of [125I]AT-1012 at the rat α3β4 nAChR was 1.4 nM, with a Bmax of 10.3 pmol/mg protein, similar to what was determined using [3H]epibatidine. Saturation isotherms suggested that [125I]AT-1012 binds to a single site on the α3β4 nAChR. Similar high binding affinity was also observed for [125I]AT-1012 at human α3β4α5 nAChR in a human α3β4aα nAChR transfected cell line. [125I]AT-1012 did not bind with high affinity to membranes from α4β2 nAChR-transfected HEK cells, and [3H]epibatidine binding studies showed that AT-1012 had over 100-fold binding selectivity for the α3β4 over α4β2 nAChR. Ki values determined for known nAChR compounds using [125I]AT-1012 as radioligand were comparable to those obtained with [3H]epibatidine. [125I]AT-1012 was also used to label the α3β4 nAChR in rat brain slices in vitro using autoradiography which showed highly localized binding of the radioligand in brain regions consistent with the discreet localization of the α3β4 nAChR. We

  12. Exhaust gas purification device

    SciTech Connect

    Fujiwara, H.; Hibi, T.; Sayo, S.; Sugiura, Y.; Ueda, K.

    1980-02-19

    The exhaust gas purification device includes an exhaust manifold , a purification cylinder connected with the exhaust manifold through a first honey-comb shaped catalyst, and a second honeycomb shaped catalyst positioned at the rear portion of the purification cylinder. Each catalyst is supported by steel wool rings including coarse and dense portions of steel wool. The purification device further includes a secondary air supplying arrangement.

  13. Calcium-dependent association of a protein complex with the lymphocyte plasma membrane: probable identity with calmodulin-calcineurin

    PubMed Central

    1985-01-01

    A protein complex is shown to participate in a calcium-dependent association with plasma membranes purified either from pig mesenteric lymph node lymphocytes or from human lymphoblastoid cell lines. Plasma membranes prepared in the presence of calcium possess this complex; those prepared in the absence of calcium (5 mM EGTA) do not. The complex associates itself with the inner cytoplasmic surface of the plasma membrane. This complex is referred to as the "acidic protein band" because of its location during migration upon alkaline-urea gel electrophoresis. The complex dissociates from the plasma membrane during electrophoresis on 8-M urea gels, irrespective of calcium levels during electrophoresis; at intermediate urea concentrations (4-6 M), the complex is not dissociated in the presence of calcium. Upon purification of the acidic protein band, SDS acrylamide gel electrophoresis, immunoblotting, and radioimmunoassay techniques suggest that the acidic protein band is composed of at least four peptides (designated 68K, 59K, 20K, 20K): two of these (68K, 20K) are immunopositive for calcineurin and one (20K) is immunopositive for calmodulin. Immunoblots of urea gels also indicate that the calcineurin heavy chain (68K) can also appear at three different locations on the urea gel. Patches and caps induced in human peripheral blood lymphocytes by fluorescein-conjugated goat anti-human IgG are not coincident with the location of calcineurin, which remains distributed throughout the cell. PMID:2989299

  14. High-affinity Anticalins with aggregation-blocking activity directed against the Alzheimer β-amyloid peptide

    PubMed Central

    Rauth, Sabine; Hinz, Dominik; Börger, Michael; Uhrig, Markus; Mayhaus, Manuel; Riemenschneider, Matthias; Skerra, Arne

    2016-01-01

    Amyloid beta (Aβ) peptides, in particular Aβ42 and Aβ40, exert neurotoxic effects and their overproduction leads to amyloid deposits in the brain, thus constituting an important biomolecular target for treatments of Alzheimer's disease (AD). We describe the engineering of cognate Anticalins as a novel type of neutralizing protein reagent based on the human lipocalin scaffold. Phage display selection from a genetic random library comprising variants of the human lipocalin 2 (Lcn2) with mutations targeted at 20 exposed amino acid positions in the four loops that form the natural binding site was performed using both recombinant and synthetic target peptides and resulted in three different Anticalins. Biochemical characterization of the purified proteins produced by periplasmic secretion in Escherichia coli revealed high folding stability in a monomeric state, with Tm values ranging from 53.4°C to 74.5°C, as well as high affinities for Aβ40, between 95 pM and 563 pM, as measured by real-time surface plasmon resonance analysis. The central linear VFFAED epitope within the Aβ sequence was mapped using a synthetic peptide array on membranes and was shared by all three Anticalins, despite up to 13 mutual amino acid differences in their binding sites. All Anticalins had the ability–with varying extent–to inhibit Aβ aggregation in vitro according to the thioflavin-T fluorescence assay and, furthermore, they abolished Aβ42-mediated toxicity in neuronal cell culture. Thus, these Anticalins provide not only useful protein reagents to study the molecular pathology of AD but they also show potential as alternative drug candidates compared with antibodies. PMID:27029347

  15. The high affinity dopamine uptake inhibitor, JHW 007, blocks cocaine-induced reward, locomotor stimulation and sensitization.

    PubMed

    Velázquez-Sánchez, C; Ferragud, A; Murga, J; Cardá, M; Canales, J J

    2010-07-01

    The discovery and evaluation of high affinity dopamine transport inhibitors with low abuse liability is an important step toward the development of efficacious medications for cocaine addiction. We examined in mice the behavioural effects of (N-(n-butyl)-3alpha-[bis(4'-fluorophenyl)methoxy]-tropane) (JHW 007), a benztropine (BZT) analogue that blocks dopamine uptake, and assessed its potential to influence the actions of cocaine in clinically-relevant models of cocaine addiction. In the conditioned place preference (CPP) paradigm, JHW 007 exposure did not produce place conditioning within an ample dose range but effectively blocked the CPP induced by cocaine administration. Similarly, in the CPP apparatus JHW 007 treatment failed to stimulate locomotor activity at any dose but dose-dependently suppressed the hyperactivity evoked by cocaine treatment. In locomotor sensitization assays performed in the open field, JHW 007 did not produce sensitized locomotor behaviour when given alone, but it prevented the sensitized component of the locomotor response elicited by subchronic (8-day) cocaine exposure. In the elevated plus maze (EPM), acute treatment with JHW 007, cocaine and combinations of the BZT analogue and cocaine produced an anxiogenic-like profile. Re-test in the EPM following subchronic (8-day) exposure enhanced the anxiogenic-like effect of the same drug treatments. The present findings indicate that JHW 007 exposure counteracts some critical behavioural correlates of cocaine treatment, including conditioned reward, locomotor stimulation and sensitization, and lend support to the further development of BZT analogues as potential replacement medications in cocaine addiction.

  16. Mutational Analysis of the High-Affinity Zinc Binding Site Validates a Refined Human Dopamine Transporter Homology Model

    PubMed Central

    Stockner, Thomas; Montgomery, Therese R.; Kudlacek, Oliver; Weissensteiner, Rene; Ecker, Gerhard F.; Freissmuth, Michael; Sitte, Harald H.

    2013-01-01

    The high-resolution crystal structure of the leucine transporter (LeuT) is frequently used as a template for homology models of the dopamine transporter (DAT). Although similar in structure, DAT differs considerably from LeuT in a number of ways: (i) when compared to LeuT, DAT has very long intracellular amino and carboxyl termini; (ii) LeuT and DAT share a rather low overall sequence identity (22%) and (iii) the extracellular loop 2 (EL2) of DAT is substantially longer than that of LeuT. Extracellular zinc binds to DAT and restricts the transporter‚s movement through the conformational cycle, thereby resulting in a decrease in substrate uptake. Residue H293 in EL2 praticipates in zinc binding and must be modelled correctly to allow for a full understanding of its effects. We exploited the high-affinity zinc binding site endogenously present in DAT to create a model of the complete transmemberane domain of DAT. The zinc binding site provided a DAT-specific molecular ruler for calibration of the model. Our DAT model places EL2 at the transporter lipid interface in the vicinity of the zinc binding site. Based on the model, D206 was predicted to represent a fourth co-ordinating residue, in addition to the three previously described zinc binding residues H193, H375 and E396. This prediction was confirmed by mutagenesis: substitution of D206 by lysine and cysteine affected the inhibitory potency of zinc and the maximum inhibition exerted by zinc, respectively. Conversely, the structural changes observed in the model allowed for rationalizing the zinc-dependent regulation of DAT: upon binding, zinc stabilizes the outward-facing state, because its first coordination shell can only be completed in this conformation. Thus, the model provides a validated solution to the long extracellular loop and may be useful to address other aspects of the transport cycle. PMID:23436987

  17. A human β-III-spectrin spinocerebellar ataxia type 5 mutation causes high-affinity F-actin binding

    PubMed Central

    Avery, Adam W.; Crain, Jonathan; Thomas, David D.; Hays, Thomas S.

    2016-01-01

    Spinocerebellar ataxia type 5 (SCA5) is a human neurodegenerative disease that stems from mutations in the SPTBN2 gene encoding the protein β-III-spectrin. Here we investigated the molecular consequence of a SCA5 missense mutation that results in a L253P substitution in the actin-binding domain (ABD) of β-III-spectrin. We report that the L253P substitution in the isolated β-III-spectrin ABD causes strikingly high F-actin binding affinity (Kd = 75.5 nM) compared to the weak F-actin binding affinity of the wild-type ABD (Kd = 75.8 μM). The mutation also causes decreased thermal stability (Tm = 44.6 °C vs 59.5 °C). Structural analyses indicate that leucine 253 is in a loop at the interface of the tandem calponin homology (CH) domains comprising the ABD. Leucine 253 is predicted to form hydrophobic contacts that bridge the CH domains. The decreased stability of the mutant indicates that these bridging interactions are probably disrupted, suggesting that the high F-actin binding affinity of the mutant is due to opening of the CH domain interface. These results support a fundamental role for leucine 253 in regulating opening of the CH domain interface and binding of the ABD to F-actin. This study indicates that high-affinity actin binding of L253P β-III-spectrin is a likely driver of neurodegeneration. PMID:26883385

  18. High-affinity no-carrier-added 99mTc-labeled chemotactic peptides for studies of inflammation in vivo.

    PubMed

    Baidoo, K E; Scheffel, U; Stathis, M; Finley, P; Lever, S Z; Zhan, Y; Wagner, H N

    1998-01-01

    Nalpha-for-Nle-Leu-Phe-Nle-Tyr-Lys, a chemotactic peptide that binds with high affinity to the chemoattractant receptor on granulocytes and monocytes, was labeled with 99mTc using the diaminedithiol (DADT) chelating system to coordinate the Tc. 99mTc labeling of the DADT-coupled peptide was accomplished in 84% overall yield (room temperature for 10 min) using [99mTc]glucoheptonate as the donor of prereduced Tc. HPLC analysis showed two major 99mTc-labeled peptide peaks, 99mTc-DADT-Pep-I and 99mTc-DADT-Pep-II, were obtained in a ratio of 1:0.85. Using an iodoacetamide-derivatized gel to remove unlabeled peptide from the 99mTc labeling mixtures, essentially no-carrier-added (nca) high-specific activity 99mTc-labeled chemotactic peptides were obtained. The 99Tc analogues of the peptides were synthesized (72% yield) in a similar fashion and correlated with 99mTc complexes I and II by HPLC. In vitro competitive receptor binding assays of the isolated 99Tc analogues were performed against the tritiated chemotactic peptide [3H]N-for-Met-Leu-Phe ([3H]fMLF) using isolated granulocytes. The 99Tc-derivatized peptides showed similar binding affinities to the chemoattractant receptor as the unlabeled Nalpha-for-Nle-Leu-Phe-Nle-Tyr-Lys. The nca 99mTc-labeled peptides gave high contrast images of experimental inflammation in rabbits without causing neutropenia. Thus, it is feasible to attach the Tc-DADT chelate to low-molecular weight receptor binding chemotactic peptides and retain substantial binding to the receptor. Chemotactic peptides labeled with 99mTc via the DADT ligand system have the potential for imaging focal sites of inflammation without toxic effects, an important consideration in the successful utilization of chemotactic peptide agonists.

  19. Development of BODIPY FL Vindoline as a Novel and High-Affinity Pregnane X Receptor Fluorescent Probe

    PubMed Central

    2015-01-01

    The pregnane X receptor (PXR) regulates the metabolism and excretion of xenobiotics and endobiotics by regulating the expression of drug-metabolizing enzymes and transporters. The unique structure of PXR allows it to bind many drugs and drug leads, possibly causing undesired drug–drug interactions. Therefore, it is crucial to evaluate whether chemicals or drugs bind to PXR. Fluorescence-based assays are preferred because of their sensitivity and nonradioactive nature. On the basis of our previously characterized 4 (BODIPY FL vinblastine), a high-affinity PXR probe, we developed 20 (BODIPY FL vindoline) and showed that it is a novel and potent PXR fluorescent probe with Kd of 256 nM in a time-resolved fluorescence resonance energy transfer (TR-FRET) binding assay with PXR. By using 20 (BODIPY FL vindoline) in the PXR TR-FRET assay, we obtained a more than 7-fold signal-to-background ratio and high signal stability (signal was stable for at least 120 min, and Z′-factor > 0.85 from 30 to 240 min). The assay can tolerate DMSO up to 2%. This assay has been used to evaluate a panel of PXR ligands for their PXR-binding affinities. The performance of 20 (BODIPY FL vindoline) in the PXR TR-FRET assay makes it an ideal PXR fluorescent probe, and the newly developed PXR TR-FRET assay with 20 (BODIPY FL vindoline) as a fluorescent probe is suitable for high-throughput screening to identify PXR-binding ligands. PMID:25133934

  20. Plasticity in the High Affinity Menaquinone Binding Site of the Cytochrome aa3-600 Menaquinol Oxidase from Bacillus subtilis.

    PubMed

    Yi, Sophia M; Taguchi, Alexander T; Samoilova, Rimma I; O'Malley, Patrick J; Gennis, Robert B; Dikanov, Sergei A

    2015-08-18

    Cytochrome aa3-600 is a terminal oxidase in the electron transport pathway that contributes to the electrochemical membrane potential by actively pumping protons. A notable feature of this enzyme complex is that it uses menaquinol as its electron donor instead of cytochrome c when it reduces dioxygen to water. The enzyme stabilizes a menasemiquinone radical (SQ) at a high affinity site that is important for catalysis. One of the residues that interacts with the semiquinone is Arg70. We have made the R70H mutant and have characterized the menasemiquinone radical by advanced X- and Q-band EPR. The bound SQ of the R70H mutant exhibits a strong isotropic hyperfine coupling (a(14)N ≈ 2.0 MHz) with a hydrogen bonded nitrogen. This nitrogen originates from a histidine side chain, based on its quadrupole coupling constant, e(2)qQ/h = 1.44 MHz, typical for protonated imidazole nitrogens. In the wild-type cyt aa3-600, the SQ is instead hydrogen bonded with Nε from the Arg70 side chain. Analysis of the (1)H 2D electron spin echo envelope modulation (ESEEM) spectra shows that the mutation also changes the number and strength of the hydrogen bonds between the SQ and the surrounding protein. Despite the alterations in the immediate environment of the SQ, the R70H mutant remains catalytically active. These findings are in contrast to the equivalent mutation in the close homologue, cytochrome bo3 ubiquinol oxidase from Escherichia coli, where the R71H mutation eliminates function.

  1. Patterned enzymatic degradation of poly(ε-caprolactone) by high-affinity microcontact printing and polymer pen lithography.

    PubMed

    Ganesh, Manoj; Nachman, Jonathan; Mao, Zhantong; Lyons, Alan; Rafailovich, Miriam; Gross, Richard

    2013-08-12

    This paper reports deposition of Candida antarctica Lipase B (CALB) on relatively thick poly(ε-caprolactone) (PCL) films (300-500 nm) to create well-defined patterns using two different writing techniques: high-affinity microcontact (HA-μCL) and polymer pen (PPL) lithography. For both, an aqueous CALB ink is absorbed onto a polydimethylsiloxane (PDMS) writing implement (PDMS stamp or a PDMS pen tip), which is transferred to a spun-cast PCL film. HA-μCL experiments demonstrated the importance of applied pressure to obtain high-resolution patterns since uniform contact is needed between raised 20 μm parallel line regions of the PDMS stamp and the surface. AFM imaging shows pattern formation evolves gradually over incubation time only in areas stamped with CALB cutting through spherulites without apparent influence by grain boundaries. Strong binding of CALB to PCL is postulated as the mechanism by which lateral diffusion is limited. PPL enables formation of an arbitrary image by appropriate programming of the robot. The PDMS pen tips were coated with an aqueous CALB solution and then brought into contact with the PCL film to transfer CALB onto the surface. By repeating the ink transfer step multiple times where pen tips are brought into contact with the PCL film at a different locations, a pattern of dots is formed. After printing, patterns were developed at 37 °C and 95% RH. Over a 7-day period, CALB progressively etched the PCL down to the silicon wafer on which it was spun (350 nm) giving round holes with diameters about 10 μm. AFM images show the formation of steep PCL walls indicating CALB degraded the PCL film in areas to which it was applied. This work demonstrates that high-resolution patterns can be achieved without immobilizing the enzyme on the surface of polymeric stamps that limits the depth of features obtained as well as the throughput of the process. PMID:23808571

  2. Role of the human high-affinity copper transporter in copper homeostasis regulation and cisplatin sensitivity in cancer chemotherapy.

    PubMed

    Kuo, Macus Tien; Fu, Siqing; Savaraj, Niramol; Chen, Helen H W

    2012-09-15

    The high-affinity copper transporter (Ctr1; SCLC31A1) plays an important role in regulating copper homeostasis because copper is an essential micronutrient and copper deficiency is detrimental to many important cellular functions, but excess copper is toxic. Recent research has revealed that human copper homeostasis is tightly controlled by interregulatory circuitry involving copper, Sp1, and human (hCtr1). This circuitry uses Sp1 transcription factor as a copper sensor in modulating hCtr1 expression, which in turn controls cellular copper and Sp1 levels in a 3-way mutual regulatory loop. Posttranslational regulation of hCtr1 expression by copper stresses has also been described in the literature. Because hCtr1 can also transport platinum drugs, this finding underscores the important role of hCtr1 in platinum-drug sensitivity in cancer chemotherapy. Consistent with this notion is the finding that elevated hCtr1 expression was associated with favorable treatment outcomes in cisplatin-based cancer chemotherapy. Moreover, cultured cell studies showed that elevated hCtr1 expression can be induced by depleting cellular copper levels, resulting in enhanced cisplatin uptake and its cell-killing activity. A phase I clinical trial using a combination of trientine (a copper chelator) and carboplatin has been carried out with encouraging results. This review discusses new insights into the role of hCtr1 in regulating copper homeostasis and explains how modulating cellular copper availability could influence treatment efficacy in platinum-based cancer chemotherapy through hCtr1 regulation.

  3. Competitive Inhibition of High-Affinity Oryzalin Binding to Plant Tubulin by the Phosphoric Amide Herbicide Amiprophos-Methyl.

    PubMed Central

    Murthy, J. V.; Kim, H. H.; Hanesworth, V. R.; Hugdahl, J. D.; Morejohn, L. C.

    1994-01-01

    Amiprophos-methyl (APM), a phosphoric amide herbicide, was previously reported to inhibit the in vitro polymerization of isolated plant tubulin (L.C. Morejohn, D.E. Fosket [1984] Science 224: 874-876), yet little other biochemical information exists concerning this compound. To characterize further the mechanism of action of APM, its interactions with tubulin and microtubules purified from cultured cells of tobacco (Nicotiana tabacum cv Bright Yellow-2) were investigated. Low micromolar concentrations of APM depolymerized preformed, taxol-stabilized tobacco microtubules. Remarkably, at the lowest APM concentration examined, many short microtubules were redistributed into fewer but 2.7-fold longer microtubules without a substantial decrease in total polymer mass, a result consistent with an end-to-end annealing of microtubules with enhanced kinetic properties. Quasi-equilibrium binding measurements showed that tobacco tubulin binds [14C]oryzalin with high affinity to produce a tubulin-oryzalin complex having a dissociation constant (Kd) = 117 nM (pH 6.9; 23[deg]C). Also, an estimated maximum molar binding stoichiometry of 0.32 indicates pharamacological heterogeneity of tobacco dimers and may be related to structural heterogeneity of tobacco tubulin subunits. APM inhibits competitively the binding of [14C]oryzalin to tubulin with an inhibition constant (Ki) = 5 [mu]M, indicating the formation of a moderate affinity tubulin-APM complex that may interact with the ends of microtubules. APM concentrations inhibiting tobacco cell growth were within the threshold range of APM concentrations that depolymerized cellular microtubules, indicating that growth inhibition is caused by microtubules depolymerization. APM had no apparent effect on microtubules in mouse 3T3 fibroblasts. Because cellular microtubules were depolymerized at APM and oryzalin concentrations below their respective Ki and Kd values, both herbicides are proposed to depolymerize microtubules by a

  4. Transmembrane-truncated alphavbeta3 integrin retains high affinity for ligand binding: evidence for an 'inside-out' suppressor?

    PubMed Central

    Mehta, R J; Diefenbach, B; Brown, A; Cullen, E; Jonczyk, A; Güssow, D; Luckenbach, G A; Goodman, S L

    1998-01-01

    The molecular mechanisms of alphavbeta3 integrin affinity regulation have important biological implications in tumour development, wound repair and angiogenesis. We expressed, purified and characterized recombinant forms of human alphavbeta3 (r-alphavbeta3) and compared the activation state of these with alphavbeta3 in its cellular environment. The ligand specificity and selectivity of recombinant full-length and double transmembrane truncations of r-alphavbeta3 cloned in BacPAK6 vectors and expressed in Sf9 and High Five insect cells were compared with those of native placental alphavbeta3 and the receptor in situ on the cell surface. r-alphavbeta3 integrins were purified by affinity chromatography from detergent extracts of cells (full-length), and from the culture medium of cells expressing double-truncated r-alphavbeta3. r-alphavbeta3 had the same epitopes, ligand-binding specificities, bivalent cation requirements and susceptibility to RGD-containing peptides as native alphavbeta3. On M21-L4 melanoma cells, alphavbeta3 mediated binding to vitronectin, but not to fibrinogen unless activated with Mn2+. Non-activated alphaIIbbeta3 integrin as control in M21-L-IIb cells had the opposite profile, mediating binding to fibrinogen, but not to vitronectin unless activated with Mn2+. Thus these receptors had moderate to low ligand affinity. In marked contrast, purified alphavbeta3 receptors, with or without transmembrane and cytoplasmic domains, were constitutively of high affinity and able to bind strongly to vitronectin, fibronectin and fibrinogen under physiological conditions. Our data suggest that, in contrast with the positive regulation of alphaIIbbeta3 in situ, intracellular controls lower the affinity of alphavbeta3, and the cytoplasmic domains may act as a target for negative regulators of alphavbeta3 activity. PMID:9480902

  5. Basis of the 1:1 stoichiometry of the high affinity receptor Fc epsilon RI-IgE complex.

    PubMed

    Keown, M B; Ghirlando, R; Mackay, G A; Sutton, B J; Gould, H J

    1997-01-01

    A soluble fragment of the high-affinity IgE receptor Fc epsilon RI alpha-chain (sFc epsilon RI alpha) binds to the Fc fragment of IgE (IgE-Fc) as a 1:1 complex. IgE-Fc consists of a dimer of the C epsilon 2, C epsilon 3 and C epsilon 4 domains of the epsilon-heavy chain of IgE. This region of IgE has been modelled on the crystal structure of the Fc region of IgG1, which exhibits twofold rotational symmetry. This implies that IgE should be divalent with respect to its ligands. X-ray scattering studies reveal however that the twofold rotational symmetry of IgE-Fc is perturbed by a bend in the linker region between the C epsilon 2 and C epsilon 3 domains. The 1:1 stoichiometry could then arise from the conformational asymmetry or from steric occlusion of one of the sites by the overhanging C epsilon 2 domains. To test this hypothesis we have expressed a recombinant epsilon-chain fragment containing C epsilon 3 and C epsilon 4. This product, Fc epsilon 3-4, is secreted from cells as a disulphide linked dimer and binds with higher affinity than either IgE or IgE-Fc to cell surface Fc epsilon RI. Titration experiments, together with molecular mass measurements of the Fc epsilon 3-4/sFc epsilon RI alpha complex, reveal that Fc epsilon 3-4 binds only a single receptor molecule. This excludes the possibility that steric hindrance by C epsilon 2 accounts for the unexpected stoichiometry.

