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Sample records for binding protein dependent

  1. Human neutrophil calmodulin-binding proteins: identification of the calmodulin-dependent protein phosphatase

    International Nuclear Information System (INIS)

    Blackburn, W.D.; Tallant, E.A.; Wallace, R.W.

    1986-01-01

    The molecular events in linking neutrophil activation and ligand binding to specific membrane receptors are mediated in part by an increase in intracellular Ca 2+ . One mechanism by which Ca 2+ may trigger neutrophil activation is through Ca 2+ /calmodulin (CaM)-regulated proteins and enzymes. To determine which Ca 2+ /CaM-regulated enzymes may be present in the neutrophil, they have used Western blotting techniques and 125 I-CaM to identify neutrophil CaM-binding proteins. Eleven proteins with molecular weights ranging from 230K to 13.5K bound 125 I-CaM in a Ca 2+ -dependent manner. One predominant region of 125 I-Cam binding was to a 59K protein; a protein with an identical mobility was labeled by an antisera against brain CaM-dependent phosphatase. Ca 2+ -dependent phosphatase activity, which was inhibited by the CaM antagonist trifluoperazine, was detected in a neutrophil extract; a radioimmunoassay for the phosphatase indicated that it was present in the extract at approximately 0.2 μg/mg protein. Most of the CaM-binding proteins, including the 59K protein, were rapidly degraded upon lysis of the neutrophil. There was a close correlation between the degradation of the 59K protein and the loss of Ca 2+ -dependent phosphatase activity in the neutrophil extract. Thus, human neutrophils contain numerous CaM-binding proteins which are presumably Ca 2+ /calmodulin-regulated enzymes and proteins; the 59K protein is a CaM-dependent phosphatase

  2. A robust assay to measure DNA topology-dependent protein binding affinity.

    Science.gov (United States)

    Litwin, Tamara R; Solà, Maria; Holt, Ian J; Neuman, Keir C

    2015-04-20

    DNA structure and topology pervasively influence aspects of DNA metabolism including replication, transcription and segregation. However, the effects of DNA topology on DNA-protein interactions have not been systematically explored due to limitations of standard affinity assays. We developed a method to measure protein binding affinity dependence on the topology (topological linking number) of supercoiled DNA. A defined range of DNA topoisomers at equilibrium with a DNA binding protein is separated into free and protein-bound DNA populations using standard nitrocellulose filter binding techniques. Electrophoretic separation and quantification of bound and free topoisomers combined with a simple normalization procedure provide the relative affinity of the protein for the DNA as a function of linking number. Employing this assay we measured topology-dependent DNA binding of a helicase, a type IB topoisomerase, a type IIA topoisomerase, a non-specific mitochondrial DNA binding protein and a type II restriction endonuclease. Most of the proteins preferentially bind negatively supercoiled DNA but the details of the topology-dependent affinity differ among proteins in ways that expose differences in their interactions with DNA. The topology-dependent binding assay provides a robust and easily implemented method to probe topological influences on DNA-protein interactions for a wide range of DNA binding proteins. Published by Oxford University Press on behalf of Nucleic Acids Research 2014. This work is written by US Government employees and is in the public domain in the US.

  3. CHARACTERIZATION OF A BINDING PROTEIN-DEPENDENT GLUTAMATE TRANSPORT-SYSTEM OF RHODOBACTER-SPHAEROIDES

    NARCIS (Netherlands)

    Jacobs, M.H J; Driessen, A.J.M.; Konings, W.N

    The mechanism of L-glutamate uptake was studied in Rhodobacter sphaeroides. Uptake of L-glutamate is mediated by a high-affinity (K-t of 1.2 mu M), shock-sensitive transport system that is inhibited by vanadate and dependent on the internal pH. From the shock fluid, an L-glutamate-binding protein

  4. Characterization of a Binding Protein-Dependent Glutamate Transport System of Rhodobacter sphaeroides

    NARCIS (Netherlands)

    Jacobs, Mariken H.J.; Driessen, Arnold J.M.; Konings, Wil N.

    1995-01-01

    The mechanism of L-glutamate uptake was studied in Rhodobacter sphaeroides. Uptake of L-glutamate is mediated by a high-affinity (Kt of 1.2 µM), shock-sensitive transport system that is inhibited by vanadate and dependent on the internal pH. From the shock fluid, an L-glutamate-binding protein was

  5. Human platelet calmodulin-binding proteins: Ca2+-dependent proteolysis upon platelet activation

    International Nuclear Information System (INIS)

    Wallace, R.W.; Tallant, E.A.; McManus, M.C.