  6. Identification and Tumour-Binding Properties of a Peptide with High Affinity to the Disialoganglioside GD2

    PubMed Central

    Müller, Jan; Reichel, Robin; Vogt, Sebastian; Müller, Stefan P.; Sauerwein, Wolfgang; Brandau, Wolfgang; Eggert, Angelika

    2016-01-01

    Neuroectodermal tumours are characterized by aberrant processing of disialogangliosides concomitant with high expression of GD2 or GD3 on cell surfaces. Antibodies targeting GD2 are already in clinical use for therapy of neuroblastoma, a solid tumour of early childhood. Here, we set out to identify peptides with high affinity to human disialoganglioside GD2. To this end, we performed a combined in vivo and in vitro screen using a recombinant phage displayed peptide library. We isolated a phage displaying the peptide sequence WHWRLPS that specifically binds to the human disialoganglioside GD2. Binding specificity was confirmed by mutational scanning and by comparative analyses using structurally related disialogangliosides. In vivo, significant enrichment of phage binding to xenografts of human neuroblastoma cells in mice was observed. Tumour-specific phage accumulation could be blocked by intravenous coinjection of the corresponding peptide. Comparative pharmacokinetic analyses revealed higher specific accumulation of 68Ga-labelled GD2-binding peptide compared to 111In-labelled peptide in xenografts of human neuroblastoma. In contrast to 124I-MIBG, which is currently evaluated as a neuroblastoma marker in PET/CT, 68Ga-labelled GD2-specific peptide spared the thyroid but was enriched in the kidneys, which could be partially blocked by infusion of amino acids.In summary, we here report on a novel tumour-homing peptide that specifically binds to the disialoganglioside GD2, accumulates in xenografts of neuroblastoma cells in mice and bears the potential for tumour detection using PET/CT. Thus, this peptide may serve as a new scaffold for diagnosing GD2-positive tumours of neuroectodermal origin. PMID:27716771

  7. New Regulators of a High Affinity Ca2+ Influx System Revealed through a Genome-wide Screen in Yeast*

    PubMed Central

    Martin, D. Christian; Kim, Hyemin; Mackin, Nancy A.; Maldonado-Báez, Lymarie; Evangelista, Carlos C.; Beaudry, Veronica G.; Dudgeon, Drew D.; Naiman, Daniel Q.; Erdman, Scott E.; Cunningham, Kyle W.

    2011-01-01

    The bakers' yeast Saccharomyces cerevisiae utilizes a high affinity Ca2+ influx system (HACS) to survive assaults by mating pheromones, tunicamycin, and azole-class antifungal agents. HACS consists of two known subunits, Cch1 and Mid1, that are homologous and analogous to the catalytic α-subunits and regulatory α2δ-subunits of mammalian voltage-gated calcium channels, respectively. To search for additional subunits and regulators of HACS, a collection of gene knock-out mutants was screened for abnormal uptake of Ca2+ after exposure to mating pheromone or to tunicamycin. The screen revealed that Ecm7 is required for HACS function in most conditions. Cycloheximide chase experiments showed that Ecm7 was stabilized by Mid1, and Mid1 was stabilized by Cch1 in non-signaling conditions, suggesting they all interact. Ecm7 is a member of the PMP-22/EMP/MP20/Claudin superfamily of transmembrane proteins that includes γ-subunits of voltage-gated calcium channels. Eleven additional members of this superfamily were identified in yeast, but none was required for HACS activity in response to the stimuli. Remarkably, many dozens of genes involved in vesicle-mediated trafficking and protein secretion were required to prevent spontaneous activation of HACS. Taken together, the findings suggest that HACS and calcineurin monitor performance of the membrane trafficking system in yeasts and coordinate compensatory processes. Conservation of this quality control system in Candida glabrata suggests that many pathogenic species of fungi may utilize HACS and calcineurin to resist azoles and other compounds that target membrane biosynthesis. PMID:21252230

  8. Piperazine imidazo[1,5-a]quinoxaline ureas as high-affinity GABAA ligands of dual functionality.

    PubMed

    Jacobsen, E J; Stelzer, L S; TenBrink, R E; Belonga, K L; Carter, D B; Im, H K; Im, W B; Sethy, V H; Tang, A H; VonVoigtlander, P F; Petke, J D; Zhong, W Z; Mickelson, J W

    1999-04-01

    A series of imidazo[1,5-a]quinoxaline piperazine ureas appended with a tert-butyl ester side chain at the 3-position was developed. Analogues within this series have high affinity for the gamma-aminobutyric acid A (GABAA)/benzodiazepine receptor complex with efficacies ranging from inverse agonists to full agonists. Many analogues were found to be partial agonists as indicated by [35S]TBPS and Cl- current ratios. Uniquely, a number of these analogues were found to have a bell-shaped dose-response profile in the alpha1 beta2 gamma2 subtype as determined by whole cell patch-clamp technique, where in vitro efficacy was found to decrease with increasing drug concentration. Many of the compounds from this series were effective in antagonizing metrazole-induced seizures, consistent with anticonvulsant and possibly anxiolytic activity. Additionally, several analogues were also effective in lowering cGMP levels (to control values) after applied stress, also consistent with anxiolytic-like properties. The most effective compounds in these screens were also active in animal models of anxiety such as the Vogel and Geller assays. The use of the piperazine substituent allowed for excellent drug levels and a long duration of action in the central nervous system for many of the quinoxalines, as determined by ex vivo assay. Pharmacokinetic analysis of several compounds indicated excellent oral bioavailability and a reasonable half-life in rats. From this series emerged two partial agonists (55, 91) which had good activity in anxiolytic models, acceptable pharmacokinetics, and minimal benzodiazepine-type side effects.

  9. Functional mapping and implications of substrate specificity of the yeast high-affinity leucine permease Bap2.

    PubMed

    Usami, Yuki; Uemura, Satsohi; Mochizuki, Takahiro; Morita, Asami; Shishido, Fumi; Inokuchi, Jin-ichi; Abe, Fumiyoshi

    2014-07-01

    Leucine is a major amino acid in nutrients and proteins and is also an important precursor of higher alcohols during brewing. In Saccharomyces cerevisiae, leucine uptake is mediated by multiple amino acid permeases, including the high-affinity leucine permease Bap2. Although BAP2 transcription has been extensively analyzed, the mechanisms by which a substrate is recognized and moves through the permease remain unknown. Recently, we determined 15 amino acid residues required for Tat2-mediated tryptophan import. Here we introduced homologous mutations into Bap2 amino acid residues and showed that 7 residues played a role in leucine import. Residues I109/G110/T111 and E305 were located within the putative α-helix break in TMD1 and TMD6, respectively, according to the structurally homologous Escherichia coli arginine/agmatine antiporter AdiC. Upon leucine binding, these α-helix breaks were assumed to mediate a conformational transition in Bap2 from an outward-open to a substrate-binding occluded state. Residues Y336 (TMD7) and Y181 (TMD3) were located near I109 and E305, respectively. Bap2-mediated leucine import was inhibited by some amino acids according to the following order of severity: phenylalanine, leucine>isoleucine>methionine, tyrosine>valine>tryptophan; histidine and asparagine had no effect. Moreover, this order of severity clearly coincided with the logP values (octanol-water partition coefficients) of all amino acids except tryptophan. This result suggests that the substrate partition efficiency to the buried Bap2 binding pocket is the primary determinant of substrate specificity rather than structural amino acid side chain recognition.

  10. High affinity FRβ-specific CAR T cells eradicate AML and normal yeloid lineage without HSC toxicity

    PubMed Central

    Lynn, Rachel C; Feng, Yang; Schutsky, Keith; Poussin, Mathilde; Kalota, Anna; Dimitrov, Dimiter S; Powell, Daniel J

    2016-01-01

    Acute myeloid leukemia (AML) is an aggressive malignancy, and development of new treatments to prolong remissions is warranted. Chimeric antigen receptor (CAR) T-cell therapies appear promising but on-target, off-tumor recognition of antigen in healthy tissues remains a concern. Here, we isolated a high affinity (HA) folate receptor beta (FRβ)-specific scFv (2.48nM KD) for optimization of FRβ-redirected CAR T-cell therapy for AML. T-cells stably expressing the HA-FRβ CAR exhibited greatly enhanced antitumor activity against FRβ+ AML in vitro and in vivo compared to a low affinity (LA) FRβ CAR (54.3nM KD). Using the HA-FRβ IgG, FRβ expression was detectable in myeloid-lineage hematopoietic cells; however, expression in CD34+ hematopoietic stem cells (HSCs) was nearly undetectable. Accordingly, HA-FRβ CAR T-cells lysed mature CD14+ monocytes, while HSC colony formation was unaffected. Because of the potential for elimination of mature myeloid lineage, mRNA CAR electroporation for transient CAR expression was evaluated. mRNA-electroporated HA-FRβ CAR T-cells retained effective anti-tumor activity in vitro and in vivo. Together, our results highlight the importance of antibody affinity in target protein detection and CAR development and suggest that transient delivery of potent HA-FRβ CAR T-cells is highly effective against AML and reduces the risk for long-term myeloid toxicity. PMID:26898190

  11. Regulation of the high-affinity choline transporter activity and trafficking by its association with cholesterol-rich lipid rafts.

    PubMed

    Cuddy, Leah K; Winick-Ng, Warren; Rylett, Rebecca Jane

    2014-03-01

    The sodium-coupled, hemicholinium-3-sensitive, high-affinity choline transporter (CHT) is responsible for transport of choline into cholinergic nerve terminals from the synaptic cleft following acetylcholine release and hydrolysis. In this study, we address regulation of CHT function by plasma membrane cholesterol. We show for the first time that CHT is concentrated in cholesterol-rich lipid rafts in both SH-SY5Y cells and nerve terminals from mouse forebrain. Treatment of SH-SY5Y cells expressing rat CHT with filipin, methyl-β-cyclodextrin (MβC) or cholesterol oxidase significantly decreased choline uptake. In contrast, CHT activity was increased by addition of cholesterol to membranes using cholesterol-saturated MβC. Kinetic analysis of binding of [(3)H]hemicholinium-3 to CHT revealed that reducing membrane cholesterol with MβC decreased both the apparent binding affinity (KD) and maximum number of binding sites (Bmax ); this was confirmed by decreased plasma membrane CHT protein in lipid rafts in cell surface protein biotinylation assays. Finally, the loss of cell surface CHT associated with lipid raft disruption was not because of changes in CHT internalization. In summary, we provide evidence that CHT association with cholesterol-rich rafts is critical for transporter function and localization. Alterations in plasma membrane cholesterol cholinergic nerve terminals could diminish cholinergic transmission by reducing choline availability for acetylcholine synthesis. The sodium-coupled choline transporter CHT moves choline into cholinergic nerve terminals to serve as substrate for acetylcholine synthesis. We show for the first time that CHT is concentrated in cholesterol-rich lipid rafts, and decreasing membrane cholesterol significantly reduces both choline uptake activity and cell surface CHT protein levels. CHT association with cholesterol-rich rafts is critical for its function, and alterations in plasma membrane cholesterol could diminish cholinergic

  12. Evaluation of the full evaporation technique for quantitative analysis of high boiling compounds with high affinity for apolar matrices.

    PubMed

    van Boxtel, Niels; Wolfs, Kris; van Schepdael, Ann; Adams, Erwin

    2014-06-27

    In order to reduce inaccuracies due to possible matrix effects in conventional static headspace-gas chromatography (sHS-GC), it is standard practice to match the composition of calibration standards towards the composition of the sample to be analysed by adding blank matrix. However, the latter is not always available and in that case the full evaporation technique (FET) could be a solution. With FET a small sample volume is introduced in a HS vial and compounds of interest are completely evaporated. Hence no equilibrium between the condensed phase and vapour phase exists. Without the existence of an equilibrium, matrix effects are less likely to occur. Another issue often encountered with sHS-sampling is that low vapour pressure compounds with a high affinity for the dilution medium show a limited sensitivity. FET has proven to be an appropriate solution to address this problem too. In this work, the applicability of FET for the quantitative analysis of high boiling compounds in different complex apolar matrices is examined. Data show that FET is an excellent tool to overcome matrix effects often encountered with conventional sHS analysis. The tested method shows excellent accuracy with recovery values around 100% as well as repeatability with RSD values around 1% for the quantification of high boiling compounds (bp>200°C) such as camphor, menthol, methyl salicylate and ethyl salicylate in various matrices. LOQ values were found to be around 0.3μg per vial. Following validation of the technique, several topical pharmaceutical formulations like ThermoCream(®), Reflexspray(®), Vicks Vaporub(®) and Radosalil(®) were examined. For the latter, a comparison has been made with a sHS-method described in literature.

  13. Dephosphorylation and quantification of organic phosphorus in poultry litter by purified phytic-acid high affinity Aspergillus phosphohydrolases.

    PubMed

    Dao, Thanh H; Hoang, Khanh Q

    2008-08-01

    Extracellular phosphohydrolases mediate the dephosphorylation of phosphoesters and influence bioavailability and loss of agricultural P to the environment to pose risks of impairment of sensitive aquatic ecosystems. Induction and culture of five strains of Aspergillus were conducted to develop a source of high-affinity and robust phosphohydrolases for detecting environmental P and quantifying bioactive P pools in heterogeneous environmental specimens. Enzyme stability and activity against organic P in poultry litter were evaluated in 71 samples collected across poultry producing regions of Arkansas, Maryland, and Oklahoma of the US Differences existed in strains' adaptability to fermentation medium as they showed a wide range of phytate-degrading activity. Phosphohydrolases from Aspergillus ficuum had highest activity when the strain was cultured on a primarily chemical medium, compared to Aspergillus oryzae which preferred a wheat bran-based organic medium. Kinetics parameters of A. ficuum enzymes (K(m)=210 microM; V(max) of 407 nmol s(-1)) indicated phytic acid-degrading potential equivalent to that of commercial preparations. Purified A. ficuum phosphohydrolases effectively quantified litter bioactive P pools, showing that organic P occurred at an average of 54 (+/-14)% of total P, compared to inorganic phosphates, which averaged 41 (+/-12)%. Litter management and land application options must consider the high water-extractable and organic P concentrations and the biological availability of the organic enzyme-labile P pool. Robustness of A. ficuum enzymes and simplicity of the in situ ligand-based enzyme assay may thus increase routine assessment of litter bioactive P composition to sense for on-farm accumulation of such environmentally-sensitive P forms. PMID:18555509

  14. Characterization of a common high-affinity receptor for reovirus serotypes 1 and 3 on endothelial cells.

    PubMed Central

    Verdin, E M; King, G L; Maratos-Flier, E

    1989-01-01

    During viremia, viruses may be cleared from the bloodstream and taken up by specific organs. The uptake of virus from the bloodstream is dependent on the association of viral particles with endothelial cells that line the luminal surfaces of large and small blood vessels. To understand the nature of this interaction, we have studied the binding of reovirus serotypes 1 and 3 to these cells in vitro. Both serotypes of reovirus productively infected endothelial cells. By using [35S]methionine-biolabeled reovirus as a tracer ligand, we found that both viruses rapidly bind to endothelial cells and that equilibrium is reached after 4 h. The binding of the radiolabeled viruses was saturable and mediated by a homogeneous population of cellular receptors with very high affinity (Kd = 0.5 nM) for the virus ligands. Both serotypes bind to the same receptor, since the attachment of each radiolabeled serotype is inhibited by both the homologous and heterologous unlabeled virus. Exposure of labeled virus to monoclonal antibodies directed against the viral hemagglutinin (sigma 1 protein) inhibited binding, demonstrating that the attachment of reovirus to endothelial cells is mediated by the hemagglutinin for both serotypes. By using a novel ligand-blotting assay, the binding of both viruses to a 54,000-dalton protein could be demonstrated. The binding of each radiolabeled serotype to this protein was inhibited by the homologous and heterologous unlabeled serotype. By using cell fractionation after homogenization, we demonstrated that this 54-kilodalton protein is a membrane protein, in agreement with its proposed role as a cell surface receptor for reovirus serotypes 1 and 3. Images PMID:2915382

  15. Tethering of Epidermal Growth Factor (EGF) to Beta Tricalcium Phosphate (βTCP) via Fusion to a High Affinity, Multimeric βTCP-Binding Peptide: Effects on Human Multipotent Stromal Cells/Connective Tissue Progenitors

    PubMed Central

    Stockdale, Linda; Saini, Sunil; Lee, Richard T.; Griffith, Linda G.

    2015-01-01

    Transplantation of freshly-aspirated autologous bone marrow, together with a scaffold, is a promising clinical alternative to harvest and transplantation of autologous bone for treatment of large defects. However, survival proliferation, and osteogenic differentiation of the marrow-resident stem and progenitor cells with osteogenic potential can be limited in large defects by the inflammatory microenvironment. Previous studies using EGF tethered to synthetic polymer substrates have demonstrated that surface-tethered EGF can protect human bone marrow-derived osteogenic stem and progenitor cells from pro-death inflammatory cues and enhance their proliferation without detriment to subsequent osteogenic differentiation. The objective of this study was to identify a facile means of tethering EGF to clinically-relevant βTCP scaffolds and to demonstrate the bioactivity of EGF tethered to βTCP using stimulation of the proliferative response of human bone-marrow derived mesenchymal stem cells (hBMSC) as a phenotypic metric. We used a phage display library and panned against βTCP and composites of βTCP with a degradable polyester biomaterial, together with orthogonal blocking schemes, to identify a 12-amino acid consensus binding peptide sequence, LLADTTHHRPWT, with high affinity for βTCP. When a single copy of this βTCP-binding peptide sequence was fused to EGF via a flexible peptide tether domain and expressed recombinantly in E. coli together with a maltose-binding domain to aid purification, the resulting fusion protein exhibited modest affinity for βTCP. However, a fusion protein containing a linear concatamer containing 10 repeats of the binding motif the resulting fusion protein showed high affinity stable binding to βTCP, with only 25% of the protein released after 7 days at 37oC. The fusion protein was bioactive, as assessed by its abilities to activate kinase signaling pathways downstream of the EGF receptor when presented in soluble form, and to enhance

  16. Fast-onset lidocaine block of rat NaV1.4 channels suggests involvement of a second high-affinity open state.

    PubMed

    Gingrich, Kevin J; Wagner, Larry E

    2016-06-01

    Local anesthetics (LAs) block resting, open, and inactivated states of voltage-gated Na(+) channels where inactivated states are thought to bind with highest affinity. However, reports of fast-onset block occurring over milliseconds hint at high-affinity block of open channels. Movement of voltage-sensor domain IV-segment 4 (DIVS4) has been associated with high affinity LA block termed voltage-sensor block (VSB) that also leads to a second open state. These observations point to a second high-affinity open state that may underlie fast-onset block. To test for this state, we analyzed the modulation of Na(+) currents by lidocaine and its quaternary derivative (QX222) from heterologously expressed (Xenopus laevis oocytes) rat skeletal muscle μ1 NaV1.4 (rSkM1) with β1 (WT-β1), and a mutant form (IFM-QQQ mutation in the III-IV interdomain, QQQ) lacking fast inactivation, in combination with Markov kinetic gating models. 100 μM lidocaine induced fast-onset (τonset≈2 ms), long-lived (τrecovery≈120 ms) block of WT-β1 macroscopic currents. Lidocaine blocked single-channel and macroscopic QQQ currents in agreement with our previously described mechanism of dual, open-channel block (DOB mechanism). A DOB kinetic model reproduced lidocaine effects on QQQ currents. The DOB model was extended to include trapping fast-inactivation and activation gates, and a second open state (OS2); the latter arising from DIVS4 translocation that precedes inactivation and exhibits high-affinity, lidocaine binding (apparent Kd=25 μM) that accords with VSB (DOB-S2VSB mechanism). The DOB-S2VSB kinetic model predicted fast-onset block of WT-β1. The findings support the involvement of a second, high-affinity, open state in lidocaine modulation of Na(+) channels.

  17. Purification, crystallization and preliminary X-ray crystallographic analysis of mammalian MSS4–Rab8 GTPase protein complex

    SciTech Connect

    Itzen, Aymelt; Bleimling, Nathalie; Ignatev, Alexander; Pylypenko, Olena; Rak, Alexey

    2006-02-01

    The MSS4 (mammalian suppressor of Sec4) protein in complex with nucleotide-free Rab8 GTPase has been purified and crystallized in a form suitable for structure analysis and a complete data set has been collected to 2 Å resolution. Rab GTPases function as ubiquitous key regulators of membrane-vesicle transport in eukaryotic cells. MSS4 is an evolutionarily conserved protein that binds to exocytotic Rabs and facilitates nucleotide release. The MSS4 protein in complex with nucleotide-free Rab8 GTPase has been purified and crystallized in a form suitable for structure analysis. The crystals belonged to space group P1, with unit-cell parameters a = 40.92, b = 49.85, c = 83.48 Å, α = 102.88, β = 97.46, γ = 90.12°. A complete data set has been collected to 2 Å resolution.

  18. Fluorescent actin analogs with a high affinity for profilin in vitro exhibit an enhanced gradient of assembly in living cells

    PubMed Central

    1994-01-01

    Constitutive centripetal transport of the actin-based cytoskeleton has been detected in cells spreading on a substrate, locomoting fibroblasts and keratocytes, and non-locomoting serum-deprived fibroblasts. These results suggest a gradient of actin assembly, highest in the cortex at the cytoplasm-membrane interface and lowest in the non-cortical perinuclear cytoplasm. We predicted that such a gradient would be maintained in part by phosphoinositide-regulated actin binding proteins because the intracellular free Ca2+ and pH are low and spatially constant in serum-deprived cells. The cytoplasm-membrane interface presents one surface where the assembly of actin is differentially regulated relative to the non-cortical cytoplasm. Several models, based on in vitro biochemistry, propose that phosphoinositide-regulated actin binding proteins are involved in local actin assembly. To test these models in living cells using imaging techniques, we prepared a new fluorescent analog of actin that bound profilin, a protein that interacts with phosphoinositides and actin-monomers in a mutually exclusive manner, with an order of magnitude greater affinity (Kd = 3.6 microM) than cys-374-labeled actin (Kd > 30 microM), yet retained the ability to inhibit DNase I. Hence, we were able to directly compare the distribution and activity of a biochemical mutant of actin with an analog possessing closer to wild-type activity. Three-dimensional fluorescence microscopy of the fluorescent analog of actin with a high affinity for profilin revealed that it incorporated into cortical cytoplasmic fibers and was also distributed diffusely in the non- cortical cytoplasm consistent with a bias of actin assembly near the surface of the cell. Fluorescence ratio imaging revealed that serum- deprived and migrating fibroblasts concentrated the new actin analog into fibers up to four-fold in the periphery and leading edge of these cells, respectively, relative to a soluble fluorescent dextran volume marker

  19. Human Eosinophils Express the High Affinity IgE Receptor, FcεRI, in Bullous Pemphigoid

    PubMed Central

    Messingham, Kelly N.; Holahan, Heather M.; Frydman, Alexandra S.; Fullenkamp, Colleen; Srikantha, Rupasree; Fairley, Janet A.

    2014-01-01

    Bullous pemphigoid (BP) is an autoimmune blistering disease mediated by autoantibodies targeting BP180 (type XVII collagen). Patient sera and tissues typically have IgG and IgE autoantibodies and elevated eosinophil numbers. Although the pathogenicity of the IgE autoantibodies is established in BP, their contribution to the disease process is not well understood. Our aims were two-fold: 1) To establish the clinical relationships between total and BP180-specific IgE, eosinophilia and other markers of disease activity; and 2) To determine if eosinophils from BP patients express the high affinity IgE receptor, FcεRI, as a potential mechanism of action for IgE in BP. Our analysis of 48 untreated BP patients revealed a correlation between BP180 IgG and both BP180 IgE and peripheral eosinophil count. Additionally, we established a correlation between total IgE concentration and both BP180 IgE levels and eosinophil count. When only sera from patients (n = 16) with total IgE≥400 IU/ml were analyzed, BP180 IgG levels correlated with disease severity, BP230 IgG, total circulating IgE and BP180 IgE. Finally, peripheral eosinophil count correlated more strongly with levels of BP180 IgE then with BP180 IgG. Next, eosinophil FcεRI expression was investigated in the blood and skin using several methods. Peripheral eosinophils from BP patients expressed mRNA for all three chains (α, β and γ) of the FcεRI. Surface expression of the FcεRIα was confirmed on both peripheral and tissue eosinophils from most BP patients by immunostaining. Furthermore, using a proximity ligation assay, interaction of the α- and β-chains of the FcεRI was observed in some biopsy specimens, suggesting tissue expression of the trimeric receptor form in some patients. These studies provide clinical support for the relevance of IgE in BP disease and provide one mechanism of action of these antibodies, via binding to the FcεRI on eosinophils. PMID:25255430

  20. Human eosinophils express the high affinity IgE receptor, FcεRI, in bullous pemphigoid.

    PubMed

    Messingham, Kelly N; Holahan, Heather M; Frydman, Alexandra S; Fullenkamp, Colleen; Srikantha, Rupasree; Fairley, Janet A

    2014-01-01

    Bullous pemphigoid (BP) is an autoimmune blistering disease mediated by autoantibodies targeting BP180 (type XVII collagen). Patient sera and tissues typically have IgG and IgE autoantibodies and elevated eosinophil numbers. Although the pathogenicity of the IgE autoantibodies is established in BP, their contribution to the disease process is not well understood. Our aims were two-fold: 1) To establish the clinical relationships between total and BP180-specific IgE, eosinophilia and other markers of disease activity; and 2) To determine if eosinophils from BP patients express the high affinity IgE receptor, FcεRI, as a potential mechanism of action for IgE in BP. Our analysis of 48 untreated BP patients revealed a correlation between BP180 IgG and both BP180 IgE and peripheral eosinophil count. Additionally, we established a correlation between total IgE concentration and both BP180 IgE levels and eosinophil count. When only sera from patients (n = 16) with total IgE ≥ 400 IU/ml were analyzed, BP180 IgG levels correlated with disease severity, BP230 IgG, total circulating IgE and BP180 IgE. Finally, peripheral eosinophil count correlated more strongly with levels of BP180 IgE then with BP180 IgG. Next, eosinophil FcεRI expression was investigated in the blood and skin using several methods. Peripheral eosinophils from BP patients expressed mRNA for all three chains (α, β and γ) of the FcεRI. Surface expression of the FcεRIα was confirmed on both peripheral and tissue eosinophils from most BP patients by immunostaining. Furthermore, using a proximity ligation assay, interaction of the α- and β-chains of the FcεRI was observed in some biopsy specimens, suggesting tissue expression of the trimeric receptor form in some patients. These studies provide clinical support for the relevance of IgE in BP disease and provide one mechanism of action of these antibodies, via binding to the FcεRI on eosinophils. PMID:25255430

  1. Identification of selective, high affinity [125I]-angiotensin and [125I]-bradykinin binding sites in rat intestinal epithelia.

    PubMed Central

    Cox, H. M.; Munday, K. A.; Poat, J. A.

    1986-01-01

    Specific [125I]-angiotensin II (AII) and [125I]-bradykinin (Bk) binding sites have been identified within epithelial membranes from rat jejunum and descending colon. These high affinity intestinal sites exhibited KD values of 0.64 +/- 0.16 nM for [125I]-AII and 0.69 +/- 0.13 nM for [125I]-Bk, which were similar to those for [125I]-AII (0.85 nM) and [125I]-Bk binding sites (1.03 nM) previously identified in renal cortex epithelia. Specific [125I]-AII binding capacity was only 19.77 +/- 2.74 fmol mg-1 in small intestine and 11.31 +/- 2.66 fmol mg-1 in descending colon epithelia while a larger population, 332.0 +/- 72.9 fmol-mg-1 of specific [125I]-Bk sites were identified in epithelial membranes from small intestine. Significant hydrolysis of both free [125I]-AII and [125I]-Bk was observed while membrane bound peptides remained relatively resistant to degradation. Whilst no corrections have been made to the observed values of KD and Bmax quoted above, one may assume that the calculated reductions in the free hormone concentration will result in a decrease of the KD value for both peptides. Loss of membrane bound peptide, particularly of [125I]-AII, may indicate that the calculated Bmax value is an underestimation. Despite the rapid degradation of unbound [125I]-AII and [125I]-Bk during incubations the kinetics of specific peptide binding were reversible and highly selective. The order of potency for specific [125I]-AII binding was [Sar1, Leu8]-AII greater than or equal to [Sar1, Thr8]-AII greater than or equal to AII greater than [Sar1, Ile8]-AII greater than or equal to [Des, Asp1, Ile8] AII greater than AIII. Specific [125I]-Bk binding was also highly selective, the order of potency being Phe8-Bk greater than or equal to Tyr8-Bk greater than or equal to Lys-Bk much greater than Des, Arg1-Bk. AII exhibited an IC50 of greater than 1mM for specific [125I]-Bk binding and likewise Phe8-Bk for specific [125I]-AII binding. PMID:2869810

  2. Safety, Pharmacokinetics, Pharmacodynamics, and Activity of Navitoclax, a Targeted High Affinity Inhibitor of BCL-2, in Lymphoid Malignancies

    PubMed Central

    Wilson, Wyndham H.; O’Connor, Owen A.; Czuczman, Myron S.; LaCasce, Ann S.; Gerecitano, John F.; Leonard, John P.; Tulpule, Anil; Dunleavy, Kieron; Xiong, Hao; Chiu, Yi-Lin; Cui, Yue; Busman, Todd; Elmore, Steven W.; Rosenberg, Saul H.; Krivoshik, Andrew P.; Enschede, Sari H.; Humerickhouse, Rod A.

    2010-01-01

    SUMMARY Background BCL-2 family proteins play a central role in regulating clonal selection and survival of lymphocytes and are frequently over expressed in lymphomas. Navitoclax (ABT-263) is a targeted high-affinity small molecule that occupies the BH3 binding groove of BCL-2 and BCL-XL and inhibits their anti-apoptotic activity. Experimentally, navitoclax kills cells in a BAX/BAK-dependent manner and results in regression of lymphoid tumors in xenograft models. Methods This is a phase I dose-escalation study of navitoclax in patients with relapsed or refractory lymphoid malignancies. Study endpoints included safety, maximum tolerated dose (MTD), pharmacokinetic profile and clinical activity. In addition, mechanism-based pharmacodynamic effects on platelets and lymphocytes were assessed. Navitoclax was orally administered and assessed on an intermittent schedule of once daily for 14 days followed by 7 days off (14/21 days) or on a continuous once daily schedule (21/21 days). This trial is registered with ClinicalTrials.gov, number NCT00406809. Findings Fifty-five patients were enrolled, (median age 59 years, IQR 51–67), of whom two did not complete the first cycle and were not evaluable for assessment of dose-limiting toxicity (DLT). Common toxicities included grade 1/2 diarrhea and fatigue in 31 and 21 patients, respectively. Thrombocytopenia and neutropenia were the serious common toxicities with grade 3/4 observed in 29 and 17 patients, respectively. On the intermittent schedule (14/21), 5 DLT’s were observed; two due to hospitalizations for bronchitis and pleural effusion, and one each due to grade 3 transaminase elevation, grade 4 thrombocytopenia and grade 3 cardiac arrhythmia. Navitoclax caused a rapid and dose-dependent decline in peripheral platelets following initial drug exposure, followed by a rebound. To reduce the platelet nadir associated with intermittent dosing, a lead-in dose followed by continuous dosing (21/21 schedule) was examined. Three

  3. Structure of the high-affinity binding site for noncompetitive blockers of the acetylcholine receptor: serine-262 of the delta subunit is labeled by [3H]chlorpromazine.

    PubMed Central

    Giraudat, J; Dennis, M; Heidmann, T; Chang, J Y; Changeux, J P

    1986-01-01

    The membrane-bound acetylcholine receptor from Torpedo marmorata was photolabeled by the noncompetitive channel blocker [3H]chlorpromazine under equilibrium conditions in the presence of agonist. Incorporation of radioactivity into all subunits occurred and was reduced by addition of phencyclidine, a specific ligand for the high-affinity site for noncompetitive blockers. The delta subunit was purified and digested with trypsin, and the resulting fragments were fractionated by reversed-phase HPLC. The labeled peptide could not be purified to homogeneity because of its marked hydrophobic character, but a combination of differential CNBr subcleavage and cosequencing of partially purified fragments enabled us to identify Ser-262 as being labeled by [3H]chlorpromazine. The labeling of this particular residue was prevented by phencyclidine and thus took place at the level of, or in proximity to, the high-affinity site for noncompetitive blockers. Ser-262 is located in a hydrophobic and potentially transmembrane segment termed MII. Images PMID:3085104

  4. Cloning of chrysanthemum high-affinity nitrate transporter family (CmNRT2) and characterization of CmNRT2.1.

    PubMed

    Gu, Chunsun; Song, Aiping; Zhang, Xiaoxue; Wang, Haibin; Li, Ting; Chen, Yu; Jiang, Jiafu; Chen, Fadi; Chen, Sumei

    2016-01-01

    The family of NITRATE TRANSPORTER 2 (NRT2) proteins belongs to the high affinity transport system (HATS) proteins which acts at low nitrate concentrations. The relevant gene content of the chrysanthemum genome was explored here by isolating the full length sequences of six distinct CmNRT2 genes. One of these (CmNRT2.1) was investigated at the functional level. Its transcription level was inducible by low concentrations of both nitrate and ammonium. A yeast two hybrid assay showed that CmNRT2.1 interacts with CmNAR2, while a BiFC assay demonstrated that the interaction occurs at the plasma membrane. Arabidopsis thaliana plants heterologously expressing CmNRT2.1 displayed an enhanced rate of labeled nitrogen uptake, suggesting that CmNRT2.1 represents a high affinity root nitrate transporter. PMID:27004464

  5. High-affinity K(+) transport in Arabidopsis: AtHAK5 and AKT1 are vital for seedling establishment and postgermination growth under low-potassium conditions.

    PubMed

    Pyo, Young Jae; Gierth, Markus; Schroeder, Julian I; Cho, Myeon Haeng

    2010-06-01

    Potassium (K(+)) is a major plant nutrient required for growth and development. It is generally accepted that plant roots absorb K(+) through uptake systems operating at low concentrations (high-affinity transport) and/or high external concentrations (low-affinity transport). To understand the molecular basis of high-affinity K(+) uptake in Arabidopsis (Arabidopsis thaliana), we analyzed loss-of-function mutants in AtHAK5 and AKT1, two transmembrane proteins active in roots. Compared with the wild type under NH(4)(+)-free growth conditions, athak5 mutant plants exhibited growth defects at 10 mum K(+), but at K(+) concentrations of 20 mum and above, athak5 mutants were visibly indistinguishable from the wild type. While germination, scored as radicle emergence, was only slightly decreased in athak5 akt1 double mutants on low-K(+) medium, double mutants failed to grow on medium containing up to 100 mum K(+) and growth was impaired at concentrations up to 450 mum K(+). Moreover, transfer of 3-d-old plants from high to low K(+) concentrations led to growth defects and leaf chlorosis at 10 mum K(+) in athak5 akt1 double mutant plants. Determination of Rb(+)(K(+)) uptake kinetics in wild-type and mutant roots using rubidium ((86)Rb(+)) as a tracer for K(+) revealed that high-affinity Rb(+)(K(+)) uptake into roots is almost completely abolished in double mutants and impaired in single mutants. These results strongly indicate that AtHAK5 and AKT1 are the two major, physiologically relevant molecular entities mediating high-affinity K(+) uptake into roots during seedling establishment and postgermination growth and that residual Rb(+)(K(+)) uptake measured in athak5 akt1 double mutant roots is insufficient to enable plant growth. PMID:20413648

  6. Solubilization of high affinity corticotropin-releasing factor receptors from rat brain: Characterization of an active digitonin-solubilized receptor complex

    SciTech Connect

    Grigoriadis, D.E.; Zaczek, R.; Pearsall, D.M.; De Souza, E.B. )

    1989-12-01

    The binding characteristics of CRF receptors in rat frontal cerebral cortex membranes solubilized in 1% digitonin were determined. The binding of (125I)Tyro-ovine CRF ((125I)oCRF) to solubilized membrane proteins was dependent on incubation time, temperature, and protein concentration, was saturable and of high affinity, and was absent in boiled tissue. The solubilized receptors retained their high affinity for (125I) oCRF in the solubilized state, exhibiting a dissociation constant (KD) of approximately 200 pM, as determined by direct binding saturation isotherms. Solubilized CRF receptors maintained the rank order of potencies for various related and unrelated CRF peptides characteristic of the membrane CRF receptor: rat/human CRF congruent to ovine CRF congruent to Nle21,38-rat CRF greater than alpha-helical oCRF-(9-41) greater than oCRF-(7-41) much greater than vasoactive intestinal peptide, arginine vasopressin, or the substance-P antagonist. Furthermore, the absolute potencies (Ki values) for the various CRF-related peptides in solubilized receptors were almost identical to those observed in the membrane preparations, indicating that the CRF receptor retained its high affinity binding capacity in the digitonin-solubilized state. Chemical affinity cross-linking of digitonin-solubilized rat cortical membrane proteins revealed a specifically labeled protein with an apparent mol wt of 58,000 which was similar to the labeled protein in native membrane homogenates. Although solubilized CRF receptors retained their high affinity for agonists, their sensitivity for guanine nucleotide was lost. Size exclusion chromatography substantiated these results, demonstrating that in the presence or absence of guanine nucleotides, (125I)oCRF labeled the same size receptor complex.

  7. Synthesis, Structure-affinity Relationships and Radiolabeling of Selective High-affinity 5-HT4 Receptor Ligands as Prospective Imaging Probes for PET

    PubMed Central

    Xu, Rong; Hong, Jinsoo; Morse, Cheryl L.; Pike, Victor W.

    2010-01-01

    In a search for high-affinity receptor ligands that might serve for development as radioligands for the imaging of brain 5-HT4 receptors in vivo with positron emission tomography (PET), structural modifications were made to the high-affinity 5-HT4 antagonist, (1-butylpiperidin-4-yl)methyl 8-amino-7-iodo-2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (1, SB 207710). These modifications were made mainly on the aryl side of the ester bond to permit possible rapid labeling of the carboxylic acid component with a positron-emitter, either carbon-11 (t1/2 = 20.4 min) or fluorine-18 (t1/2 = 109.7 min), and included, i) replacement of the iodine atom with a small substituent such as nitrile, methyl or fluoro, ii) methylation of the 8-amino group, iii) opening of the dioxan ring, and iv) alteration of the length of the N-alkyl goup. High-affinity ligands were discovered for recombinant human 5-HT4 receptors with amenability to labeling with a positron-emitter and potential for development as imaging probes. The ring-opened radioligand, (([methoxy-11C]1-butylpiperidin-4-yl)methyl 4-amino-3-methoxybenzoate; [11C]13), showed an especially favorable array of properties for future evaluation as a PET radioligand for brain 5-HT4 receptors. PMID:20812727

  8. Identification of a PutP proline permease gene homolog from Staphylococcus aureus by expression cloning of the high-affinity proline transport system in Escherichia coli.

    PubMed Central

    Wengender, P A; Miller, K J

    1995-01-01

    The important food-borne pathogen Staphylococcus aureus is distinguished by its ability to grow at low water activity values. Previous work in our laboratory and by others has revealed that proline accumulation via transport is an important osmoregulatory strategy employed by this bacterium. Furthermore, proline uptake by this bacterium has been shown to be mediated by two distinct transport systems: a high-affinity system and a low-affinity system (J.-H. Bae, and K. J. Miller, Appl. Environ. Microbiol. 58:471-475, 1992; D. E. Townsend and B. J. Wilkinson, J. Bacteriol. 174:2702-2710, 1992). In the present study, we report the cloning of the high-affinity proline transport system of S. aureus by functional expression in an Escherichia coli host. The sequence of the staphylococcal proline permease gene was predicted to encode a protein of 497 amino acids which shares 49% identity with the PutP high-affinity proline permease of E. coli. Analysis of hydropathy also indicated a common overall structure for these proteins. PMID:7887605

  9. Excited protein states of human tear lipocalin for low- and high-affinity ligand binding revealed by functional AB loop motion.

    PubMed

    Gasymov, Oktay K; Abduragimov, Adil R; Glasgow, Ben J

    2010-06-01

    Human tear lipocalin (TL), a prominent member of lipocalin family, exhibits functional and structural promiscuity. The plasticity of loop regions modulates entry to the ligand pocket at the "open" end of the eight-stranded beta-barrel. Site-directed multi-distance measurements using fluorescence resonance energy transfer between functional loops register two excited protein states for low- and high-affinity ligand binding. At low pH, the longest loop AB adopts the conformation of the low-affinity excited protein state that matches the crystal structure of holo-TL at pH 8. A "crankshaft" like movement is detected for the loop AB in a low pH transition. At pH 7.3 the holo-protein assumes a high-affinity excited protein state, in which the loop AB is more compact (RMS=3.1A). In the apo-holo transition, the reporter Trp 28 moves about 4.5A that reflects a decrease in distance between Glu27 and Lys108. This interaction fixes the loop AB conformation for the high-affinity mode. No such movement is detected at low pH, where Glu27 is protonated. Data strongly indicate that the protonation state of Glu27 modulates the conformation of the loop AB for high- and low-affinity binding. PMID:20439130

  10. Synthesis, structure-affinity relationships, and radiolabeling of selective high-affinity 5-HT4 receptor ligands as prospective imaging probes for positron emission tomography.

    PubMed

    Xu, Rong; Hong, Jinsoo; Morse, Cheryl L; Pike, Victor W

    2010-10-14

    In a search for high-affinity receptor ligands that might serve for development as radioligands for the imaging of brain 5-HT(4) receptors in vivo with positron emission tomography (PET), structural modifications were made to the high-affinity 5-HT(4) antagonist (1-butylpiperidin-4-yl)methyl 8-amino-7-iodo-2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (1, SB 207710). These modifications were made mainly on the aryl side of the ester bond to permit possible rapid labeling of the carboxylic acid component with a positron emitter, either carbon-11 (t(1/2) = 20.4 min) or fluorine-18 (t(1/2) = 109.7 min), and included (i) replacement of the iodine atom with a small substituent such as nitrile, methyl, or fluoro, (ii) methylation of the 8-amino group, (iii) opening of the dioxan ring, and (iv) alteration of the length of the N-alkyl goup. High-affinity ligands were discovered for recombinant human 5-HT(4) receptors with amenability to labeling with a positron emitter and potential for development as imaging probes. The ring-opened radioligand, (([methoxy-(11)C]1-butylpiperidin-4-yl)methyl 4-amino-3-methoxybenzoate; [(11)C]13), showed an especially favorable array of properties for future evaluation as a PET radioligand for brain 5-HT(4) receptors. PMID:20812727

  11. The presence of high-affinity, low-capacity estradiol-17β binding in rainbow trout scale indicates a possible endocrine route for the regulation of scale resorption

    USGS Publications Warehouse

    Persson, Petra; Shrimpton, J.M.; McCormick, S.D.; Bjornsson, Bjorn Thrandur

    2000-01-01

    High-affinity, low-capacity estradiol-17β (E2) binding is present in rainbow trout scale. The Kd and Bmax of the scale E2 binding are similar to those of the liver E2 receptor (Kd is 1.6 ± 0.1 and 1.4 ± 0.1 nM, and Bmax is 9.1 ± 1.2 and 23.1 ± 2.2 fmol x mg protein-1, for scale and liver, respectively), but different from those of the high-affinity, low-capacity E2 binding in plasma (Kd is 4.0 ± 0.4 nM and Bmax is 625.4 ± 63.1 fmol x mg protein-1). The E2 binding in scale was displaced by testosterone, but not by diethylstilbestrol. Hence, the ligand binding specificity is different from that of the previously characterized liver E2 receptor, where E2 is displaced by diethylstilbestrol, but not by testosterone. The putative scale E2 receptor thus appears to bind both E2 and testosterone, and it is proposed that the increased scale resorption observed during sexual maturation in both sexes of several salmonid species may be mediated by this receptor. No high-affinity, low-capacity E2 binding could be detected in rainbow trout gill or skin.

  12. B cell expression of the SH2-containing inositol 5-phosphatase (SHIP-1) is required to establish anergy to high affinity, proteinacious autoantigens.

    PubMed

    Akerlund, Janie; Getahun, Andrew; Cambier, John C

    2015-08-01

    Many self-reactive B cells exist in the periphery in a rapidly reversible state of unresponsiveness referred to as anergy. Reversibility of anergy indicates that chronically occupied BCR must transduce non-durable regulatory signals that maintain unresponsiveness. Consistent with such a mechanism, studies of immunoglobulin transgenic, as well as naturally occurring polyclonal autoreactive B cells demonstrate activation of the inositol 5-phosphatase SHIP-1 in anergic cells, and low affinity chromatin autoantigen-reactive B cells have been shown to require expression of this phosphatase to maintain anergy. However, it has been reported that anergy of B cells recognizing high affinity soluble antigen may not require SHIP-1, and is instead mediated by upregulation of the inositol 3-phosphatase PTEN. To further explore this apparent difference in mechanism we analyzed the effect of B cell-targeted SHIP-1 deletion on immune tolerance of high affinity anti-HEL B cells in mice expressing soluble HEL (MD4.ML-5). We report that SHIP-1 functions to dampen responses of naïve and low-dose antigen-primed B cells in vitro, and is required for induction of B cell tolerance. Thus, while anergy of B cells reactive with low affinity and likely polyvalent chromatin antigens is maintained by activation of inhibitory signaling circuitry involving SHIP-1, anergy of B cells recognizing soluble self antigen with high affinity also requires increased activity of SHIP-1.

  13. Hepatotoxicity of high affinity gapmer antisense oligonucleotides is mediated by RNase H1 dependent promiscuous reduction of very long pre-mRNA transcripts

    PubMed Central

    Burel, Sebastien A.; Hart, Christopher E.; Cauntay, Patrick; Hsiao, Jill; Machemer, Todd; Katz, Melanie; Watt, Andy; Bui, Huynh-hoa; Younis, Husam; Sabripour, Mahyar; Freier, Susan M.; Hung, Gene; Dan, Amy; Prakash, T.P.; Seth, Punit P.; Swayze, Eric E.; Bennett, C. Frank; Crooke, Stanley T.; Henry, Scott P.

    2016-01-01

    High affinity antisense oligonucleotides (ASOs) containing bicylic modifications (BNA) such as locked nucleic acid (LNA) designed to induce target RNA cleavage have been shown to have enhanced potency along with a higher propensity to cause hepatotoxicity. In order to understand the mechanism of this hepatotoxicity, transcriptional profiles were collected from the livers of mice treated with a panel of highly efficacious hepatotoxic or non-hepatotoxic LNA ASOs. We observed highly selective transcript knockdown in mice treated with non-hepatotoxic LNA ASOs, while the levels of many unintended transcripts were reduced in mice treated with hepatotoxic LNA ASOs. This transcriptional signature was concurrent with on-target RNA reduction and preceded transaminitis. Remarkably, the mRNA transcripts commonly reduced by toxic LNA ASOs were generally not strongly associated with any particular biological process, cellular component or functional group. However, they tended to have much longer pre-mRNA transcripts. We also demonstrate that the off-target RNA knockdown and hepatotoxicity is attenuated by RNase H1 knockdown, and that this effect can be generalized to high affinity modifications beyond LNA. This suggests that for a certain set of ASOs containing high affinity modifications such as LNA, hepatotoxicity can occur as a result of unintended off-target RNase H1 dependent RNA degradation. PMID:26553810

  14. ConPlex: a server for the evolutionary conservation analysis of protein complex structures.

    PubMed

    Choi, Yoon Sup; Han, Seong Kyu; Kim, Jinho; Yang, Jae-Seong; Jeon, Jouhyun; Ryu, Sung Ho; Kim, Sanguk

    2010-07-01

    Evolutionary conservation analyses are important for the identification of protein-protein interactions. For protein complex structures, sequence conservation has been applied to determine protein oligomerization states, to characterize native interfaces from non-specific crystal contacts, and to discriminate near-native structures from docking artifacts. However, a user-friendly web-based service for evolutionary conservation analysis of protein complexes has not been available. Therefore, we developed ConPlex (http://sbi.postech.ac.kr/ConPlex/) a web application that enables evolutionary conservation analyses of protein interactions within protein quaternary structures. Users provide protein complex structures; ConPlex automatically identifies protein interfaces and carries out evolutionary conservation analyses for the interface regions. Moreover, ConPlex allows the results of the residue-specific conservation analysis to be displayed on the protein complex structure and provides several options to customize the display output to fit each user's needs. We believe that ConPlex offers a convenient platform to analyze protein complex structures based on evolutionary conservation of protein-protein interface residues.