    1986-01-01

    Calmodulin (CaM)-binding proteins have been identified in human platelets using Western blotting techniques and 125 I-CaM. Ten distinct proteins with molecular weights of 245, 225K, 175K, 150K, 90K, 82K(2), 60K and 41K(2) bound 125 I-CaM in a Ca 2+ -dependent manner; the binding was blocked by both trifluoperazine and nonradiolabeled CaM. The 225K and 90K proteins were labeled by antisera against myosin light chain kinase (MLCK); the 60K and one of the 82K proteins were identified as the CaM-dependent phosphatase and caldesmon. The remaining proteins have not yet been identified. Most of the CaM-binding proteins were degraded upon addition of Ca 2+ to a platelet homogenate; the degradation could be blocked by either EGTA, leupeptin or N-ethyl-maleimide which suggests that it was due to a Ca 2+ -dependent protease. Activation of intact platelets by thrombin, ADP, collagen and the Ca 2+ -ionophores A23187 and ionomycin under conditions which promote platelet aggregation (i.e. stirring with extracellular Ca 2+ ) also resulted in limited proteolysis of CaM-binding proteins including those labeled with anti-MLCK and the phosphatase. Many Ca 2+ /CaM-regulated enzymes have been shown to be irreversibly activated in vitro by limited proteolysis. Their data indicates that limited proteolysis also occurs in vivo; under certain conditions proteolysis may be an important physiological mechanism for irreversibly activating Ca 2+ /CaM-regulated enzymes

  6. Sequence-selective DNA binding to the regulatory subunit of cAMP-dependent protein kinase.

    Science.gov (United States)

    Wu, J C; Wang, J H

    1989-06-15

    The fluorescence of Trp-226 in the regulatory subunit of bovine type II cAMP-dependent protein kinase is unaffected by the binding of cAMP, but is quenched by the binding of 2'-dansyl-cAMP (DNS-cAMP). Up to 67% of the fluorescence of Trp-226 can be quenched by resonant energy transfer to the DNS-cAMP bound to the first site, and 96% of the fluorescence can be quenched by saturating both sites with DNS-cAMP. The observed efficiencies of energy transfer gave a distance of 16 A between Trp-226 and the DNS-cAMP bound at the first site and a distance of 12.7 A between Trp-226 and the DNS-cAMP bound at second site. The fluorescence of Trp-226 was suppressed by incubation of RII with the self-complementary octanucleotide TGACGTCA (CRE) due to binding of the oligonucleotide to RII. A detailed study of the binding equilibrium showed that each RII(cAMP)2 molecule binds 1 molecule of CRE with Kd = 80 nM. The corresponding Kd value for cAMP-depleted RII was found to be 25-fold higher. RII was also found to bind randomly selected DNA fragments with an average Kd value much higher than that of CRE. These observations show for the first time that the binding of oligonucleotide to RII is cAMP-enhanced and sequence-selective.

  7. The NAP motif of activity-dependent neuroprotective protein (ADNP) regulates dendritic spines through microtubule end binding proteins.

    Science.gov (United States)

    Oz, S; Kapitansky, O; Ivashco-Pachima, Y; Malishkevich, A; Giladi, E; Skalka, N; Rosin-Arbesfeld, R; Mittelman, L; Segev, O; Hirsch, J A; Gozes, I

    2014-10-01

    The NAP motif of activity-dependent neuroprotective protein (ADNP) enhanced memory scores in patients suffering from mild cognitive impairment and protected activities of daily living in schizophrenia patients, while fortifying microtubule (MT)-dependent axonal transport, in mice and flies. The question is how does NAP fortify MTs? Our sequence analysis identified the MT end-binding protein (EB1)-interacting motif SxIP (SIP, Ser-Ile-Pro) in ADNP/NAP and showed specific SxIP binding sites in all members of the EB protein family (EB1-3). Others found that EB1 enhancement of neurite outgrowth is attenuated by EB2, while EB3 interacts with postsynaptic density protein 95 (PSD-95) to modulate dendritic plasticity. Here, NAP increased PSD-95 expression in dendritic spines, which was inhibited by EB3 silencing. EB1 or EB3, but not EB2 silencing inhibited NAP-mediated cell protection, which reflected NAP binding specificity. NAPVSKIPQ (SxIP=SKIP), but not NAPVAAAAQ mimicked NAP activity. ADNP, essential for neuronal differentiation and brain formation in mouse, a member of the SWI/SNF chromatin remodeling complex and a major protein mutated in autism and deregulated in schizophrenia in men, showed similar EB interactions, which were enhanced by NAP treatment. The newly identified shared MT target of NAP/ADNP is directly implicated in synaptic plasticity, explaining the breadth and efficiency of neuroprotective/neurotrophic capacities.