  15. Neomycin-neomycin dimer: an all-carbohydrate scaffold with high affinity for AT-rich DNA duplexes.

    PubMed

    Kumar, Sunil; Xue, Liang; Arya, Dev P

    2011-05-18

    A dimeric neomycin-neomycin conjugate 3 with a flexible linker, 2,2'-(ethylenedioxy)bis(ethylamine), has been synthesized and characterized. Dimer 3 can selectively bind to AT-rich DNA duplexes with high affinity. Biophysical studies have been performed between 3 and different nucleic acids with varying base composition and conformation by using ITC (isothermal calorimetry), CD (circular dichroism), FID (fluorescent intercalator displacement), and UV (ultraviolet) thermal denaturation experiments. A few conclusions can be drawn from this study: (1) FID assay with 3 and polynucleotides demonstrates the preference of 3 toward AT-rich sequences over GC-rich sequences. (2) FID assay and UV thermal denaturation experiments show that 3 has a higher affinity for the poly(dA)·poly(dT) DNA duplex than for the poly(dA)·2poly(dT) DNA triplex. Contrary to neomycin, 3 destabilizes poly(dA)·2poly(dT) triplex but stabilizes poly(dA)·poly(dT) duplex, suggesting the major groove as the binding site. (3) UV thermal denaturation studies and ITC experiments show that 3 stabilizes continuous AT-tract DNA better than DNA duplexes with alternating AT bases. (4) CD and FID titration studies show a DNA binding site size of 10-12 base pairs/drug, depending upon the structure/sequence of the duplex for AT-rich DNA duplexes. (5) FID and ITC titration between 3 and an intramolecular DNA duplex [d(5'-A(12)-x-T(12)-3'), x = hexaethylene glycol linker] results in a binding stoichiometry of 1:1 with a binding constant ∼10(8) M(-1) at 100 mM KCl. (6) FID assay using 3 and 512 hairpin DNA sequences that vary in their AT base content and placement also show a higher binding selectivity of 3 toward continuous AT-rich than toward DNA duplexes with alternate AT base pairs. (7) Salt-dependent studies indicate the formation of three ion pairs during binding of the DNA duplex d[5'-A(12)-x-T(12)-3'] and 3. (8) ITC-derived binding constants between 3 and DNA duplexes have the following order: AT

  16. Protein complex prediction via improved verification methods using constrained domain-domain matching.

    PubMed

    Zhao, Yang; Hayashida, Morihiro; Nacher, Jose C; Nagamochi, Hiroshi; Akutsu, Tatsuya

    2012-01-01

    Identification of protein complexes within protein-protein interaction networks is one of the important objectives in functional genomics. Ozawa et al. proposed a verification method of protein complexes by introducing a structural constraint. In this paper, we propose an improved integer programming-based method based on the idea that a candidate complex should not be divided into many small complexes, and combination methods with maximal components and extreme sets. The results of computational experiments suggest that our methods outperform the method by Ozawa et al. We prove that the verification problems are NP-hard, which justifies the use of integer programming. PMID:22961452

  17. On the Efficiency of NHS Ester Cross-Linkers for Stabilizing Integral Membrane Protein Complexes

    NASA Astrophysics Data System (ADS)

    Chen, Fan; Gerber, Sabina; Korkhov, Volodymyr M.; Mireku, Samantha; Bucher, Monika; Locher, Kaspar P.; Zenobi, Renato

    2015-03-01

    We have previously presented a straightforward approach based on high-mass matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) to study membrane proteins. In addition, the stoichiometry of integral membrane protein complexes could be determined by MALDI-MS, following chemical cross-linking via glutaraldehyde. However, glutaraldehyde polymerizes in solution and reacts nonspecifically with various functional groups of proteins, limiting its usefulness for structural studies of protein complexes. Here, we investigated the capability of N-hydroxysuccinimide (NHS) esters, which react much more specifically, to cross-link membrane protein complexes such as PglK and BtuC2D2. We present clear evidence that NHS esters are capable of stabilizing membrane protein complexes in situ, in the presence of detergents such as DDM, C12E8, and LDAO. The stabilization efficiency strongly depends on the membrane protein structure (i.e, the number of primary amine groups and the distances between primary amines). A minimum number of primary amine groups is required, and the distances between primary amines govern whether a cross-linker with a specific spacer arm length is able to bridge two amine groups.

  18. Node sampling for protein complex estimation in bait-prey graphs.

    PubMed

    Scholtens, Denise M; Spencer, Bruce D

    2015-08-01

    In cellular biology, node-and-edge graph or "network" data collection often uses bait-prey technologies such as co-immunoprecipitation (CoIP). Bait-prey technologies assay relationships or "interactions" between protein pairs, with CoIP specifically measuring protein complex co-membership. Analyses of CoIP data frequently focus on estimating protein complex membership. Due to budgetary and other constraints, exhaustive assay of the entire network using CoIP is not always possible. We describe a stratified sampling scheme to select baits for CoIP experiments when protein complex estimation is the main goal. Expanding upon the classic framework in which nodes represent proteins and edges represent pairwise interactions, we define generalized nodes as sets of adjacent nodes with identical adjacency outside the set and use these as strata from which to select the next set of baits. Strata are redefined at each round of sampling to incorporate accumulating data. This scheme maintains user-specified quality thresholds for protein complex estimates and, relative to simple random sampling, leads to a marked increase in the number of correctly estimated complexes at each round of sampling. The R package seqSample contains all source code and is available at http://vault.northwestern.edu/~dms877/Rpacks/.

  19. Coarse-Grained Structure-Based Model for RNA-Protein Complexes Developed by Fluctuation Matching.

    PubMed

    Hori, Naoto; Takada, Shoji

    2012-09-11

    RNA and RNA-protein complexes have recently been intensively studied in experiments, but the corresponding molecular simulation work is much less abundant, primarily due to its large system size and the long time scale involved. Here, to overcome these bottlenecks, we develop a coarse-grained (CG) structure-based simulation model for RNA and RNA-protein complexes and test it for several molecular systems. The CG model for RNA contains three particles per nucleotide, each for phosphate, sugar, and a base. Focusing on RNA molecules that fold to well-defined native structures, we employed a structure-based potential, which is similar to the Go-like potential successfully used in CG modeling of proteins. In addition, we tested three means to approximate electrostatic interactions. Many parameters involved in the CG potential were determined via a multiscale method: We matched the native fluctuation of the CG model with that by all-atom simulations for 16 RNA molecules and 10 RNA-protein complexes, from which we derived a generic set of CG parameters. We show that the derived parameters can reproduce native fluctuations well for four RNA and two RNA-protein complexes. For tRNA, the native fluctuation in solution includes large-amplitude motions that reach conformations nearly corresponding to the hybrid state P/E and EF-Tu-bound state A/T seen in the complexes with ribosome. Finally, large-amplitude modes of ribosome are briefly described.

  20. Envelope protein complexes of Mycobacterium avium subsp. paratuberculosis and their antigenicity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s disease, a chronic enteric disease of ruminant animals. In the present study, blue native PAGE electrophoresis and 2D SDS-PAGE were used to separate MAP envelope protein complexes, followed by mass spectrometry (MS) ...

  1. Detecting Protein Complexes in Protein Interaction Networks Modeled as Gene Expression Biclusters

    PubMed Central

    Hanna, Eileen Marie; Zaki, Nazar; Amin, Amr

    2015-01-01

    Developing suitable methods for the detection of protein complexes in protein interaction networks continues to be an intriguing area of research. The importance of this objective originates from the fact that protein complexes are key players in most cellular processes. The more complexes we identify, the better we can understand normal as well as abnormal molecular events. Up till now, various computational methods were designed for this purpose. However, despite their notable performance, questions arise regarding potential ways to improve them, in addition to ameliorative guidelines to introduce novel approaches. A close interpretation leads to the assent that the way in which protein interaction networks are initially viewed should be adjusted. These networks are dynamic in reality and it is necessary to consider this fact to enhance the detection of protein complexes. In this paper, we present “DyCluster”, a framework to model the dynamic aspect of protein interaction networks by incorporating gene expression data, through biclustering techniques, prior to applying complex-detection algorithms. The experimental results show that DyCluster leads to higher numbers of correctly-detected complexes with better evaluation scores. The high accuracy achieved by DyCluster in detecting protein complexes is a valid argument in favor of the proposed method. DyCluster is also able to detect biologically meaningful protein groups. The code and datasets used in the study are downloadable from https://github.com/emhanna/DyCluster. PMID:26641660

  2. Identification of Essential Proteins Based on a New Combination of Local Interaction Density and Protein Complexes

    PubMed Central

    Luo, Jiawei; Qi, Yi

    2015-01-01

    Background Computational approaches aided by computer science have been used to predict essential proteins and are faster than expensive, time-consuming, laborious experimental approaches. However, the performance of such approaches is still poor, making practical applications of computational approaches difficult in some fields. Hence, the development of more suitable and efficient computing methods is necessary for identification of essential proteins. Method In this paper, we propose a new method for predicting essential proteins in a protein interaction network, local interaction density combined with protein complexes (LIDC), based on statistical analyses of essential proteins and protein complexes. First, we introduce a new local topological centrality, local interaction density (LID), of the yeast PPI network; second, we discuss a new integration strategy for multiple bioinformatics. The LIDC method was then developed through a combination of LID and protein complex information based on our new integration strategy. The purpose of LIDC is discovery of important features of essential proteins with their neighbors in real protein complexes, thereby improving the efficiency of identification. Results Experimental results based on three different PPI(protein-protein interaction) networks of Saccharomyces cerevisiae and Escherichia coli showed that LIDC outperformed classical topological centrality measures and some recent combinational methods. Moreover, when predicting MIPS datasets, the better improvement of performance obtained by LIDC is over all nine reference methods (i.e., DC, BC, NC, LID, PeC, CoEWC, WDC, ION, and UC). Conclusions LIDC is more effective for the prediction of essential proteins than other recently developed methods. PMID:26125187

  3. Cyclic peptide ligand with high binding capacity for affinity purification of immunoglobulin G.

    PubMed

    Kang, Hyo Jin; Choe, Weonu; Min, Jeong-Ki; Lee, Young-Mi; Kim, B Moon; Chung, Sang J

    2016-09-30

    The rapidly increasing implementation of antibodies in therapeutic and diagnostic applications has necessitated the development of antibody production and purification technologies for both academic and industrial usage. Bacterial Protein A and Protein G are known to bind antibodies with high affinity and have facilitated the isolation and purification thereof. Recently, small peptide ligands (i.e. IgG Fc domain-binding peptides, FcBP) that specifically bind to the Fc-domain of antibodies were reported. In the present study we describe the development of a reusable high affinity column for antibody purification utilizing immobilized FcBP, comprising 13 amino acids residues, on a sepharose resin. In addition to FcBP, Cys to Ser substituted FcBP (FcBP-Ser), reduced FcBP (FcBP-Red), commercial Protein A and Protein G resins, packed into columns, were evaluated for antibody purification. All these columns except the FcBP-Ser one showed good binding capacity for a humanized IgG (trastuzumab) and a chimeric IgG (cetuximab). The column packed with FcBP-Red allowed antibody purification at a less acidic pH (pH 4.8) than was required for the other ligand affinity columns used in our experiments (i.e., pH 3.2 for Protein G and FcBP columns, and pH 3.5 for Protein A column, respectively). Utilizing the FcBP column, antibodies from swine human sera were isolated with a purity of 95%. Interestingly, the FcBP column could be easily regenerated and operated without loss of efficiency for up to 60 runs, the maximum number of runs performed in the present study.

  4. Mechanism of activation of cholera toxin by ADP-ribosylation factor (ARF): both low- and high-affinity interactions of ARF with guanine nucleotides promote toxin activation.

    PubMed

    Bobak, D A; Bliziotes, M M; Noda, M; Tsai, S C; Adamik, R; Moss, J

    1990-01-30

    Activation of adenylyl cyclase by cholera toxin A subunit (CT-A) results from the ADP-ribosylation of the stimulatory guanine nucleotide binding protein (GS alpha). This process requires GTP and an endogenous guanine nucleotide binding protein known as ADP-ribosylation factor (ARF). One membrane (mARF) and two soluble forms (sARF I and sARF II) of ARF have been purified from bovine brain. Because the conditions reported to enhance the binding of guanine nucleotides by ARF differ from those observed to promote optimal activity, we sought to characterize the determinants influencing the functional interaction of guanine nucleotides with ARF. High-affinity GTP binding by sARF II (apparent KD of approximately 70 nM) required Mg2+, DMPC, and sodium cholate. sARF II, in DMPC/cholate, also enhanced CT-A ADP-ribosyltransferase activity (apparent EC50 for GTP of approximately 50 nM), although there was a delay before achievement of a maximal rate of sARF II stimulated toxin activity. The delay was abolished by incubation of sARF II with GTP at 30 degrees C before initiation of the assay. In contrast, a maximal rate of activation of toxin by sARF II, in 0.003% SDS, occurred without delay (apparent EC50 for GTP of approximately 5 microM). High-affinity GTP binding by sARF II was not detectable in SDS. Enhancement of CT-A ADP-ribosyltransferase activity by sARF II, therefore, can occur under conditions in which sARF II exhibits either a relatively low affinity or a relatively high affinity for GTP. The interaction of GTP with ARF under these conditions may reflect ways in which intracellular membrane and cytosolic environments modulate GTP-mediated activation of ARF.

  5. /sup 19/F nuclear magnetic resonance measurement of the distance between the E-site GTP and the high-affinity Mg/sup 2 +/ in tubulin

    SciTech Connect

    Monasterio, O.

    1987-09-22

    The distance separating the divalent metal ion high-affinity binding site and the exchangeable nucleotide binding site on tubulin was evaluated by using high-resolution /sup 19/F NMR. The /sup 31/P and /sup 19/F NMR spectra of guanosine 5'-(..gamma..-fluorotriphosphate) (GTP(..gamma..F)) were studied. Both the fluorine and the ..gamma..-phosphate were split into a doublet with a coupling constant of 936 Hz. Tubulin purified according to the method of Weisenberg was incubated with 1 mM Mn/sup 2 +/. After one cycle of assembly, Mn/sup 2 +/ only partially, i.e., 60% at the high-affinity binding site. After colchicine treatment of tubulin to stabilize it, GTP(..gamma..F) was added, and the 254-MHz fluorine-19 relaxation rates were measured within the first 4 h. Longitudinal and transversal relaxation rates were determined at two concentrations of GTP(..gamma..F) and variable concentrations of colchicine-tubulin-Mn(II) (paramagnetic complex) or the ternary complex with magnesium diamagnetic complex). The analysis of the relaxation data indicates that the rate of exchange of GTP(..gamma..F) from the exchangeable nucleotide site has a lower limit of 8.7 x 10/sup 4/ s/sup -1/ and the metal and exchangeable nucleotide binding sites are separated by an upper distance between 6 and 8 A. These data confirm that the high-affinity divalent cation site is situated in the same locus as that of the exchangeable nucleotide, forming a metal-nucleotide complex.

  6. Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter for pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs

    PubMed Central

    Munday, Jane C.; Eze, Anthonius A.; Baker, Nicola; Glover, Lucy; Clucas, Caroline; Aguinaga Andrés, David; Natto, Manal J.; Teka, Ibrahim A.; McDonald, Jennifer; Lee, Rebecca S.; Graf, Fabrice E.; Ludin, Philipp; Burchmore, Richard J. S.; Turner, C. Michael R.; Tait, Andy; MacLeod, Annette; Mäser, Pascal; Barrett, Michael P.; Horn, David; De Koning, Harry P.

    2014-01-01

    Objectives Trypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs. Methods The TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants. Results All MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to pentamidine and >1000-fold to melaminophenyl arsenicals and induced a high-affinity pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to pentamidine and an arsenical. Conclusions TbAQP2 mediates high-affinity uptake of pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporter. PMID:24235095

  7. The presence of high-affinity, low-capacity estradiol-17β binding in rainbow trout scale indicates a possible endocrine route for the regulation of scale resorption

    USGS Publications Warehouse

    Persson, Petra; Shrimpton, J. Mark; McCormick, Stephen D.; Bjornsson, Bjorn Thrandur

    2000-01-01

    High-affinity, low-capacity estradiol-17β (E2) binding is present in rainbow trout scale. The Kd and Bmax of the scale E2 binding are similar to those of the liver E2 receptor (Kd is 1.6 ± 0.1 and 1.4 ± 0.1 nM, and Bmax is 9.1 ± 1.2 and 23.1 ± 2.2 fmol × mg protein-1, for scale and liver, respectively), but different from those of the high-affinity, low-capacity E2 binding in plasma (Kd is 4.0 ± 0.4 nM and Bmax is 625.4 ± 63.1 fmol × mg protein−1). The E2 binding in scale was displaced by testosterone, but not by diethylstilbestrol. Hence, the ligand binding specificity is different from that of the previously characterized liver E2 receptor, where E2 is displaced by diethylstilbestrol, but not by testosterone. The putative scale E2 receptor thus appears to bind both E2 and testosterone, and it is proposed that the increased scale resorption observed during sexual maturation in both sexes of several salmonid species may be mediated by this receptor. No high-affinity, low-capacity E2 binding could be detected in rainbow trout gill or skin.

  8. Structure of FcγRI in complex with Fc reveals the importance of glycan recognition for high-affinity IgG binding.

    PubMed

    Lu, Jinghua; Chu, Jonathan; Zou, Zhongcheng; Hamacher, Nels B; Rixon, Mark W; Sun, Peter D

    2015-01-20

    Fc gamma receptor I (FcγRI) contributes to protective immunity against bacterial infections, but exacerbates certain autoimmune diseases. The sole high-affinity IgG receptor, FcγRI plays a significant role in immunotherapy. To elucidate the molecular mechanism of its high-affinity IgG binding, we determined the crystal structure of the extracellular domains of human FcγRI in complex with the Fc domain of human IgG1. FcγRI binds to the Fc in a similar mode as the low-affinity FcγRII and FcγRIII receptors. In addition to many conserved contacts, FcγRI forms additional hydrogen bonds and salt bridges with the lower hinge region of Fc. Unique to the high-affinity receptor-Fc complex, however, is the conformation of the receptor D2 domain FG loop, which enables a charged KHR motif to interact with proximal carbohydrate units of the Fc glycans. Both the length and the charge of the FcγRI FG loop are well conserved among mammalian species. Ala and Glu mutations of the FG loop KHR residues showed significant contributions of His-174 and Arg-175 to antibody binding, and the loss of the FG loop-glycan interaction resulted in an ∼ 20- to 30-fold decrease in FcγRI affinity to all three subclasses of IgGs. Furthermore, deglycosylation of IgG1 resulted in a 40-fold loss in FcγRI binding, demonstrating involvement of the receptor FG loop in glycan recognition. These results highlight a unique glycan recognition in FcγRI function and open potential therapeutic avenues based on antibody glycan engineering or small molecular glycan mimics to target FcγRI for certain autoimmune diseases.

  9. Expression and Functional Properties of an Anti-Triazophos High-Affinity Single-Chain Variable Fragment Antibody with Specific Lambda Light Chain.

    PubMed

    Liu, Rui; Liang, Xiao; Xiang, Dandan; Guo, Yirong; Liu, Yihua; Zhu, Guonian

    2016-01-01

    Triazophos is a widely used organophosphorous insecticide that has potentially adverse effects to organisms. In the present study, a high-affinity single-chain variable fragment (scFv) antibody with specific lambda light chain was developed for residue monitoring. First, the specific variable regions were correctly amplified from a hybridoma cell line 8C10 that secreted monoclonal antibody (mAb) against triazophos. The regions were then assembled as scFv via splicing by overlap extension polymerase chain reaction. Subsequently, the recombinant anti-triazophos scFv-8C10 was successfully expressed in Escherichia coli strain HB2151 in soluble form, purified through immobilized metal ion affinity chromatography, and verified via Western blot and peptide mass fingerprinting analyses. Afterward, an indirect competitive enzyme-linked immunosorbent assay was established based on the purified anti-triazophos scFv-8C10 antibody. The assay exhibited properties similar to those based on the parent mAb, with a high sensitivity (IC50 of 1.73 ng/mL) to triazophos and no cross reaction for other organophosphorus pesticides; it was reliable in detecting triazophos residues in spiked water samples. Moreover, kinetic measurement using a surface plasmon resonance biosensor indicated that the purified scFv-8C10 antibody had a high affinity of 1.8 × 10(-10) M and exhibited good binding stability. Results indicated that the recombinant high-affinity scFv-8C10 antibody was an effective detection material that would be promising for monitoring triazophos residues in environment samples. PMID:27338340

  10. Expression and Functional Properties of an Anti-Triazophos High-Affinity Single-Chain Variable Fragment Antibody with Specific Lambda Light Chain

    PubMed Central

    Liu, Rui; Liang, Xiao; Xiang, Dandan; Guo, Yirong; Liu, Yihua; Zhu, Guonian

    2016-01-01

    Triazophos is a widely used organophosphorous insecticide that has potentially adverse effects to organisms. In the present study, a high-affinity single-chain variable fragment (scFv) antibody with specific lambda light chain was developed for residue monitoring. First, the specific variable regions were correctly amplified from a hybridoma cell line 8C10 that secreted monoclonal antibody (mAb) against triazophos. The regions were then assembled as scFv via splicing by overlap extension polymerase chain reaction. Subsequently, the recombinant anti-triazophos scFv-8C10 was successfully expressed in Escherichia coli strain HB2151 in soluble form, purified through immobilized metal ion affinity chromatography, and verified via Western blot and peptide mass fingerprinting analyses. Afterward, an indirect competitive enzyme-linked immunosorbent assay was established based on the purified anti-triazophos scFv-8C10 antibody. The assay exhibited properties similar to those based on the parent mAb, with a high sensitivity (IC50 of 1.73 ng/mL) to triazophos and no cross reaction for other organophosphorus pesticides; it was reliable in detecting triazophos residues in spiked water samples. Moreover, kinetic measurement using a surface plasmon resonance biosensor indicated that the purified scFv-8C10 antibody had a high affinity of 1.8 × 10−10 M and exhibited good binding stability. Results indicated that the recombinant high-affinity scFv-8C10 antibody was an effective detection material that would be promising for monitoring triazophos residues in environment samples. PMID:27338340

  11. Activated G-protein releases cGMP from high affinity binding sites on PDE from toad rod outer segments (ROS)

    SciTech Connect

    Yuen, P.S.T.; Walseth, T.F.; Panter, S.S.; Sundby, S.R.; Graeff, R.M.; Goldberg, N.D.

    1987-05-01

    cGMP binding proteins in toad ROS were identified by direct photoaffinity labeling (PAL) with /sup 32/P-cGMP and quantified by retention of complexes on nitrocellulose filters. By PAL, high affinity sites were present on the ..cap alpha.. and ..beta.. subunits of the cGMP-specific phosphodiesterase (PDE) which have MW/sub app/ of 94 and 90 kDa. A doublet was deduced from its photolabeling properties to represent PDE/sub ..gamma../ photocrosslinked with PDE/sub ..cap alpha../ or PDE/sub ..beta../, respectively. cGMP prebound to these high affinity sites was released by light-activated G-protein or its ..cap alpha.. subunit complexed with GTP..gamma..S; this inhibition of cGMP binding to PDE did not result from decreased cGMP availability due to enhanced hydrolysis. A low affinity cGMP binding component identified by PAL is tightly associated with ROS membranes. Apparent ATP/light-dependent stimulation of cGMP binding was shown to result from light activated cGMP hydrolysis in conjunction with ATP-promoted conversion of GMP to GDP/GTP and increased GDP/GTP binding. These findings coincide with a model for light-related regulation of cGMP binding and metabolism predicted from intact and cellfree kinetic measurements: in the dark state the cGMP hydrolic rate is constrained by the availability of cGMP because of its binding to high affinity sites on PDE. Light activated G-protein releases cGMP from these sites and allows for its redistribution to lower affinity sites represented by PDE catalytic site(s) and possible cGMP-dependent membrane cation channels.

  12. Assignment of the gene coding for the human high-affinity glutamate transporter EAAC1 to 9p24: Potential role in dicarboxylic aminoaciduria and neurodegenerative disorders

    SciTech Connect

    Smith, C.P.; Kanai, Y.; Stelzner, M.; Hediger, M.A.; Weremowicz, S.; Morton, C.C. )

    1994-03-15

    Functional defects of high-affinity glutamate transporters have been implicated in the pathophysiology of neurodegenerative diseases such as amyotrophic lateral sclerosis. In small intestine and kidney, in which the high-affinity glutamate transporter mediates net absorption of glutamate and aspartate across epithelial cells, an inborn error of glutamate transport is thought to cause dicarboxylic aminoaciduria. This disorder is characterized by increased urinary excretion of glutamate and aspartate and is, in general, associated with neurologic and developmental abnormalities. Recently, the authors isolated a cDNA encoding a high-affinity glutamate transporter (EAAC1) that also transports aspartate but not other amino acids. EAAC1 is ubiquitously expressed throughout the body, particularly in brain (neurons), intestine, and kidney. Here, the authors present mapping of the chromosome location of EAAC1 using Southern analysis of a panel of human/rodent somatic cell hybrids and fluorescence in situ hybridization (FISH). Southern analysis of EcoRI-digested DNA gave bands at 6.5, 5.6, 5.1, and 1.2 kb for human genomic DNA; 7.5 kb for mouse genomic DNA; and 7.3, 3.2, and 1 kb for hamster genomic DNA. All four human EAAC1-specific bands were observed in the lane corresponding to the human/Chinese hamster hybrid containing chromosome 9 but not in lanes corresponding to any other hybrid. Because the human/Chinese hamster hybrid is the only one retaining chromosome 9, this result unambiguously assigns human EAAC1 to chromosome 9. For precise chromosome assignment of the human EAAC1 gene, they employed FISH. Map position of the EAAC1 probe was assigned by visual inspection of the fluorescent signal on the DAPI-stained metaphase chromosomes. The human EAAC1 gene was assigned to 9p24.

  13. Protein complex detection via weighted ensemble clustering based on Bayesian nonnegative matrix factorization.

    PubMed

    Ou-Yang, Le; Dai, Dao-Qing; Zhang, Xiao-Fei

    2013-01-01

    Detecting protein complexes from protein-protein interaction (PPI) networks is a challenging task in computational biology. A vast number of computational methods have been proposed to undertake this task. However, each computational method is developed to capture one aspect of the network. The performance of different methods on the same network can differ substantially, even the same method may have different performance on networks with different topological characteristic. The clustering result of each computational method can be regarded as a feature that describes the PPI network from one aspect. It is therefore desirable to utilize these features to produce a more accurate and reliable clustering. In this paper, a novel Bayesian Nonnegative Matrix Factorization (NMF)-based weighted Ensemble Clustering algorithm (EC-BNMF) is proposed to detect protein complexes from PPI networks. We first apply different computational algorithms on a PPI network to generate some base clustering results. Then we integrate these base clustering results into an ensemble PPI network, in the form of weighted combination. Finally, we identify overlapping protein complexes from this network by employing Bayesian NMF model. When generating an ensemble PPI network, EC-BNMF can automatically optimize the values of weights such that the ensemble algorithm can deliver better results. Experimental results on four PPI networks of Saccharomyces cerevisiae well verify the effectiveness of EC-BNMF in detecting protein complexes. EC-BNMF provides an effective way to integrate different clustering results for more accurate and reliable complex detection. Furthermore, EC-BNMF has a high degree of flexibility in the choice of base clustering results. It can be coupled with existing clustering methods to identify protein complexes.

  14. Description and control of dissociation channels in gas-phase protein complexes

    NASA Astrophysics Data System (ADS)

    Thachuk, Mark; Fegan, Sarah K.; Raheem, Nigare

    2016-08-01

    Using molecular dynamics simulations of a coarse-grained model of the charged apo-hemoglobin protein complex, this work expands upon our initial report [S. K. Fegan and M. Thachuk, J. Am. Soc. Mass Spectrom. 25, 722-728 (2014)] about control of dissociation channels in the gas phase using specially designed charge tags. Employing a charge hopping algorithm and a range of temperatures, a variety of dissociation channels are found for activated gas-phase protein complexes. At low temperatures, a single monomer unfolds and becomes charge enriched. At higher temperatures, two additional channels open: (i) two monomers unfold and charge enrich and (ii) two monomers compete for unfolding with one eventually dominating and the other reattaching to the complex. At even higher temperatures, other more complex dissociation channels open with three or more monomers competing for unfolding. A model charge tag with five sites is specially designed to either attract or exclude charges. By attaching this tag to the N-terminus of specific monomers, the unfolding of those monomers can be decidedly enhanced or suppressed. In other words, using charge tags to direct the motion of charges in a protein complex provides a mechanism for controlling dissociation. This technique could be used in mass spectrometry experiments to direct forces at specific attachment points in a protein complex, and hence increase the diversity of product channels available for quantitative analysis. In turn, this could provide insight into the function of the protein complex in its native biological environment. From a dynamics perspective, this system provides an interesting example of cooperative behaviour involving motions with differing time scales.

  15. BiCAMWI: A Genetic-Based Biclustering Algorithm for Detecting Dynamic Protein Complexes

    PubMed Central

    Lakizadeh, Amir; Jalili, Saeed

    2016-01-01

    Considering the roles of protein complexes in many biological processes in the cell, detection of protein complexes from available protein-protein interaction (PPI) networks is a key challenge in the post genome era. Despite high dynamicity of cellular systems and dynamic interaction between proteins in a cell, most computational methods have focused on static networks which cannot represent the inherent dynamicity of protein interactions. Recently, some researchers try to exploit the dynamicity of PPI networks by constructing a set of dynamic PPI subnetworks correspondent to each time-point (column) in a gene expression data. However, many genes can participate in multiple biological processes and cellular processes are not necessarily related to every sample, but they might be relevant only for a subset of samples. So, it is more interesting to explore each subnetwork based on a subset of genes and conditions (i.e., biclusters) in a gene expression data. Here, we present a new method, called BiCAMWI to employ dynamicity in detecting protein complexes. The preprocessing phase of the proposed method is based on a novel genetic algorithm that extracts some sets of genes that are co-regulated under some conditions from input gene expression data. Each extracted gene set is called bicluster. In the detection phase of the proposed method, then, based on the biclusters, some dynamic PPI subnetworks are extracted from input static PPI network. Protein complexes are identified by applying a detection method on each dynamic PPI subnetwork and aggregating the results. Experimental results confirm that BiCAMWI effectively models the dynamicity inherent in static PPI networks and achieves significantly better results than state-of-the-art methods. So, we suggest BiCAMWI as a more reliable method for protein complex detection. PMID:27462706

  16. BiCAMWI: A Genetic-Based Biclustering Algorithm for Detecting Dynamic Protein Complexes.

    PubMed

    Lakizadeh, Amir; Jalili, Saeed

    2016-01-01

    Considering the roles of protein complexes in many biological processes in the cell, detection of protein complexes from available protein-protein interaction (PPI) networks is a key challenge in the post genome era. Despite high dynamicity of cellular systems and dynamic interaction between proteins in a cell, most computational methods have focused on static networks which cannot represent the inherent dynamicity of protein interactions. Recently, some researchers try to exploit the dynamicity of PPI networks by constructing a set of dynamic PPI subnetworks correspondent to each time-point (column) in a gene expression data. However, many genes can participate in multiple biological processes and cellular processes are not necessarily related to every sample, but they might be relevant only for a subset of samples. So, it is more interesting to explore each subnetwork based on a subset of genes and conditions (i.e., biclusters) in a gene expression data. Here, we present a new method, called BiCAMWI to employ dynamicity in detecting protein complexes. The preprocessing phase of the proposed method is based on a novel genetic algorithm that extracts some sets of genes that are co-regulated under some conditions from input gene expression data. Each extracted gene set is called bicluster. In the detection phase of the proposed method, then, based on the biclusters, some dynamic PPI subnetworks are extracted from input static PPI network. Protein complexes are identified by applying a detection method on each dynamic PPI subnetwork and aggregating the results. Experimental results confirm that BiCAMWI effectively models the dynamicity inherent in static PPI networks and achieves significantly better results than state-of-the-art methods. So, we suggest BiCAMWI as a more reliable method for protein complex detection. PMID:27462706

  17. Discovery of high affinity anti-ricin antibodies by B cell receptor sequencing and by yeast display of combinatorial VH:VL libraries from immunized animals.

    PubMed

    Wang, Bo; Lee, Chang-Han; Johnson, Erik L; Kluwe, Christien A; Cunningham, Josephine C; Tanno, Hidetaka; Crooks, Richard M; Georgiou, George; Ellington, Andrew D

    2016-01-01

    Ricin is a toxin that could potentially be used as a bioweapon. We identified anti-ricin A chain antibodies by sequencing the antibody repertoire from immunized mice and by selecting high affinity antibodies using yeast surface display. These methods led to the isolation of multiple antibodies with high (sub-nanomolar) affinity. Interestingly, the antibodies identified by the 2 independent approaches are from the same clonal lineages, indicating for the first time that yeast surface display can identify native antibodies. The new antibodies represent well-characterized reagents for biodefense diagnostics and therapeutics development.

  18. Discovery of high affinity anti-ricin antibodies by B cell receptor sequencing and by yeast display of combinatorial VH:VL libraries from immunized animals.

    PubMed

    Wang, Bo; Lee, Chang-Han; Johnson, Erik L; Kluwe, Christien A; Cunningham, Josephine C; Tanno, Hidetaka; Crooks, Richard M; Georgiou, George; Ellington, Andrew D

    2016-01-01

    Ricin is a toxin that could potentially be used as a bioweapon. We identified anti-ricin A chain antibodies by sequencing the antibody repertoire from immunized mice and by selecting high affinity antibodies using yeast surface display. These methods led to the isolation of multiple antibodies with high (sub-nanomolar) affinity. Interestingly, the antibodies identified by the 2 independent approaches are from the same clonal lineages, indicating for the first time that yeast surface display can identify native antibodies. The new antibodies represent well-characterized reagents for biodefense diagnostics and therapeutics development. PMID:27224530

  19. High-affinity labeling and tracking of individual histidine-tagged proteins in live cells using Ni2+ tris-nitrilotriacetic acid quantum dot conjugates.

    PubMed

    Roullier, Victor; Clarke, Samuel; You, Changjiang; Pinaud, Fabien; Gouzer, G Géraldine; Schaible, Dirk; Marchi-Artzner, Valérie; Piehler, Jacob; Dahan, Maxime

    2009-03-01

    Investigation of many cellular processes using fluorescent quantum dots (QDs) is hindered by the nontrivial requirements for QD surface functionalization and targeting. To address these challenges, we designed, characterized and applied QD-trisNTA, which integrates tris-nitrilotriacetic acid, a small and high-affinity recognition unit for the ubiquitous polyhistidine protein tag. Using QD-trisNTA, we demonstrate two-color QD tracking of the type-1 interferon receptor subunits in live cells, potentially enabling direct visualization of protein-protein interactions at the single molecule level. PMID:19216518

  20. Chemical synthesis and X-ray structure of a heterochiral {D-protein antagonist plus vascular endothelial growth factor} protein complex by racemic crystallography

    SciTech Connect

    Mandal, Kalyaneswar; Uppalapati, Maruti; Ault-Riché, Dana; Kenney, John; Lowitz, Joshua; Sidhu, Sachdev S.; Kent, Stephen B.H.

    2012-10-23

    Total chemical synthesis was used to prepare the mirror image (D-protein) form of the angiogenic protein vascular endothelial growth factor (VEGF-A). Phage display against D-VEGF-A was used to screen designed libraries based on a unique small protein scaffold in order to identify a high affinity ligand. Chemically synthesized D- and L- forms of the protein ligand showed reciprocal chiral specificity in surface plasmon resonance binding experiments: The L-protein ligand bound only to D-VEGF-A, whereas the D-protein ligand bound only to L-VEGF-A. The D-protein ligand, but not the L-protein ligand, inhibited the binding of natural VEGF{sub 165} to the VEGFR1 receptor. Racemic protein crystallography was used to determine the high resolution X-ray structure of the heterochiral complex consisting of {l_brace}D-protein antagonist + L-protein form of VEGF-A{r_brace}. Crystallization of a racemic mixture of these synthetic proteins in appropriate stoichiometry gave a racemic protein complex of more than 73 kDa containing six synthetic protein molecules. The structure of the complex was determined to a resolution of 1.6 {angstrom}. Detailed analysis of the interaction between the D-protein antagonist and the VEGF-A protein molecule showed that the binding interface comprised a contact surface area of approximately 800 {angstrom}{sup 2} in accord with our design objectives, and that the D-protein antagonist binds to the same region of VEGF-A that interacts with VEGFR1-domain 2.

  1. Novel high-affinity and selective biaromatic 4-substituted gamma-hydroxybutyric acid (GHB) analogues as GHB ligands: design, synthesis, and binding studies.

    PubMed

    Høg, Signe; Wellendorph, Petrine; Nielsen, Birgitte; Frydenvang, Karla; Dahl, Ivar F; Bräuner-Osborne, Hans; Brehm, Lotte; Frølund, Bente; Clausen, Rasmus P

    2008-12-25

    Gamma-hydroxybutyrate (GHB) is a metabolite of gamma-aminobutyric acid (GABA) and has been proposed to function as a neurotransmitter or neuromodulator. GHB is used in the treatment of narcolepsy and is a drug of abuse. GHB binds to both GABA(B) receptors and specific high-affinity GHB sites in brain, of which the latter have not been linked unequivocally to function, but are speculated to be GHB receptors. In this study, a series of biaromatic 4-substituted GHB analogues, including 4'-phenethylphenyl, 4'-styrylphenyl, and 4'-benzyloxyphenyl GHB analogues, were synthesized and characterized pharmacologically in a [3H](E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene)acetic acid ([3H]NCS-382) binding assay and in GABA(A) and GABA(B) receptor binding assays. The compounds were selective for the high-affinity GHB binding sites and several displayed Ki values below 100 nM. The affinity of the 4-[4'-(2-iodobenzyloxy)phenyl] GHB analogue 17b was shown to reside predominantly with the R-enantiomer (Ki = 22 nM), which has higher affinity than previously reported GHB ligands.

  2. Effect of P Availability on Temporal Dynamics of Carbon Allocation and Glomus intraradices High-Affinity P Transporter Gene Induction in Arbuscular Mycorrhiza

    PubMed Central

    Olsson, Pål Axel; Hansson, Maria C.; Burleigh, Stephen H.

    2006-01-01

    Arbuscular mycorrhizal (AM) fungi depend on a C supply from the plant host and simultaneously provide phosphorus to the colonized plant. We therefore evaluated the influence of external P on C allocation in monoxenic Daucus carota-Glomus intraradices cultures in an AM symbiosis. Fungal hyphae proliferated from a solid minimal medium containing colonized roots into a C-free liquid minimal medium with high or low P availability. Roots and hyphae were harvested periodically, and the flow of C from roots to fungus was measured by isotope labeling. We also measured induction of a G. intraradices high-affinity P transporter to estimate fungal P demand. The prevailing hypothesis is that high P availability reduces mycorrhizal fungal growth, but we found that C flow to the fungus was initially highest at the high P level. Only at later harvests, after 100 days of in vitro culture, were C flow and fungal growth limited at high P availability. Thus, AM fungi can benefit initially from P-enriched environments in terms of plant C allocation. As expected, the P transporter induction was significantly greater at low P availability and greatest in very young mycelia. We found no direct link between C flow to the fungus and the P transporter transcription level, which indicates that a good C supply is not essential for induction of the high-affinity P transporter. We describe a mechanism by which P regulates symbiotic C allocation, and we discuss how this mechanism may have evolved in a competitive environment. PMID:16751522

  3. Identification of the High-affinity Substrate-binding Site of the Multidrug and Toxic Compound Extrusion (MATE) Family Transporter from Pseudomonas stutzeri.

    PubMed

    Nie, Laiyin; Grell, Ernst; Malviya, Viveka Nand; Xie, Hao; Wang, Jingkang; Michel, Hartmut

    2016-07-22

    Multidrug and toxic compound extrusion (MATE) transporters exist in all three domains of life. They confer multidrug resistance by utilizing H(+) or Na(+) electrochemical gradients to extrude various drugs across the cell membranes. The substrate binding and the transport mechanism of MATE transporters is a fundamental process but so far not fully understood. Here we report a detailed substrate binding study of NorM_PS, a representative MATE transporter from Pseudomonas stutzeri Our results indicate that NorM_PS is a proton-dependent multidrug efflux transporter. Detailed binding studies between NorM_PS and 4',6-diamidino-2-phenylindole (DAPI) were performed by isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and spectrofluorometry. Two exothermic binding events were observed from ITC data, and the high-affinity event was directly correlated with the extrusion of DAPI. The affinities are about 1 μm and 0.1 mm for the high and low affinity binding, respectively. Based on our homology model of NorM_PS, variants with mutations of amino acids that are potentially involved in substrate binding, were constructed. By carrying out the functional characterization of these variants, the critical amino acid residues (Glu-257 and Asp-373) for high-affinity DAPI binding were determined. Taken together, our results suggest a new substrate-binding site for MATE transporters. PMID:27235402

  4. A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders.

    PubMed

    Cui, Naiwen; Zhang, Huidan; Schneider, Nils; Tao, Ye; Asahara, Haruichi; Sun, Zhiyi; Cai, Yamei; Koehler, Stephan A; de Greef, Tom F A; Abbaspourrad, Alireza; Weitz, David A; Chong, Shaorong

    2016-03-04

    Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-and-read drop-IVT2H method to screen a random DNA library. Drop-IVT2H was based on the correlation between the binding affinity of two interacting protein domains and transcriptional activation of a fluorescent reporter. A DNA library encoding potential peptide binders was encapsulated with IVT2H such that single DNA molecules were distributed in individual drops. We validated drop-IVT2H by screening a three-random-residue library derived from a high-affinity MDM2 inhibitor PMI. The current drop-IVT2H platform is ideally suited for affinity screening of small-to-medium-sized libraries (10(3)-10(6)). It can obtain hits within a single day while consuming minimal amounts of reagents. Drop-IVT2H simplifies and accelerates the drop-based microfluidics workflow for screening random DNA libraries, and represents a novel alternative method for protein engineering and in vitro directed protein evolution.

  5. A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders

    PubMed Central

    Cui, Naiwen; Zhang, Huidan; Schneider, Nils; Tao, Ye; Asahara, Haruichi; Sun, Zhiyi; Cai, Yamei; Koehler, Stephan A.; de Greef, Tom F. A.; Abbaspourrad, Alireza; Weitz, David A.; Chong, Shaorong

    2016-01-01

    Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-and-read drop-IVT2H method to screen a random DNA library. Drop-IVT2H was based on the correlation between the binding affinity of two interacting protein domains and transcriptional activation of a fluorescent reporter. A DNA library encoding potential peptide binders was encapsulated with IVT2H such that single DNA molecules were distributed in individual drops. We validated drop-IVT2H by screening a three-random-residue library derived from a high-affinity MDM2 inhibitor PMI. The current drop-IVT2H platform is ideally suited for affinity screening of small-to-medium-sized libraries (103–106). It can obtain hits within a single day while consuming minimal amounts of reagents. Drop-IVT2H simplifies and accelerates the drop-based microfluidics workflow for screening random DNA libraries, and represents a novel alternative method for protein engineering and in vitro directed protein evolution. PMID:26940078

  6. RraAS2 requires both scaffold domains of RNase ES for high-affinity binding and inhibitory action on the ribonucleolytic activity.

    PubMed

    Heo, Jihune; Kim, Daeyoung; Joo, Minju; Lee, Boeun; Seo, Sojin; Lee, Jaejin; Song, Saemee; Yeom, Ji-Hyun; Ha, Nam-Chul; Lee, Kangseok

    2016-10-01

    RraA is a protein inhibitor of RNase E (Rne), which catalyzes the endoribonucleolytic cleavage of a large proportion of RNAs in Escherichia coli. The antibiotic-producing bacterium Streptomyces coelicolor also contains homologs of RNase E and RraA, designated as RNase ES (Rns), RraAS1, and RraAS2, respectively. Here, we report that RraAS2 requires both scaffold domains of RNase ES for high-affinity binding and inhibitory action on the ribonucleolytic activity. Analyses of the steady-state level of RNase E substrates indicated that coexpression of RraAS2 in E. coli cells overproducing Rns effectively inhibits the ribonucleolytic activity of full-length RNase ES, but its inhibitory effects were moderate or undetectable on other truncated forms of Rns, in which the N- or/and C-terminal scaffold domain was deleted. In addition, RraAS2 more efficiently inhibited the in vitro ribonucleolytic activity of RNase ES than that of a truncated form containing the catalytic domain only. Coimmunoprecipitation and in vivo cross-linking experiments further showed necessity of both scaffold domains of RNase ES for high-affinity binding of RraAS2 to the enzyme, resulting in decreased RNA-binding capacity of RNase ES. Our results indicate that RraAS2 is a protein inhibitor of RNase ES and provide clues to how this inhibitor affects the ribonucleolytic activity of RNase ES. PMID:27687228

  7. The novel VIP-like hypothalamic polypeptide PACAP interacts with high affinity receptors in the human neuroblastoma cell line NB-OK

    SciTech Connect

    Cauvin, A.; Buscail, L.; Gourlet, P.; De Neef, P.; Gossen, D.; Arimura, A.; Miyata, A.; Coy, D.H.; Robberecht, P.; Christophe, J. )

    1990-07-01

    We investigated the ability of two forms of Pituitary Adenylate Cyclase Activating Polypeptide (PACAP-38, the 38 amino acid peptide isolated from ovine hypothalamus, and PACAP-27, a shorter N-terminal (1-27) amidated version) to interact with specific receptors in membranes from the human neuroblastoma cell line NB-OK. ({sup 125}I)PACAP-27 bound rapidly and specifically to one class of high affinity sites (Kd 0.5 nM). VIP inhibited ({sup 125}I)PACAP-27 binding 300- to 1000-fold less potently than PACAP-27 and PACAP-38. One microM PHI prevented tracer binding only partially and secretin, glucagon and GRF(1-29)NH2 were ineffective in this respect. PACAP-27 and PACAP-38 stimulated adenylate cyclase activity dose dependently and with similar efficacy (Kact 0.2-0.3 nM), this activation being compatible with the occupancy of specific high affinity PACAP receptor. VIP was markedly less potent and less efficient on this enzyme than PACAP. Chemical cross-linking of ({sup 125}I)PACAP-27 followed by SDS-PAGE and autoradiography revealed specific cross-linking with a 68 kDa protein.