  8. Binding-protein-dependent sugar transport by Agrobacterium radiobacter and A. tumefaciens grown in continuous culture.

    Science.gov (United States)

    Cornish, A; Greenwood, J A; Jones, C W

    1989-11-01

    Binding-protein-dependent sugar transport has been investigated in Agrobacterium radiobacter and A. tumefaciens. A. radiobacter contained two high-affinity glucose-binding proteins (GBP1 and GBP2) that additionally bound D-galactose (KD 0.26 microM) and D-xylose (KD 0.04 microM) respectively and were involved in the transport of these sugars. Partial sequencing of GBP1 and GBP2 showed that GBP2 exhibited significant homology with both the arabinose-binding protein (ABP) and the galactose-binding protein (GalBP) from Escherichia coli, whereas GBP1 exhibited significant homology only with ABP. Antiserum raised against GBP1 cross-reacted with GBP1 but not with GBP2, and vice versa. Anti-GBP1 and anti-GBP2 also cross-reacted with proteins corresponding to GBP1 and GBP2 respectively in A. tumefaciens, but little or no cross-reaction was observed with selected members of the Enterobacteriaceae, Rhizobiaceae and Pseudomonadaceae families grown under glucose limitation. GBP1 was less strongly repressed than GBP2 following batch growth of A. radiobacter on various carbon sources. The growth of A. radiobacter for more than approximately 10 generations in continuous culture under galactose or xylose limitation (D 0.045 h-1) led to the emergence of new strains which exhibited increased rates of glucose/galactose or glucose/xylose uptake, and which respectively hyperproduced GBP1 (strain AR18a) or GBP2 (strain AR9a). Similarly, growth of A. tumefaciens for more than approximately 15 generations under glucose or galactose limitation produced new strains which exhibited increased rates of glucose/xylose or glucose/galactose uptake and which respectively hyperproduced proteins analogous to GBP2 (strain AT9) or GBP1 (strain AT18a). It is concluded that growth of Agrobacterium species under carbon-limited conditions leads to the predictable emergence of new strains which specifically hyperproduce the transport system for the limiting nutrient. The GBP1-dependent system of A

  9. Calcium-dependent and -independent binding of the pentraxin serum amyloid P component to glycosaminoglycans and amyloid proteins

    DEFF Research Database (Denmark)

    Danielsen, B; Sørensen, I J; Nybo, Mads

    1997-01-01

    and beta2M) by ELISA. An increase in the dose-dependent binding of SAP to heparan sulfate, AA-protein and beta2M was observed as the pH decreased from 8.0 to 5.0. Furthermore, a lower, but significant Ca2(+)-independent binding of SAP to heparan sulfate, dermatan sulfate, AA protein and the amyloid...... precursor protein beta2M was observed. This binding was also enhanced at slightly acid pH, most pronounced at pH 5.0. The results of this study indicate that SAP can exhibit both Ca2(+)-dependent and -independent binding to ligands involved in amyloid fibril formation and that the binding is enhanced under...

  10. GTP-dependent binding and nuclear transport of RNA polymerase II by Npa3 protein

    DEFF Research Database (Denmark)

    Staresincic, Lidija; Walker, Jane; Dirac-Svejstrup, A Barbara

    2011-01-01

    transport of RNAPII. Surprisingly, we were unable to detect interactions between Npa3 and proteins in the classical importin a/ß pathway for nuclear import. Interestingly, Npa3-RNAPII binding is significantly increased by the addition of GTP or its slowly hydrolyzable analogue guanosine 5'-3-O......-(thio)triphosphate (GTP¿S). Moreover, the Npa3 mutant that binds GTP, but cannot hydrolyze it, binds RNAPII even in the absence of added GTP, whereas the mutant that cannot bind GTP is unable to bind the polymerase. Together, our data suggest that Npa3 defines an unconventional pathway for nuclear import of RNAPII, which...