  8. Identification of base and backbone contacts used for DNA sequence recognition and high-affinity binding by LAC9, a transcription activator containing a C6 zinc finger

    SciTech Connect

    Halvorsen, Yuan-Di C.; Nandabalan, K.; Dickson, R.C. )

    1991-04-01

    The LAC9 protein of Kluyveromyces lactis is a transcriptional regulator of genes in the lactose-galactose regulon. To regulate transcription, LAC9 must bind to 17-bp upstream activator sequences (UASs) located in front of each target gene. LAC9 is homologous to the GAL4 protein of Saccharomyces cerevisiae, and the two proteins must bind DNA in a very similar manner. In this paper the authors show that high-affinity, sequence-specific binding by LAC9 dimers is mediated primarily by 3 bp at each end of the UAS. In addition, at least one half of the UAS must have a GC or CG base pair at position 1 for high-affinity binding; LAC9k binds preferentially to the half containing the GC base pair. Hydroxyl radical footprinting shows that a LAC9 dimer binds an unusually broad region on one face of the DNA helix. Because of the data, they suggest that LAC9 contacts positions 6, 7, and 8, both plus and minus, of the UAS, which are separated by more than one turn of the DNA helix, and twists part way around the DNA, thus protecting the broad region of the minor groove between the major-groove contacts.

  9. Binding of L-(/sup 3/H)nicotine to a single class of high affinity sites in rat brain membranes

    SciTech Connect

    Lippiello, P.M.; Fernandes, K.G.

    1986-05-01

    The binding of optically pure L-(/sup 3/H)nicotine to rat brain membrane preparations was studied using a rapid filtration method. The binding properties observed depended on the method used for tissue isolation. The most consistent results were obtained with membranes prepared in the presence of protease inhibitors, without divalent cations. Binding was saturable, reversible, and stereospecific. Scatchard analysis revealed a single class of high affinity sites with an average KD of 2 nM and a Bmax of approximately 200 fmol/mg of protein. The Hill coefficient was near unity. The KD calculated from the kinetic rate constants for association (k1 = 0.012 min-1 nM-1) and dissociation (k-1 = 0.04 min-1) was around 3 nM, in good agreement with the dissociation constant determined from equilibrium binding. In competition studies, cholinergic agonists were generally the most effective in inhibiting L-(/sup 3/H)nicotine binding, whereas antagonists were relatively ineffective. The D-isomer of nicotine was about 60-fold less potent than the L-isomer in inhibiting binding. The results were unaffected by temperature, with the exception that Bmax was somewhat lower at 37 degrees. The equilibrium binding properties of these sites were essentially identical in adult male and female brain. However, Bmax was lower in fetal brain tissue. The present findings are consistent with the idea that there is a single class of high affinity nicotinic binding sites in rat brain with cholinoceptive properties.

  10. In silico investigation of PARP-1 catalytic domains in holo and apo states for the design of high-affinity PARP-1 inhibitors.

    PubMed

    Salmas, Ramin Ekhteiari; Unlu, Ayhan; Yurtsever, Mine; Noskov, Sergei Y; Durdagi, Serdar

    2016-01-01

    The rational design of high-affinity inhibitors of poly-ADP-ribose polymerase-1 (PARP-1) is at the heart of modern anti-cancer drug design. While relevance of enzyme to DNA repair processes in cellular environment is firmly established, the structural and functional understanding of the main determinants for high-affinity ligands controlling PARP-1 activity is still lacking. The conserved active site of PARP-1 represents an ideal target for inhibitors and may offer a novel target at the treatment of breast cancer. To fill the gap in the structural knowledge, we report on the combination of molecular dynamics (MD) simulations, principal component analysis (PCA), and conformational analysis that analyzes in great details novel binding mode for a number of inhibitors at the PARP-1. While optimization of the binding affinity for original target is an important goal in the drug design, many of the promising molecules for treatment of the breast cancer are plagued by significant cardiotoxicity. One of the most common side-effects reported for a number of polymerase inhibitors is its off-target interactions with cardiac ion channels and hERG1 channel, in particular. Thus, selected candidate PARP-1 inhibitors were also screened in silico at the central cavities of hERG1 potassium ion channel.

  11. Specificity of Bacillus thuringiensis endotoxins is correlated with the presence of high-affinity binding sites in the brush border membrane of target insect midguts

    SciTech Connect

    Hofmann, C.; Vanderbruggen, H.; Hoefte, H.; Van Rie, J.; Jansens, S.; Van Mellaert, H. )

    1988-11-01

    Binding studies were performed with two {sup 125}I-labeled Bacillus thuringiensis {delta}-endotoxins on brush border membrane vesicles prepared from the larval midgut of the tobacco hornworm Manduca sexta or the cabbage butterfly Pieris brassicae. One {delta}-endotoxin, Bt2-protoxin, is a 130-kDa recombinant crystalline protein from B. thuringiensis subsp. berliner. It kills larvae of both insect species. The active Bt2-toxin is a 60-kDa proteolytic fragment of the Bt2-protoxin. It binds saturably and with high affinity to brush border membrane vesicles from the midgut of both species. The other {delta}-endotoxin, Bt4412-protoxin, is a 136-kDa crystalline protein from B. thuringiensis subsp. thuringiensis, which is highly toxic for P. brassicae, but not for M. sexta larvae. Bt4412-toxin, obtained after proteolytic activation of Bt4412-protoxin, shows high-affinity saturable binding to P. brassicae vesicles but not to M. sexta vesicles. The correlation between toxicity and specific binding is further strengthened by competition studies. Other B. thuringiensis {delta}-endotoxins active against M. sexta compete for binding of {sup 125}I-labeled Bt2-toxin to M. sexta vesicles, whereas toxins active against dipteran or coleopteran larvae do not compete. Bt2-toxin and Bt4412-toxin bind to different sites on P. brassicae vesicles.

  12. Deep Sequencing-guided Design of a High Affinity Dual Specificity Antibody to Target Two Angiogenic Factors in Neovascular Age-related Macular Degeneration.

    PubMed

    Koenig, Patrick; Lee, Chingwei V; Sanowar, Sarah; Wu, Ping; Stinson, Jeremy; Harris, Seth F; Fuh, Germaine

    2015-09-01

    The development of dual targeting antibodies promises therapies with improved efficacy over mono-specific antibodies. Here, we engineered a Two-in-One VEGF/angiopoietin 2 antibody with dual action Fab (DAF) as a potential therapeutic for neovascular age-related macular degeneration. Crystal structures of the VEGF/angiopoietin 2 DAF in complex with its two antigens showed highly overlapping binding sites. To achieve sufficient affinity of the DAF to block both angiogenic factors, we turned to deep mutational scanning in the complementarity determining regions (CDRs). By mutating all three CDRs of each antibody chain simultaneously, we were able not only to identify affinity improving single mutations but also mutation pairs from different CDRs that synergistically improve both binding functions. Furthermore, insights into the cooperativity between mutations allowed us to identify fold-stabilizing mutations in the CDRs. The data obtained from deep mutational scanning reveal that the majority of the 52 CDR residues are utilized differently for the two antigen binding function and permit, for the first time, the engineering of several DAF variants with sub-nanomolar affinity against two structurally unrelated antigens. The improved variants show similar blocking activity of receptor binding as the high affinity mono-specific antibodies against these two proteins, demonstrating the feasibility of generating a dual specificity binding surface with comparable properties to individual high affinity mono-specific antibodies.

  13. The high-affinity maltose switch MBP317-347 has low affinity for glucose: implications for targeting tumors with metabolically directed enzyme prodrug therapy.

    PubMed

    Valdes, Gilmer; Schulte, Reinhard W; Ostermeier, Marc; Iwamoto, Keisuke S

    2014-03-01

    Development of agents with high affinity and specificity for tumor-specific markers is an important goal of molecular-targeted therapy. Here, we propose a shift in paradigm using a strategy that relies on low affinity for fundamental metabolites found in different concentrations in cancerous and non-cancerous tissues: glucose and lactate. A molecular switch, MBP317-347, originally designed to be a high-affinity switch for maltose and maltose-like polysaccharides, was demonstrated to be a low-affinity switch for glucose, that is, able to be activated by high concentrations (tens of millimolar) of glucose. We propose that such a low-affinity glucose switch could be used as a proof of concept for a new prodrug therapy strategy denominated metabolically directed enzyme prodrug therapy (MDEPT) where glucose or, preferably, lactate serves as the activator. Accordingly, considering the typical differential concentrations of lactate found in tumors and in healthy tissues, a low-affinity lactate-binding switch analogous to the low-affinity glucose-binding switch MBP317-347 would be an order of magnitude more active in tumors than in normal tissues and therefore can work as a differential activator of anticancer drugs in tumors.

  14. The single Cys2-His2 zinc finger domain of the GAGA protein flanked by basic residues is sufficient for high-affinity specific DNA binding.

    PubMed

    Pedone, P V; Ghirlando, R; Clore, G M; Gronenborn, A M; Felsenfeld, G; Omichinski, J G

    1996-04-01

    Specific DNA binding to the core consensus site GAGAGAG has been shown with an 82-residue peptide (residues 310-391) taken from the Drosophila transcription factor GAGA. Using a series of deletion mutants, it was demonstrated that the minimal domain required for specific binding (residues 310-372) includes a single zinc finger of the Cys2-His2 family and a stretch of basic amino acids located on the N-terminal end of the zinc finger. In gel retardation assays, the specific binding seen with either the peptide or the whole protein is zinc dependent and corresponds to a dissociation constant of approximately 5 x 10(-9) M for the purified peptide. It has previously been thought that a single zinc finger of the Cys2-His2 family is incapable of specific, high-affinity binding to DNA. The combination of an N-terminal basic region with a single Cys2-His2 zinc finger in the GAGA protein can thus be viewed as a novel DNA binding domain. This raises the possibility that other proteins carrying only one Cys2-His2 finger are also capable of high-affinity specific binding to DNA. PMID:8610125

  15. Identification and affinity of very high affinity binding sites for the phenylalkylamine series of Ca/sup +/ channel blockers in the Drosophila nervous system

    SciTech Connect

    Pauron, D.; Qar, J.; Barhanin, J.; Fournier, D.; Cuany, A.; Pralavorio, M.; Berge, J.B.; Lazdunski, M.

    1987-10-06

    The interaction of putative Ca/sup 2 +/ channels of Drosophila head membranes with molecules of the phenylalkylamine series was studied from binding experiments using (-)-(/sup 3/H)D888 and (+/-)-(/sup 3/H)verapamil. These ligands recognize a single class of very high affinity binding sites. The most potent molecule in the phenylalkylamine series was (-)-verapamil with a K/sub d/ value as exceptional low as 4.7 pM. Molecules in the benzothiazepine and diphenylbutylpiperidine series of Ca/sup 2 +/ channel blockers as well as bepridil inhibited (-)-(/sup 3/H)D888 binding in a competitive way with K/sub d/ values between 12 and 190 nM, suggesting a close correlation, as in the mammalian system, between these receptor sites and those recognizing phenylalkylamines. A tritiated (arylazido)phenylalkylamine with high affinity for the Drosophila head membranes, phenylalkylamine receptor was used in photoaffinity experiments. A protein of M/sub r/ 135,000 +/- 5000 was specifically labeled after ultraviolet irradiation.

  16. High-affinity ouabain binding by yeast cells expressing Na+, K(+)-ATPase alpha subunits and the gastric H+, K(+)-ATPase beta subunit.

    PubMed

    Eakle, K A; Kim, K S; Kabalin, M A; Farley, R A

    1992-04-01

    Recently, a beta subunit for the rat gastric H+,K(+)-ATPase (HK beta), which is structurally similar to the beta subunit of Na+, K(+)-ATPase, has been cloned and characterized. Using heterologous expression in yeast, we have tested the specificity of beta subunit assembly with different isoforms of the alpha subunit of Na+, K(+)-ATPase. Coexpression in yeast cells of the HK beta with both the sheep alpha 1 subunit and the rat alpha 3 subunit isoforms of Na+, K(+)-ATPase (alpha 1 and alpha 3, respectively) leads to the appearance of high-affinity ouabain-binding sites in yeast membranes. These ouabain-binding sites (alpha 1 plus HK beta, alpha 3 plus HK beta) have a high affinity for ouabain (Kd, 5-10 nM) and are expressed at levels similar to those formed with the rat beta 1 subunit of Na+, K(+)-ATPase (beta 1) (alpha 1 plus beta 1 or alpha 3 plus beta 1). Potassium acts as a specific antagonist of ouabain binding by alpha 1 plus HK beta and alpha 3 plus HK beta just like sodium pumps formed with beta 1. Sodium pumps formed with the HK beta, however, show quantitative differences in their affinity for ouabain and in the antagonism of K+ for ouabain binding. These data suggest that the structure of the beta subunit may play a role in sodium pump function. PMID:1313569

  17. The Borexino purification system

    NASA Astrophysics Data System (ADS)

    Benziger, Jay

    2014-05-01

    Purification of 278 tons of liquid scintillator and 889 tons of buffer shielding for the Borexino solar neutrino detector is performed with a system of combined distillation, water extraction, gas stripping and filtration. The purification system removed K, U and Th by distillation of the pseudocumene solvent and the PPO fluor. Noble gases, Rn, Kr and Ar were removed by gas stripping. Distillation was also employed to remove optical impurities and reduce the attenuation of scintillation light. The success of the purification system has facilitated the first time real time detection of low energy solar neutrinos.

  18. Ca2+-dependent phosphatase and Ca2+-dependent ATPase activities in plasma membranes of eel gill epithelium--III. Stimulation of branchial high-affinity Ca2+-ATPase activity during prolactin-induced hypercalcemia in American eels.

    PubMed

    Flik, G; Wendelaar Bonga, S E; Fenwick, J C

    1984-01-01

    Infusions of ovine prolactin for 10 days induced hypercalcemia in unfed American eels, Anguilla rostrata LeSueur, that tentatively was related to stimulation of branchial Ca2+-uptake mechanisms. Analysis of ATPase activities in the plasma membranes of the branchial epithelium in prolactin treated eels showed a specific stimulation of high-affinity Ca2+-ATPase. The results of this study form further evidence that the high-affinity Ca2+-ATPase activity represents the Ca2+-pump of the branchial epithelium.

  19. Binding Linkage in a Telomere DNA–Protein Complex at the Ends of Oxytricha nova Chromosomes

    PubMed Central

    Buczek, Pawel; Orr, Rochelle S.; Pyper, Sean R.; Shum, Mili; Ota, Emily Kimmel Irene; Gerum, Shawn E.; Horvath, Martin P.

    2005-01-01

    Alpha and beta protein subunits of the telomere end binding protein from Oxytricha nova (OnTEBP) combine with telomere single strand DNA to form a protective cap at the ends of chromosomes. We tested how protein–protein interactions seen in the co-crystal structure relate to DNA binding through use of fusion proteins engineered as different combinations of domains and subunits derived from OnTEBP. Joining alpha and beta resulted in a protein that bound single strand telomere DNA with high affinity (KD-DNA=1.4 nM). Another fusion protein, constructed without the C-terminal protein–protein interaction domain of alpha, bound DNA with 200-fold diminished affinity (KD-DNA=290 nM) even though the DNA-binding domains of alpha and beta were joined through a peptide linker. Adding back the alpha C-terminal domain as a separate protein restored high-affinity DNA binding. The binding behaviors of these fusion proteins and the native protein subunits are consistent with cooperative linkage between protein-association and DNA-binding equilibria. Linking DNA–protein stability to protein–protein contacts at a remote site may provide a trigger point for DNA–protein disassembly during telomere replication when the single strand telomere DNA must exchange between a very stable OnTEBP complex and telomerase. PMID:15967465

  20. The Monitoring and Affinity Purification of Proteins Using Dual Tags with Tetracysteine Motifs

    NASA Astrophysics Data System (ADS)

    Giannone, Richard J.; Liu, Yie; Wang, Yisong

    Identification and characterization of protein-protein interaction networks is essential for the elucidation of biochemical mechanisms and cellular function. Affinity purification in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a very powerful tactic for the identification of specific protein-protein interactions. In this chapter, we describe a comprehensive methodology that uses our recently developed dual-tag affinity purification system for the enrichment and identification of mammalian protein complexes. The protocol covers a series of separate but sequentially related techniques focused on the facile monitoring and purification of a dual-tagged protein of interest and its interacting partners via a system built with tetracysteine motifs and various combinations of affinity tags. Using human telomeric repeat binding factor 2 (TRF2) as an example, we demonstrate the power of the system in terms of bait protein recovery after dual-tag affinity purification, detection of bait protein subcellular localization and expression, and successful identification of known and potentially novel TRF2 interacting proteins. Although the protocol described here has been optimized for the identification and characterization of TRF2-associated proteins, it is, in principle, applicable to the study of any other mammalian protein complexes that may be of interest to the research community.

  1. The Monitoring and Affinity Purification of Proteins Using Dual-Tags with Tetracysteine Motifs

    SciTech Connect

    Giannone, Richard J; Liu, Yie; Wang, Yisong

    2009-01-01

    Identification and characterization of protein-protein interaction networks is essential for the elucidation of biochemical mechanisms and cellular function. Affinity purification in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a very powerful tactic for the identification of specific protein-protein interactions. In this chapter we describe a comprehensive methodology that utilizes our recently developed dual-tag affinity purification system for the enrichment and identification of mammalian protein complexes. The protocol covers a series of separate but sequentially related techniques focused on the facile monitoring and purification of a dual-tagged protein of interest and its interacting partners via a system built with tetracysteine motifs and various combinations of affinity tags. Using human telomeric repeat binding factor 2 (TRF2) as an example, we have demonstrated the power of the system in terms of bait protein recovery after dual-tag affinity purification, detection of bait protein subcellular localization and expression, and successful identification of known and potentially novel TRF2 interacting proteins. Although the protocol described here has been optimized for the identification and characterization of TRF2-associated proteins, it is, in principle, applicable to the study of any other mammalian protein complexes that may be of interest to the research community.

  2. On the preservation of non-covalent protein complexes during electrospray ionization.

    PubMed

    Chingin, Konstantin; Barylyuk, Konstantin; Chen, Huanwen

    2016-10-28

    The application range of electrospray ionization mass spectrometry for the quantitative determination of stoichiometries and binding constants for non-covalent protein complexes is broadly discussed. The underlying fundamental question is whether or not the original molecular equilibrium can be preserved during the ionization process and be revealed by subsequent mass spectrometry analysis. Here, we take a new look at this question by discussing recent studies in droplet chemistry.This article is part of the themed issue 'Quantitative mass spectrometry'. PMID:27644969

  3. Determination of the structure and morphology of gold nanoparticle-HSA protein complexes.

    PubMed

    Capomaccio, Robin; Jimenez, Isaac Ojea; Colpo, Pascal; Gilliland, Douglas; Ceccone, Giacomo; Rossi, François; Calzolai, Luigi

    2015-11-14

    We propose a simple method to determine the structure and morphology of nanoparticle protein complexes. By combining a separation method with online size measurements, density measurements and circular dichroism, we could identify the number of proteins bound to each nanoparticle and their secondary structure changes in the complex. This method provides much-needed experimental information on the interaction of proteins with nanoparticles and on the behavior of nanoparticles in biological systems. PMID:26462441

  4. Detecting protein complexes from active protein interaction networks constructed with dynamic gene expression profiles

    PubMed Central

    2013-01-01

    Background Protein interaction networks (PINs) are known to be useful to detect protein complexes. However, most available PINs are static, which cannot reflect the dynamic changes in real networks. At present, some researchers have tried to construct dynamic networks by incorporating time-course (dynamic) gene expression data with PINs. However, the inevitable background noise exists in the gene expression array, which could degrade the quality of dynamic networkds. Therefore, it is needed to filter out contaminated gene expression data before further data integration and analysis. Results Firstly, we adopt a dynamic model-based method to filter noisy data from dynamic expression profiles. Then a new method is proposed for identifying active proteins from dynamic gene expression profiles. An active protein at a time point is defined as the protein the expression level of whose corresponding gene at that time point is higher than a threshold determined by a standard variance involved threshold function. Furthermore, a noise-filtered active protein interaction network (NF-APIN) is constructed. To demonstrate the efficiency of our method, we detect protein complexes from the NF-APIN, compared with those from other dynamic PINs. Conclusion A dynamic model based method can effectively filter out noises in dynamic gene expression data. Our method to compute a threshold for determining the active time points of noise-filtered genes can make the dynamic construction more accuracy and provide a high quality framework for network analysis, such as protein complex prediction. PMID:24565281

  5. The solution structural ensembles of RNA kink-turn motifs and their protein complexes.

    PubMed

    Shi, Xuesong; Huang, Lin; Lilley, David M J; Harbury, Pehr B; Herschlag, Daniel

    2016-03-01

    With the growing number of crystal structures of RNA and RNA-protein complexes, a critical next step is understanding the dynamic solution behavior of these entities in terms of conformational ensembles and energy landscapes. To this end, we have used X-ray scattering interferometry (XSI) to probe the ubiquitous RNA kink-turn motif and its complexes with the canonical kink-turn binding protein L7Ae. XSI revealed that the folded kink-turn is best described as a restricted conformational ensemble. The ions present in solution alter the nature of this ensemble, and protein binding can perturb the kink-turn ensemble without collapsing it to a unique state. This study demonstrates how XSI can reveal structural and ensemble properties of RNAs and RNA-protein complexes and uncovers the behavior of an important RNA-protein motif. This type of information will be necessary to understand, predict and engineer the behavior and function of RNAs and their protein complexes. PMID:26727239

  6. Structural basis of pyrimidine specificity in the MS2 RNA hairpin-coat-protein complex.

    PubMed Central

    Grahn, E; Moss, T; Helgstrand, C; Fridborg, K; Sundaram, M; Tars, K; Lago, H; Stonehouse, N J; Davis, D R; Stockley, P G; Liljas, L

    2001-01-01

    We have determined the X-ray structures of six MS2 RNA hairpin-coat-protein complexes having five different substitutions at the hairpin loop base -5. This is a uracil in the wild-type hairpin and contacts the coat protein both by stacking on to a tyrosine side chain and by hydrogen bonding to an asparagine side chain. The RNA consensus sequence derived from coat protein binding studies with natural sequence variants suggested that the -5 base needs to be a pyrimidine for strong binding. The five -5 substituents used in this study were 5-bromouracil, pyrimidin-2-one, 2-thiouracil, adenine, and guanine. The structure of the 5-bromouracil complex was determined to 2.2 A resolution, which is the highest to date for any MS2 RNA-protein complex. All the complexes presented here show very similar conformations, despite variation in affinity in solution. The results suggest that the stacking of the -5 base on to the tyrosine side chain is the most important driving force for complex formation. A number of hydrogen bonds that are present in the wild-type complex are not crucial for binding, as they are missing in one or more of the complexes. The results also reveal the flexibility of this RNA-protein interface, with respect to functional group variation, and may be generally applicable to other RNA-protein complexes. PMID:11720290

  7. Immobilization of soluble protein complexes in MAS solid-state NMR: Sedimentation versus viscosity.

    PubMed

    Sarkar, Riddhiman; Mainz, Andi; Busi, Baptiste; Barbet-Massin, Emeline; Kranz, Maximilian; Hofmann, Thomas; Reif, Bernd

    2016-01-01

    In recent years, MAS solid-state NMR has emerged as a technique for the investigation of soluble protein complexes. It was found that high molecular weight complexes do not need to be crystallized in order to obtain an immobilized sample for solid-state NMR investigations. Sedimentation induced by sample rotation impairs rotational diffusion of proteins and enables efficient dipolar coupling based cross polarization transfers. In addition, viscosity contributes to the immobilization of the molecules in the sample. Natural Deep Eutectic Solvents (NADES) have very high viscosities, and can replace water in living organisms. We observe a considerable amount of cross polarization transfers for NADES solvents, even though their molecular weight is too low to yield significant sedimentation. We discuss how viscosity and sedimentation both affect the quality of the obtained experimental spectra. The FROSTY/sedNMR approach holds the potential to study large protein complexes, which are otherwise not amenable for a structural characterization using NMR. We show that using this method, backbone assignments of the symmetric proteasome activator complex (1.1MDa), and high quality correlation spectra of non-symmetric protein complexes such as the prokaryotic ribosome 50S large subunit binding to trigger factor (1.4MDa) are obtained. PMID:27017576

  8. Regulation of human ornithine decarboxylase expression by the c-Myc.Max protein complex.

    PubMed

    Peña, A; Reddy, C D; Wu, S; Hickok, N J; Reddy, E P; Yumet, G; Soprano, D R; Soprano, K J

    1993-12-25

    The presence of a CACGTG element within a region of the human ornithine decarboxylase (ODC) promoter located at -491 to -474 base pairs 5' to the start site of transcription suggested that the c-Myc.Max protein complex may play a role in the regulation of ODC expression during growth. Electrophoretic mobility shift assays and methylation interference analysis showed that the nuclei of WI-38 cells expressing ODC contained proteins that bound to this region of the ODC gene in a manner that correlated with growth-associated ODC expression. Also, use of antibodies against c-Myc and Max and purified recombinant c-Myc and Max protein in the electrophoretic mobility shift assay confirmed that these proteins can specifically bind this portion of the human ODC promoter. Transient transfection studies showed that increase in the level of c-Myc and/or Max led to a significant enhancement of expression of a human ODC promoter-CAT reporter construct. Moreover, treatment of actively growing WI-38 cells with an antisense oligomer to c-Myc reduced the amount of endogenous protein complex formed and the amount of endogenous ODC mRNA expressed. These studies show that the c-Myc.Max protein complex plays a role in the transcriptional regulation of human ODC in vivo.

  9. A 1 MDa protein complex containing critical components of the Escherichia coli divisome

    PubMed Central

    Trip, Erik N.; Scheffers, Dirk-Jan

    2015-01-01

    Cell division in bacteria is an essential process that is carried out at mid-cell by a group of cell division proteins referred to as the divisome. In Escherichia coli, over two dozen cell division proteins have been identified of which ten are essential. These division proteins localize sequentially and interdependently to the division site, after which constriction eventually produces two daughter cells. Various genetic and biochemical techniques have identified many interactions amongst cell division proteins, however the existence of the divisome as a large multi-protein complex has never been shown. Here, we identify a 1 MDa protein complex by native page that contains seven essential cell division proteins (FtsZ, ZipA, FtsK, FtsQ, FtsB, FtsL, and FtsN). The 1 MDa complex is present in rapidly dividing cells, but absent when cultures enter the stationary growth phase. Slight overexpression of the ftsQ D237N mutation that blocks cell division prevents formation of this 1 MDa complex. In cells depleted of FtsN, the 1 MDa complex is not assembled. Combined, our findings indicate that a large protein complex containing many different cell division proteins indeed exists. We note that this complex is very fragile and sensitive to the expression of tagged versions of FtsQ. PMID:26643979

  10. The Solution Structural Ensembles of RNA Kink-turn Motifs and Their Protein Complexes

    PubMed Central

    Huang, Lin; Lilley, David M. J.

    2015-01-01

    With the growing number of crystal structures of RNA and RNA/protein complexes, a critical next step is understanding the dynamic behavior of these entities in solution in terms of conformational ensembles and energy landscapes. To this end, we have used X-ray scattering interferometry (XSI) to probe the widespread RNA kink-turn motif and its complexes with the canonical kink-turn binding protein L7Ae. XSI revealed that the folded kink-turn is best described as a restricted conformational ensemble. The ions present in solution alter the nature of this ensemble, and protein binding can perturb the kink-turn ensemble without collapsing it to a unique state. This study demonstrates how XSI can reveal structural and ensemble properties of RNAs and RNA/protein complexes in solution and uncovers the behavior of an important RNA/protein motif. This type of information will be necessary to understand, predict, and engineer the behavior and function of RNAs and their protein complexes. PMID:26727239

  11. Discovering New Features of Protein Complexes Structures by Small-Angle X-Ray Scattering

    NASA Astrophysics Data System (ADS)

    Oliveira, C. L. P.; Vorup-Jensen, T.; Andersen, C. B. F.; Andersen, G. R.; Pedersen, J. S.

    In spite of the recent advances in the X-Ray crystallography and nuclear magnetic resonance techniques, the determination of the quaternary structure of large protein complexes is still a challenge in molecular biology and biological sciences. In this respect, small-angle X-ray scattering (SAXS) is a key technique, enabling the determination of the possible structural conformation of complexes in an almost native state. Despite of this book being devoted to scattering techniques by synchrotron radiation, in this chapter we present two examples of application of laboratory-based SAXS to protein solution. The fundaments of the technique are obviously the same and have been deeply described in Chap. 2. In this chapter, we will introduce the application of SAXS to protein solution. Special emphasis is done on data reduction and absolute units calibration. As an example to illustrate the power of this technique, two new data sets for two protein complexes will be presented. This will show how high-quality SAXS data combined with advanced model strategies enables the determination of the quaternary structure of protein complexes.

  12. Immobilization of soluble protein complexes in MAS solid-state NMR: Sedimentation versus viscosity.

    PubMed

    Sarkar, Riddhiman; Mainz, Andi; Busi, Baptiste; Barbet-Massin, Emeline; Kranz, Maximilian; Hofmann, Thomas; Reif, Bernd

    2016-01-01

    In recent years, MAS solid-state NMR has emerged as a technique for the investigation of soluble protein complexes. It was found that high molecular weight complexes do not need to be crystallized in order to obtain an immobilized sample for solid-state NMR investigations. Sedimentation induced by sample rotation impairs rotational diffusion of proteins and enables efficient dipolar coupling based cross polarization transfers. In addition, viscosity contributes to the immobilization of the molecules in the sample. Natural Deep Eutectic Solvents (NADES) have very high viscosities, and can replace water in living organisms. We observe a considerable amount of cross polarization transfers for NADES solvents, even though their molecular weight is too low to yield significant sedimentation. We discuss how viscosity and sedimentation both affect the quality of the obtained experimental spectra. The FROSTY/sedNMR approach holds the potential to study large protein complexes, which are otherwise not amenable for a structural characterization using NMR. We show that using this method, backbone assignments of the symmetric proteasome activator complex (1.1MDa), and high quality correlation spectra of non-symmetric protein complexes such as the prokaryotic ribosome 50S large subunit binding to trigger factor (1.4MDa) are obtained.

  13. Alternate dissociation pathways identified in charge-reduced protein complex ions.

    PubMed

    Pagel, Kevin; Hyung, Suk-Joon; Ruotolo, Brandon T; Robinson, Carol V

    2010-06-15

    Tandem mass spectrometry (MS) of large protein complexes has proven to be capable of assessing the stoichiometry, connectivity, and structural details of multiprotein assemblies. While the utility of tandem MS is without question, a deeper understanding of the mechanism of protein complex dissociation will undoubtedly drive the technology into new areas of enhanced utility and information content. We present here the systematic analysis of the charge state dependent decay of the noncovalently associated complex of human transthyretin, generated by collision-induced dissociation (CID). A crown ether based charge reduction approach was applied to generate intact transthyretin tetramers with charge states ranging from 15+ to 7+. These nine charge states were subsequently analyzed by means of tandem MS and ion mobility spectrometry. Three different charge-dependent mechanistic regimes were identified: (1) common asymmetric dissociation involving ejection of unfolded monomers, (2) expulsion of folded monomers from the intact tetramer, and (3) release of C-terminal peptide fragments from the intact complex. Taken together, the results presented highlight the potential of charge state modulation as a method for directing the course of gas-phase dissociation and unfolding of protein complexes.

  14. Human metabolites of synthetic cannabinoids JWH-018 and JWH-073 bind with high affinity and act as potent agonists at cannabinoid type-2 receptors

    SciTech Connect

    Rajasekaran, Maheswari; Brents, Lisa K.; Franks, Lirit N.; Moran, Jeffery H.; Prather, Paul L.

    2013-06-01

    K2 or Spice is an emerging drug of abuse that contains synthetic cannabinoids, including JWH-018 and JWH-073. Recent reports indicate that monohydroxylated metabolites of JWH-018 and JWH-073 retain high affinity and activity at cannabinoid type-1 receptors (CB{sub 1}Rs), potentially contributing to the enhanced toxicity of K2 compared to marijuana. Since the parent compounds also bind to cannabinoid type-2 receptors (CB{sub 2}Rs), this study investigated the affinity and intrinsic activity of JWH-018, JWH-073 and several monohydroxylated metabolites at human CB{sub 2}Rs (hCB{sub 2}Rs). The affinity of cannabinoids for hCB{sub 2}Rs was determined by competition binding studies employing CHO-hCB{sub 2} membranes. Intrinsic activity of compounds was assessed by G-protein activation and adenylyl cyclase (AC)-inhibition in CHO-hCB{sub 2} cells. JWH-073, JWH-018 and several of their human metabolites exhibit nanomolar affinity and act as potent agonists at hCB{sub 2}Rs. Furthermore, a major omega hydroxyl metabolite of JWH-073 (JWH-073-M5) binds to CB{sub 2}Rs with 10-fold less affinity than the parent molecule, but unexpectedly, is equipotent in regulating AC-activity when compared to the parent molecule. Finally, when compared to CP-55,940 and Δ{sup 9}-tetrahydrocannabinol (Δ{sup 9}-THC), JWH-018, JWH-018-M5 and JWH-073-M5 require significantly less CB{sub 2}R occupancy to produce similar levels of AC-inhibition, indicating that these compounds may more efficiently couple CB{sub 2}Rs to AC than the well characterized cannabinoid agonists examined. These results indicate that JWH-018, JWH-073 and several major human metabolites of these compounds exhibit high affinity and demonstrate distinctive signaling properties at CB{sub 2}Rs. Therefore, future studies examining pharmacological and toxicological properties of synthetic cannabinoids present in K2 products should consider potential actions of these drugs at both CB{sub 1} and CB{sub 2}Rs. - Highlights: • JWH-018

  15. High-affinity Manganese Coordination by Human Calprotectin is Calcium-dependent and Requires the Histidine-rich Site Formed at the Dimer Interface

    PubMed Central

    Hayden, Joshua A.; Brophy, Megan Brunjes; Cunden, Lisa S.; Nolan, Elizabeth M.

    2013-01-01

    Calprotectin (CP) is a transition metal-chelating antimicrobial protein of the calcium-binding S100 family that is produced and released by neutrophils. It inhibits the growth of various pathogenic microorganisms by sequestering the transition metal ions manganese and zinc. In this work, we investigate the manganese-binding properties of calprotectin. We demonstrate that the unusual His4 motif (site 2) formed at the S100A8/S100A9 dimer interface is the site of high-affinity Mn(II) coordination. We identify a low-temperature Mn(II) spectroscopic signal for this site consistent with an octahedral Mn(II) coordination sphere with simulated zero-field splitting parameters D = 270 MHz and E/D = 0.33 (E = 81 MHz). This analysis, combined with studies of mutant proteins, suggests that four histidine residues (H17 and H27 of S100A8; H91 and H95 of S100A9) coordinate Mn(II) in addition to two as-yet unidentified ligands. The His3Asp motif (site 1), which is also formed at the S100A8/S100A9 dimer interface, does not provide a high-affinity Mn(II) binding site. Calcium binding to the EF-hand domains of CP increases the Mn(II) affinity of the His4 site from the low-micromolar to the mid-nanomolar range. Metal-ion selectivity studies demonstrate that CP prefers to coordinate Zn(II) over Mn(II). Nevertheless, the specificity of Mn(II) for the His4 site provides CP with the propensity to form mixed Zn:Mn:CP complexes where one Zn(II) ion occupies site 1 and one Mn(II) ion occupies site 2. These studies support the notion that CP responds to physiological calcium ion gradients to become a high-affinity transition metal ion chelator in the extracellular space where it inhibits microbial growth. PMID:23276281

  16. Characterization of an AtCCX5 gene from Arabidopsis thaliana that involves in high-affinity K{sup +} uptake and Na{sup +} transport in yeast

    SciTech Connect

    Zhang, Xinxin; Zhang, Min; Takano, Tetsuo; Liu, Shenkui

    2011-10-14

    Highlights: {yields} The AtCCX5 protein coding a putative cation calcium exchanger was characterized. {yields} AtCCX5 expressed in yeast was localized in the plasma membrane and nuclear periphery. {yields} AtCCX5 protein did not show the same transport properties as the CAXs. {yields} AtCCX5 protein involves in mediating high-affinity K{sup +} uptake in yeast. {yields} AtCCX5 protein also involves in Na{sup +} transport in yeast. -- Abstract: The gene for a putative cation calcium exchanger (CCX) from Arabidopsis thaliana, AtCCX5, was cloned and its function was analyzed in yeast. Green fluorescent protein-tagged AtCCX5 expressed in yeast was localized in the plasma membrane and nuclear periphery. The yeast transformants expressing AtCCX5 were created and their growth in the presence of various cations (K{sup +}, Na{sup +}, Ca{sup 2+}, Mg{sup 2+}, Fe{sup 2+}, Cu{sup 2+}, Co{sup 2+}, Cd{sup 2+}, Mn{sup 2+}, Ba{sup 2+}, Ni{sup 2+}, Zn{sup 2+}, and Li{sup +}) were analyzed. AtCCX5 expression was found to affect the response to K{sup +} and Na{sup +} in yeast. The AtCCX5 transformant also showed a little better growth to Zn{sup 2+}. The yeast mutant 9.3 expressing AtCCX5 restored growth of the mutant on medium with low K{sup +} (0.5 mM), and also suppressed its Na{sup +} sensitivity. Ion uptake experiments showed that AtCCX5 mediated relatively high-affinity K{sup +} uptake and was also involved in Na{sup +} transport in yeast. Taken together, these findings suggest that the AtCCX5 is a novel transport protein involves in mediating high-affinity K{sup +} uptake and Na{sup +} transport in yeast.

  17. Mapping of barley alpha-amylases and outer subsite mutants reveals dynamic high-affinity subsites and barriers in the long substrate binding cleft.

    PubMed

    Kandra, Lili; Hachem, Maher Abou; Gyémánt, Gyöngyi; Kramhøft, Birte; Svensson, Birte

    2006-09-18

    Subsite affinity maps of long substrate binding clefts in barley alpha-amylases, obtained using a series of maltooligosaccharides of degree of polymerization of 3-12, revealed unfavorable binding energies at the internal subsites -3 and -5 and at subsites -8 and +3/+4 defining these subsites as binding barriers. Barley alpha-amylase 1 mutants Y105A and T212Y at subsite -6 and +4 resulted in release or anchoring of bound substrate, thus modifying the affinities of other high-affinity subsites (-2 and +2) and barriers. The double mutant Y105A-T212Y displayed a hybrid subsite affinity profile, converting barriers to binding areas. These findings highlight the dynamic binding energy distribution and the versatility of long maltooligosaccharide derivatives in mapping extended binding clefts in alpha-amylases.

  18. (I-125) 17. cap alpha. -Iodovinyl 11. beta. -methoxyestradiol: in vivo and in vitro properties of a high-affinity estrogen-receptor radiopharmaceutical

    SciTech Connect

    Jagoda, E.M.; Gibson, R.E.; Goodgold, H.; Ferreira, N.; Francis, B.E.; Reba, R.C.; Rzeszotarski, W.J.; Eckelman, W.C.

    1984-04-01

    17 ..cap alpha..-(/sup 125/I)Iodovinyl 11 ..beta..-methoxyestradiol ((I-125)MIVE/sub 2/) has been prepared with high specific activity (155-2000 Ci/mmol) and a high affinity for the estrogen receptor. In vivo distribution studies using immature rats result in high levels of activity in the uterus (20-30% dose/g) with uterus-to-plasma ratios on the order of 68 to 100. Peak activity in the uterus is obtained between 2 and 4 hr, and by 6 hr 50% of the activity has washed out. The radioactive labeling of MIVE/sub 2/ is sufficiently rapid so that (I-123)MIVE/sub 2/ has been synthesized and is currently in clinical trials. These results suggest that MIVE/sub 2/ would be an excellent agent for the study of estrogen receptors in vivo and in vitro.

  19. Evidence for the presence of a high-affinity laminin receptor-like molecule on the surface of Candida albicans yeast cells.

    PubMed Central

    López-Ribot, J L; Casanova, M; Monteagudo, C; Sepúlveda, P; Martínez, J P

    1994-01-01

    Two polypeptides of 37 and 67 kDa that bind laminin were detected in cell wall extracts of Candida albicans blastoconidia. The 37-kDa species, found only in yeast cell wall extracts, cross-reacted with a rabbit polyclonal antibody (PAb 4160) directed towards the carboxyl-terminal laminin-binding domain present in the human 67-kDa high-affinity laminin receptor (67LR) and its 37-kDa precursor (37LRP), whereas another antibody (PAb 4056), directed against internal domains of 67LR and 37LRP, recognized a 37-kDa species in wall extracts from both blastoconidia and germinated blastoconidia. Indirect immunofluorescence with PAb 4160 showed a patchy binding pattern only on yeast cells that represented about 10% of the entire blastoconidia population. Images PMID:8300236

  20. Selective targeting of the IL23 pathway: Generation and characterization of a novel high-affinity humanized anti-IL23A antibody

    PubMed Central

    Singh, Sanjaya; Kroe-Barrett, Rachel R; Canada, Keith A; Zhu, Xiang; Sepulveda, Eliud; Wu, Helen; He, Yaqin; Raymond, Ernest L; Ahlberg, Jennifer; Frego, Lee E; Amodeo, Laura M; Catron, Katrina M; Presky, David H; Hanke, Jeffrey H

    2015-01-01

    Herein, we describe the generation and characterization of BI 655066, a novel, highly potent neutralizing anti-interleukin-23 (IL23) monoclonal antibody in clinical development for autoimmune conditions, including psoriasis and Crohn's disease. IL23 is a key driver of the differentiation, maintenance, and activity of a number of immune cell subsets, including T helper 17 (Th17) cells, which are believed to mediate the pathogenesis of several immune-mediated disorders. Thus, IL23 neutralization is an attractive therapeutic approach. Designing an antibody for clinical activity and convenience for the patient requires certain properties, such as high affinity, specificity, and solubility. These properties were achieved by directed design of the immunization, lead identification, and humanization procedures. Favorable substance and pharmacokinetic properties were established by biophysical assessments and studies in cynomolgus monkeys. PMID:25905918

  1. LYR3, a high-affinity LCO-binding protein of Medicago truncatula, interacts with LYK3, a key symbiotic receptor.

    PubMed

    Fliegmann, Judith; Jauneau, Alain; Pichereaux, Carole; Rosenberg, Charles; Gasciolli, Virginie; Timmers, Antonius C J; Burlet-Schiltz, Odile; Cullimore, Julie; Bono, Jean-Jacques

    2016-05-01

    LYR3, LYK3, and NFP are lysin motif-containing receptor-like kinases (LysM-RLKs) from Medicago truncatula, involved in perception of symbiotic lipo-chitooligosaccharide (LCO) signals. Here, we show that LYR3, a high-affinity LCO-binding protein, physically interacts with LYK3, a key player regulating symbiotic interactions. In vitro, LYR3 is phosphorylated by the active kinase domain of LYK3. Fluorescence lifetime imaging/Förster resonance energy transfer (FLIM/FRET) experiments in tobacco protoplasts show that the interaction between LYR3 and LYK3 at the plasma membrane is disrupted or inhibited by addition of LCOs. Moreover, LYR3 attenuates the cell death response, provoked by coexpression of NFP and LYK3 in tobacco leaves. PMID:27129432

  2. Synthesis and in vitro autoradiographic evaluation of a novel high-affinity radioiodinated ligand for imaging brain cannabinoid subtype-1 receptors.

    PubMed

    Donohue, Sean R; Varnäs, Katarina; Jia, Zhisheng; Gulyás, Balázs; Pike, Victor W; Halldin, Christer

    2009-11-01

    There is strong interest to study the involvement of brain cannabinoid subtype-1 (CB1) receptors in neuropsychiatric disorders with single photon emission computed tomography (SPECT) and a suitable radioligand. Here we report the synthesis of a novel high-affinity radioiodinated CB1 receptor ligand ([125I]8, [125I]1-(2-iodophenyl)-4-cyano-5-(4-methoxyphenyl)-N-(piperidin-1-yl)-1H-pyrazole-3-carboxylate, [125I]SD7015). By autoradiography in vitro, [125I]8 showed selective binding to CB1 receptors on human brain postmortem cryosections and now merits labeling with iodine-123 for further evaluation as a SPECT radioligand in non-human primate. PMID:19767206

  3. Plasmid Vectors for Proteomic Analyses in Giardia: Purification of Virulence Factors and Analysis of the Proteasome

    PubMed Central

    Stadelmann, Britta; Birkestedt, Sandra; Hellman, Ulf; Svärd, Staffan G.

    2012-01-01

    In recent years, proteomics has come of age with the development of efficient tools for purification, identification, and characterization of gene products predicted by genome projects. The intestinal protozoan Giardia intestinalis can be transfected, but there is only a limited set of vectors available, and most of them are not user friendly. This work delineates the construction of a suite of cassette-based expression vectors for use in Giardia. Expression is provided by the strong constitutive ornithine carbamoyltransferase (OCT) promoter, and tagging is possible in both N- and C-terminal configurations. Taken together, the vectors are capable of providing protein localization and production of recombinant proteins, followed by efficient purification by a novel affinity tag combination, streptavidin binding peptide–glutathione S-transferase (SBP-GST). The option of removing the tags from purified proteins was provided by the inclusion of a PreScission protease site. The efficiency and feasibility of producing and purifying endogenous recombinant Giardia proteins with the developed vectors was demonstrated by the purification of active recombinant arginine deiminase (ADI) and OCT from stably transfected trophozoites. Moreover, we describe the tagging, purification by StrepTactin affinity chromatography, and compositional analysis by mass spectrometry of the G. intestinalis 26S proteasome by employing the Strep II-FLAG–tandem affinity purification (SF-TAP) tag. This is the first report of efficient production and purification of recombinant proteins in and from Giardia, which will allow the study of specific parasite proteins and protein complexes. PMID:22611020

  4. STP10 encodes a high-affinity monosaccharide transporter and is induced under low-glucose conditions in pollen tubes of Arabidopsis

    PubMed Central

    Rottmann, Theresa; Zierer, Wolfgang; Subert, Christa; Sauer, Norbert; Stadler, Ruth

    2016-01-01

    Pollen tubes are fast growing, photosynthetically inactive cells. Their energy demand is covered by specific transport proteins in the plasma membrane that mediate the uptake of sugars. Here we report on the functional characterization of AtSTP10, a previously uncharacterized member of the SUGAR TRANSPORT PROTEIN family. Heterologous expression of STP10 cDNA in yeast revealed that the encoded protein catalyses the high-affinity uptake of glucose, galactose and mannose. The transporter is sensitive to uncouplers of transmembrane proton gradients, indicating that the protein acts as a hexose–H+ symporter. Analyses of STP10 mRNA and STP10 promoter–reporter gene studies revealed a sink-specific expression pattern of STP10 in primordia of lateral roots and in pollen tubes. This restriction to sink organs is mediated by intragenic regions of STP10. qPCR analyses with cDNA of in vitro grown pollen tubes showed that STP10 expression was down-regulated in the presence of 50mM glucose. However, in pollen tubes of glucose-insensitive plants, which lack the glucose sensor hexokinase1 (HXK1), no glucose-induced down-regulation of STP10 expression was detected. A stp10 T-DNA insertion line developed normally, which may point towards functional redundancy. The data presented in this paper indicate that a high-affinity glucose uptake system is induced in growing pollen tubes under low glucose conditions and that this regulation may occur through the hexokinase pathway. PMID:26893494

  5. Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcεRI

    PubMed Central

    Sabban, Sari; Ye, Hongtu; Helm, Birgit

    2014-01-01

    The interaction of IgE with its high-affinity Fc receptor (FcεRI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and FcεRI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine FcεRIα was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine α-chain formed a functional receptor complex with the endogenous rat β- and γ-chains 1. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine FcεRIα transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using β-hexosaminidase release as a readout 2, 3. Mediator release peaked at 36.68% ± 4.88% at 100 ng ml-1 of antigen. This assay was modified from previous assays used to study human and canine allergic responses 4, 5. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease 6, 2, 3. PMID:25406512

  6. STP10 encodes a high-affinity monosaccharide transporter and is induced under low-glucose conditions in pollen tubes of Arabidopsis.

    PubMed

    Rottmann, Theresa; Zierer, Wolfgang; Subert, Christa; Sauer, Norbert; Stadler, Ruth

    2016-04-01

    Pollen tubes are fast growing, photosynthetically inactive cells. Their energy demand is covered by specific transport proteins in the plasma membrane that mediate the uptake of sugars. Here we report on the functional characterization of AtSTP10, a previously uncharacterized member of the SUGAR TRANSPORT PROTEIN family. Heterologous expression of STP10 cDNA in yeast revealed that the encoded protein catalyses the high-affinity uptake of glucose, galactose and mannose. The transporter is sensitive to uncouplers of transmembrane proton gradients, indicating that the protein acts as a hexose-H(+)symporter. Analyses of STP10 mRNA and STP10 promoter-reporter gene studies revealed a sink-specific expression pattern of STP10 in primordia of lateral roots and in pollen tubes. This restriction to sink organs is mediated by intragenic regions of STP10 qPCR analyses with cDNA of in vitro grown pollen tubes showed that STP10 expression was down-regulated in the presence of 50mM glucose. However, in pollen tubes of glucose-insensitive plants, which lack the glucose sensor hexokinase1 (HXK1), no glucose-induced down-regulation of STP10 expression was detected. A stp10T-DNA insertion line developed normally, which may point towards functional redundancy. The data presented in this paper indicate that a high-affinity glucose uptake system is induced in growing pollen tubes under low glucose conditions and that this regulation may occur through the hexokinase pathway. PMID:26893494

  7. "Velcro" engineering of high affinity CD47 ectodomain as signal regulatory protein α (SIRPα) antagonists that enhance antibody-dependent cellular phagocytosis.

    PubMed

    Ho, Chia Chi M; Guo, Nan; Sockolosky, Jonathan T; Ring, Aaron M; Weiskopf, Kipp; Özkan, Engin; Mori, Yasuo; Weissman, Irving L; Garcia, K Christopher

    2015-05-15

    CD47 is a cell surface protein that transmits an anti-phagocytic signal, known as the "don't-eat-me" signal, to macrophages upon engaging its receptor signal regulatory protein α (SIRPα). Molecules that antagonize the CD47-SIRPα interaction by binding to CD47, such as anti-CD47 antibodies and the engineered SIRPα variant CV1, have been shown to facilitate macrophage-mediated anti-tumor responses. However, these strategies targeting CD47 are handicapped by large antigen sinks in vivo and indiscriminate cell binding due to ubiquitous expression of CD47. These factors reduce bioavailability and increase the risk of toxicity. Here, we present an alternative strategy to antagonize the CD47-SIRPα pathway by engineering high affinity CD47 variants that target SIRPα, which has restricted tissue expression. CD47 proved to be refractive to conventional affinity maturation techniques targeting its binding interface with SIRPα. Therefore, we developed a novel engineering approach, whereby we augmented the existing contact interface via N-terminal peptide extension, coined "Velcro" engineering. The high affinity variant (Velcro-CD47) bound to the two most prominent human SIRPα alleles with greatly increased affinity relative to wild-type CD47 and potently antagonized CD47 binding to SIRPα on human macrophages. Velcro-CD47 synergizes with tumor-specific monoclonal antibodies to enhance macrophage phagocytosis of tumor cells in vitro, with similar potency as CV1. Finally, Velcro-CD47 interacts specifically with a subset of myeloid-derived cells in human blood, whereas CV1 binds all myeloid, lymphoid, and erythroid populations interrogated. This is consistent with the restricted expression of SIRPα compared with CD47. Herein, we have demonstrated that "Velcro" engineering is a powerful protein-engineering tool with potential applications to other systems and that Velcro-CD47 could be an alternative adjuvant to CD47-targeting agents for cancer immunotherapy.

  8. Competitive Selection from Single Domain Antibody Libraries Allows Isolation of High-Affinity Antihapten Antibodies That Are Not Favored in the llama Immune Response

    PubMed Central

    Rosa, Sofia Tabares-da; Rossotti, Martin; Carleiza, Carmen; Carrión, Federico; Pritsch, Otto; Ahn, Ki Chang; Last, Jerold A.; Hammock, Bruce D; González-Sapienza, Gualberto

    2011-01-01

    Single-domain antibodies (sdAbs) found in camelids, lack a light chain and their antigen-binding site sits completely in the heavy-chain variable domain (VHH). Their simplicity, thermostability, and ease in expression have made VHHs highly attractive. While this has been successfully exploited for macromolecular antigens, their application to the detection of small molecules is still limited to a very few reports, mostly describing low affinity VHHs. Using triclocarban (TCC) as a model hapten, we found that conventional antibodies, IgG1 fraction, reacted with free TCC with a higher relative affinity (IC50 51.0 ng/mL) than did the sdAbs (IgG2 and IgG3, 497 and 370 ng/mL, respectively). A VHH library was prepared, and by elution of phage with limiting concentrations of TCC and competitive selection of binders, we were able to isolate high-affinity clones, KD 0.98–1.37 nM (SPR) which allowed development of a competitive assay for TCC with an IC50 = 3.5 ng/mL (11 nM). This represents a 100-fold improvement with regard to the performance of the sdAb serum fraction, and it is 100-fold better than the IC50 attained with other anti-hapten VHHs reported thus far. Despite the modest overall anti-hapten sdAbs response in llamas, a small subpopulation of high affinity VHHs are generated that can be isolated by carefully design of the selection process. PMID:21827167

  9. Distribution and ultrastructure of neurons in opossum piriform cortex displaying immunoreactivity to GABA and GAD and high-affinity tritiated GABA uptake

    SciTech Connect

    Haberly, L.B.; Hansen, D.J.; Feig, S.L.; Presto, S.

    1987-12-08

    GABAergic neurons have been identified in the piriform cortex of the opossum at light and electron microscopic levels by immunocytochemical localization of GABA and the GABA-synthesizing enzyme glutamic acid decarboxylase and by autoradiographic visualization of high-affinity /sup 3/H-GABA uptake. Four major neuron populations have been distinguished on the basis of soma size, shape, and segregation at specific depths and locations: large horizontal cells in layer Ia of the anterior piriform cortex, small globular cells with thin dendrites concentrated in layers Ib and II of the posterior piriform cortex, and multipolar and fusiform cells concentrated in the deep part of layer III in anterior and posterior parts of the piriform cortex and the subjacent endopiriform nucleus. All four populations were well visualized with both antisera, but the large layer Ia horizontal cells displayed only very light /sup 3/H-GABA uptake, thus suggesting a lack of local axon collaterals or lack of high-affinity GABA uptake sites. The large, ultrastructurally distinctive somata of layer Ia horizontal cells receive a very small number of symmetrical synapses; the thin, axonlike dendrites of small globular cells are exclusively postsynaptic and receive large numbers of both symmetrical and asymmetrical synapses, in contrast to somata which receive a small number of both types; and the deep multipolar and fusiform cells receive a highly variable number of symmetrical and asymmetrical synapses on somata and proximal dendrites. Labeled puncta of axon terminal dimensions were found in large numbers in the neuropil surrounding pyramidal cell somata in layer II and in the endopiriform nucleus. Moderately large numbers of labeled puncta were found in layer I at the depth of pyramidal cell apical dendrites with greater numbers in layer Ia at the depth of distal apical segments than in layer Ib.

  10. High Affinity Pharmacological Profiling of Dual Inhibitors Targeting RET and VEGFR2 in Inhibition of Kinase and Angiogeneis Events in Medullary Thyroid Carcinoma.

    PubMed

    Dunna, Nageswara Rao; Kandula, Venkatesh; Girdhar, Amandeep; Pudutha, Amareshwari; Hussain, Tajamul; Bandaru, Srinivas; Nayarisseri, Anuraj

    2015-01-01

    Clinical evidence shows that dual inhibition of kinases as well angiogenesis provides ideal therapeutic option in the treatment of medullary thyroid carcinoma (MTC) than inhibiting either of these with the events separately. Although treatment with dual inhibitors has shown good clinical responses in patients with MTC, it has been associated with serious side effects. Some inhibitors are active agents for both angiogenesis or kinase activity. Owing to narrow therapeutic window of established inhibitors, the present study aims to identify high affinity dual inhibitors targeting RET and VEGFR2 respectively for kinase and angiogenesis activity. Established inhibitors like Vandetanib, Cabozantinib, Motesanib, PP121, RAF265 and Sunitinib served as query parent compounds for identification of structurally similar compounds by Tanimoto-based similarity searching with a threshold of 95% against the PubChem database. All the parent inhibitors and respective similar compounds were docked against RET and VEGFR2 in order to retrieve high affinity compounds with these two proteins. AGN-PC-0CUK9P PubCID: 59320403 a compound related to PPI21 showed almost equal affinity for RET and VEGFR2 and unlike other screened compounds with no apparent bias for either of the receptors. Further, AGN- PC-0CUK9P demonstrated appreciable interaction with both RET and VEGFR2 and superior kinase activity in addition to showed optimal ADMET properties and pharmacophore features. From our in silico investigation we suggest AGN-PC-0CUK9P as a superior dual inhibitor targeting RET and VEGFR2 with high efficacy which should be proposed for pharmacodynamic and pharmacokinetic studies for improved treatment of MTC.

  11. Structure-Affinity Properties of a High-Affinity Ligand of FKBP12 Studied by Molecular Simulations of a Binding Intermediate

    PubMed Central

    Olivieri, Lilian; Gardebien, Fabrice

    2014-01-01

    With a view to explaining the structure-affinity properties of the ligands of the protein FKBP12, we characterized a binding intermediate state between this protein and a high-affinity ligand. Indeed, the nature and extent of the intermolecular contacts developed in such a species may play a role on its stability and, hence, on the overall association rate. To find the binding intermediate, a molecular simulation protocol was used to unbind the ligand by gradually decreasing the biasing forces introduced. The intermediate was subsequently refined with 17 independent stochastic boundary molecular dynamics simulations that provide a consistent picture of the intermediate state. In this state, the core region of the ligand remains stable, notably because of the two anchoring oxygen atoms that correspond to recurrent motifs found in all FKBP12 ligand core structures. Besides, the non-core regions participate in numerous transient intermolecular and intramolecular contacts. The dynamic aspect of most of the contacts seems important both for the ligand to retain at least a part of its configurational entropy and for avoiding a trapped state along the binding pathway. Since the transient and anchoring contacts contribute to increasing the stability of the intermediate, as a corollary, the dissociation rate constant of this intermediate should be decreased, resulting in an increase of the affinity constant . The present results support our previous conclusions and provide a coherent rationale for explaining the prevalence in high-affinity ligands of (i) the two oxygen atoms found in carbonyl or sulfonyl groups of dissimilar core structures and of (ii) symmetric or pseudo-symmetric mobile groups of atoms found as non-core moieties. Another interesting aspect of the intermediate is the distortion of the flexible 80 s loop of the protein, mainly in its tip region, that promotes the accessibility to the bound state. PMID:25502559

  12. “Velcro” Engineering of High Affinity CD47 Ectodomain as Signal Regulatory Protein α (SIRPα) Antagonists That Enhance Antibody-dependent Cellular Phagocytosis*

    PubMed Central

    Ho, Chia Chi M.; Guo, Nan; Sockolosky, Jonathan T.; Ring, Aaron M.; Weiskopf, Kipp; Özkan, Engin; Mori, Yasuo; Weissman, Irving L.; Garcia, K. Christopher

    2015-01-01

    CD47 is a cell surface protein that transmits an anti-phagocytic signal, known as the “don't-eat-me” signal, to macrophages upon engaging its receptor signal regulatory protein α (SIRPα). Molecules that antagonize the CD47-SIRPα interaction by binding to CD47, such as anti-CD47 antibodies and the engineered SIRPα variant CV1, have been shown to facilitate macrophage-mediated anti-tumor responses. However, these strategies targeting CD47 are handicapped by large antigen sinks in vivo and indiscriminate cell binding due to ubiquitous expression of CD47. These factors reduce bioavailability and increase the risk of toxicity. Here, we present an alternative strategy to antagonize the CD47-SIRPα pathway by engineering high affinity CD47 variants that target SIRPα, which has restricted tissue expression. CD47 proved to be refractive to conventional affinity maturation techniques targeting its binding interface with SIRPα. Therefore, we developed a novel engineering approach, whereby we augmented the existing contact interface via N-terminal peptide extension, coined “Velcro” engineering. The high affinity variant (Velcro-CD47) bound to the two most prominent human SIRPα alleles with greatly increased affinity relative to wild-type CD47 and potently antagonized CD47 binding to SIRPα on human macrophages. Velcro-CD47 synergizes with tumor-specific monoclonal antibodies to enhance macrophage phagocytosis of tumor cells in vitro, with similar potency as CV1. Finally, Velcro-CD47 interacts specifically with a subset of myeloid-derived cells in human blood, whereas CV1 binds all myeloid, lymphoid, and erythroid populations interrogated. This is consistent with the restricted expression of SIRPα compared with CD47. Herein, we have demonstrated that “Velcro” engineering is a powerful protein-engineering tool with potential applications to other systems and that Velcro-CD47 could be an alternative adjuvant to CD47-targeting agents for cancer immunotherapy

  13. High Affinity Pharmacological Profiling of Dual Inhibitors Targeting RET and VEGFR2 in Inhibition of Kinase and Angiogeneis Events in Medullary Thyroid Carcinoma.

    PubMed

    Dunna, Nageswara Rao; Kandula, Venkatesh; Girdhar, Amandeep; Pudutha, Amareshwari; Hussain, Tajamul; Bandaru, Srinivas; Nayarisseri, Anuraj

    2015-01-01

    Clinical evidence shows that dual inhibition of kinases as well angiogenesis provides ideal therapeutic option in the treatment of medullary thyroid carcinoma (MTC) than inhibiting either of these with the events separately. Although treatment with dual inhibitors has shown good clinical responses in patients with MTC, it has been associated with serious side effects. Some inhibitors are active agents for both angiogenesis or kinase activity. Owing to narrow therapeutic window of established inhibitors, the present study aims to identify high affinity dual inhibitors targeting RET and VEGFR2 respectively for kinase and angiogenesis activity. Established inhibitors like Vandetanib, Cabozantinib, Motesanib, PP121, RAF265 and Sunitinib served as query parent compounds for identification of structurally similar compounds by Tanimoto-based similarity searching with a threshold of 95% against the PubChem database. All the parent inhibitors and respective similar compounds were docked against RET and VEGFR2 in order to retrieve high affinity compounds with these two proteins. AGN-PC-0CUK9P PubCID: 59320403 a compound related to PPI21 showed almost equal affinity for RET and VEGFR2 and unlike other screened compounds with no apparent bias for either of the receptors. Further, AGN- PC-0CUK9P demonstrated appreciable interaction with both RET and VEGFR2 and superior kinase activity in addition to showed optimal ADMET properties and pharmacophore features. From our in silico investigation we suggest AGN-PC-0CUK9P as a superior dual inhibitor targeting RET and VEGFR2 with high efficacy which should be proposed for pharmacodynamic and pharmacokinetic studies for improved treatment of MTC. PMID:26514495

  14. Capacity and Plasticity of Potassium Channels and High-Affinity Transporters in Roots of Barley and Arabidopsis1[C][W

    PubMed Central

    Coskun, Devrim; Britto, Dev T.; Li, Mingyuan; Oh, Saehong; Kronzucker, Herbert J.

    2013-01-01

    The role of potassium (K+) transporters in high- and low-affinity K+ uptake was examined in roots of intact barley (Hordeum vulgare) and Arabidopsis (Arabidopsis thaliana) plants by use of 42K radiotracing, electrophysiology, pharmacology, and mutant analysis. Comparisons were made between results from barley and five genotypes of Arabidopsis, including single and double knockout mutants for the high-affinity transporter, AtHAK5, and the Shaker-type channel, AtAKT1. In Arabidopsis, steady-state K+ influx at low external K+ concentration ([K+]ext = 22.5 µm) was predominantly mediated by AtAKT1 when high-affinity transport was inhibited by ammonium, whereas in barley, by contrast, K+ channels could not operate below 100 µm. Withdrawal of ammonium resulted in an immediate and dramatic stimulation of K+ influx in barley, indicating a shift from active to passive K+ uptake at low [K+]ext and yielding fluxes as high as 36 µmol g (root fresh weight)−1 h−1 at 5 mm [K+]ext, among the highest transporter-mediated K+ fluxes hitherto reported. This ammonium-withdrawal effect was also established in all Arabidopsis lines (the wild types, atakt1, athak5, and athak5 atakt1) at low [K+]ext, revealing the concerted involvement of several transport systems. The ammonium-withdrawal effect coincided with a suppression of K+ efflux and a significant hyperpolarization of the plasma membrane in all genotypes except athak5 atakt1, could be sustained over 24 h, and resulted in increased tissue K+ accumulation. We discuss key differences and similarities in K+ acquisition between two important model systems and reveal novel aspects of K+ transport in planta. PMID:23553635

  15. Histidine-rich Glycoprotein Binds Fibrin(ogen) with High Affinity and Competes with Thrombin for Binding to the γ′-Chain*

    PubMed Central

    Vu, Trang T.; Stafford, Alan R.; Leslie, Beverly A.; Kim, Paul Y.; Fredenburgh, James C.; Weitz, Jeffrey I.

    2011-01-01

    Histidine-rich glycoprotein (HRG) is an abundant protein that binds fibrinogen and other plasma proteins in a Zn2+-dependent fashion but whose function is unclear. HRG has antimicrobial activity, and its incorporation into fibrin clots facilitates bacterial entrapment and killing and promotes inflammation. Although these findings suggest that HRG contributes to innate immunity and inflammation, little is known about the HRG-fibrin(ogen) interaction. By immunoassay, HRG-fibrinogen complexes were detected in Zn2+-supplemented human plasma, a finding consistent with a high affinity interaction. Surface plasmon resonance determinations support this concept and show that in the presence of Zn2+, HRG binds the predominant γA/γA-fibrinogen and the γ-chain elongated isoform, γA/γ′-fibrinogen, with Kd values of 9 nm. Likewise, 125I-labeled HRG binds γA/γA- or γA/γ′-fibrin clots with similar Kd values when Zn2+ is present. There are multiple HRG binding sites on fibrin(ogen) because HRG binds immobilized fibrinogen fragment D or E and γ′-peptide, an analog of the COOH terminus of the γ′-chain that mediates the high affinity interaction of thrombin with γA/γ′-fibrin. Thrombin competes with HRG for γ′-peptide binding and displaces 125I-HRG from γA/γ′-fibrin clots and vice versa. Taken together, these data suggest that (a) HRG circulates in complex with fibrinogen and that the complex persists upon fibrin formation, and (b) by competing with thrombin for γA/γ′-fibrin binding, HRG may modulate coagulation. Therefore, the HRG-fibrin interaction may provide a novel link between coagulation, innate immunity, and inflammation. PMID:21757718

  16. High-affinity insulin binding to an atypical insulin-like growth factor-I receptor in human breast cancer cells.

    PubMed Central

    Milazzo, G; Yip, C C; Maddux, B A; Vigneri, R; Goldfine, I D

    1992-01-01

    We studied the nature of insulin receptor binding in MCF-7 breast cancer cells. In both intact cells and solubilized receptor preparations, high-affinity insulin binding was seen. However, unlabeled insulin-like growth factor-I (IGF-I) was five-fold more potent in inhibiting 125I-insulin binding than insulin itself. With monoclonal antibodies to the insulin receptor, 30% of 125I-insulin binding was inhibited. In contrast when alpha-IR3, a monoclonal antibody that recognizes typical IGF-I receptor, was employed over 60% of 125I-insulin binding was inhibited. The B29-MAB-125I-insulin photoprobe was then cross-linked to MCF-7 membranes. Cross-linking was inhibited by both unlabeled insulin and IGF-I. Further, the B29-MAB-125I-insulin photoprobe cross-linked to MCF-7 membranes was strongly immunoprecipitated by alpha-IR3. Employing sequential affinity chromatography with insulin-Affi-gel followed by insulin receptor monoclonal antibody agarose, atypical insulin binding activity was separated from insulin receptor binding activity. This atypical receptor had intrinsic tyrosine kinase activity. Both insulin and IGF-I stimulated the phosphorylation of the receptor's beta subunit. In MCF-7 cells both IGF-I and insulin stimulated [3H]thymidine incorporation; alpha-IR3 blocked all of the IGF-I effect but only 50-60% of the insulin effect. This study demonstrates in MCF-7 cells that, in addition to typical insulin and IGF-I receptors, there is another receptor that binds both insulin and IGF-I with high affinity. Images PMID:1311720

  17. The Interaction of Arp2/3 Complex with Actin: Nucleation, High Affinity Pointed End Capping, and Formation of Branching Networks of Filaments

    NASA Astrophysics Data System (ADS)

    Dyche Mullins, R.; Heuser, John A.; Pollard, Thomas D.

    1998-05-01

    The Arp2/3 complex is a stable assembly of seven protein subunits including two actin-related proteins (Arp2 and Arp3) and five novel proteins. Previous work showed that this complex binds to the sides of actin filaments and is concentrated at the leading edges of motile cells. Here, we show that Arp2/3 complex purified from Acanthamoeba caps the pointed ends of actin filaments with high affinity. Arp2/3 complex inhibits both monomer addition and dissociation at the pointed ends of actin filaments with apparent nanomolar affinity and increases the critical concentration for polymerization at the pointed end from 0.6 to 1.0 μ M. The high affinity of Arp2/3 complex for pointed ends and its abundance in amoebae suggest that in vivo all actin filament pointed ends are capped by Arp2/3 complex. Arp2/3 complex also nucleates formation of actin filaments that elongate only from their barbed ends. From kinetic analysis, the nucleation mechanism appears to involve stabilization of polymerization intermediates (probably actin dimers). In electron micrographs of quick-frozen, deep-etched samples, we see Arp2/3 bound to sides and pointed ends of actin filaments and examples of Arp2/3 complex attaching pointed ends of filaments to sides of other filaments. In these cases, the angle of attachment is a remarkably constant 70 ± 7 degrees. From these in vitro biochemical properties, we propose a model for how Arp2/3 complex controls the assembly of a branching network of actin filaments at the leading edge of motile cells.

  18. Histidine-rich glycoprotein binds fibrin(ogen) with high affinity and competes with thrombin for binding to the gamma'-chain.

    PubMed

    Vu, Trang T; Stafford, Alan R; Leslie, Beverly A; Kim, Paul Y; Fredenburgh, James C; Weitz, Jeffrey I

    2011-09-01

    Histidine-rich glycoprotein (HRG) is an abundant protein that binds fibrinogen and other plasma proteins in a Zn(2+)-dependent fashion but whose function is unclear. HRG has antimicrobial activity, and its incorporation into fibrin clots facilitates bacterial entrapment and killing and promotes inflammation. Although these findings suggest that HRG contributes to innate immunity and inflammation, little is known about the HRG-fibrin(ogen) interaction. By immunoassay, HRG-fibrinogen complexes were detected in Zn(2+)-supplemented human plasma, a finding consistent with a high affinity interaction. Surface plasmon resonance determinations support this concept and show that in the presence of Zn(2+), HRG binds the predominant γ(A)/γ(A)-fibrinogen and the γ-chain elongated isoform, γ(A)/γ'-fibrinogen, with K(d) values of 9 nm. Likewise, (125)I-labeled HRG binds γ(A)/γ(A)- or γ(A)/γ'-fibrin clots with similar K(d) values when Zn(2+) is present. There are multiple HRG binding sites on fibrin(ogen) because HRG binds immobilized fibrinogen fragment D or E and γ'-peptide, an analog of the COOH terminus of the γ'-chain that mediates the high affinity interaction of thrombin with γ(A)/γ'-fibrin. Thrombin competes with HRG for γ'-peptide binding and displaces (125)I-HRG from γ(A)/γ'-fibrin clots and vice versa. Taken together, these data suggest that (a) HRG circulates in comple