  11. DNA Binding in High Salt: Analysing the Salt Dependence of Replication Protein A3 from the Halophile Haloferax volcanii

    Directory of Open Access Journals (Sweden)

    Jody A. Winter

    2012-01-01

    Full Text Available Halophilic archaea maintain intracellular salt concentrations close to saturation to survive in high-salt environments and their cellular processes have adapted to function under these conditions. Little is known regarding halophilic adaptation of the DNA processing machinery, particularly intriguing since protein-DNA interactions are classically salt sensitive. To investigate such adaptation, we characterised the DNA-binding capabilities of recombinant RPA3 from Haloferax volcanii (HvRPA3. Under physiological salt conditions (3 M KCl, HvRPA3 is monomeric, binding 18 nucleotide ssDNA with nanomolar affinity, demonstrating that RPAs containing the single OB-fold/zinc finger architecture bind with broadly comparable affinity to two OB-fold/zinc finger RPAs. Reducing the salt concentration to 1 M KCl induces dimerisation of the protein, which retains its ability to bind DNA. On circular ssDNA, two concentration-dependent binding modes are observed. Conventionally, increased salt concentration adversely affects DNA binding but HvRPA3 does not bind DNA in 0.2 M KCl, although multimerisation may occlude the binding site. The single N-terminal OB-fold is competent to bind DNA in the absence of the C-terminal zinc finger, albeit with reduced affinity. This study represents the first quantitative characterisation of DNA binding in a halophilic protein in extreme salt concentrations.

  12. DNA binding in high salt: analysing the salt dependence of replication protein A3 from the halophile Haloferax volcanii.

    Science.gov (United States)

    Winter, Jody A; Patoli, Bushra; Bunting, Karen A

    2012-01-01

    Halophilic archaea maintain intracellular salt concentrations close to saturation to survive in high-salt environments and their cellular processes have adapted to function under these conditions. Little is known regarding halophilic adaptation of the DNA processing machinery, particularly intriguing since protein-DNA interactions are classically salt sensitive. To investigate such adaptation, we characterised the DNA-binding capabilities of recombinant RPA3 from Haloferax volcanii (HvRPA3). Under physiological salt conditions (3 M KCl), HvRPA3 is monomeric, binding 18 nucleotide ssDNA with nanomolar affinity, demonstrating that RPAs containing the single OB-fold/zinc finger architecture bind with broadly comparable affinity to two OB-fold/zinc finger RPAs. Reducing the salt concentration to 1 M KCl induces dimerisation of the protein, which retains its ability to bind DNA. On circular ssDNA, two concentration-dependent binding modes are observed. Conventionally, increased salt concentration adversely affects DNA binding but HvRPA3 does not bind DNA in 0.2 M KCl, although multimerisation may occlude the binding site. The single N-terminal OB-fold is competent to bind DNA in the absence of the C-terminal zinc finger, albeit with reduced affinity. This study represents the first quantitative characterisation of DNA binding in a halophilic protein in extreme salt concentrations.

  13. The potentiation of myeloperoxidase activity by the glycosaminoglycan-dependent binding of myeloperoxidase to proteins of the extracellular matrix.

    Science.gov (United States)

    Kubala, Lukáš; Kolářová, Hana; Víteček, Jan; Kremserová, Silvie; Klinke, Anna; Lau, Denise; Chapman, Anna L P; Baldus, Stephan; Eiserich, Jason P

    2013-10-01

    Myeloperoxidase (MPO) is an abundant hemoprotein expressed by neutrophil granulocytes that is recognized to play an important role in the development of vascular diseases. Upon degranulation from circulating neutrophil granulocytes, MPO binds to the surface of endothelial cells in an electrostatic-dependent manner and undergoes transcytotic migration to the underlying extracellular matrix (ECM). However, the mechanisms governing the binding of MPO to subendothelial ECM proteins, and whether this binding modulates its enzymatic functions are not well understood. We investigated MPO binding to ECM derived from aortic endothelial cells, aortic smooth muscle cells, and fibroblasts, and to purified ECM proteins, and the modulation of these associations by glycosaminoglycans. The oxidizing and chlorinating potential of MPO upon binding to ECM proteins was tested. MPO binds to the ECM proteins collagen IV and fibronectin, and this association is enhanced by the pre-incubation of these proteins with glycosaminoglycans. Correspondingly, an excess of glycosaminoglycans in solution during incubation inhibits the binding of MPO to collagen IV and fibronectin. These observations were confirmed with cell-derived ECM. The oxidizing and chlorinating potential of MPO was preserved upon binding to collagen IV and fibronectin; even the potentiation of MPO activity in the presence of collagen IV and fibronectin was observed. Collectively, the data reveal that MPO binds to ECM proteins on the basis of electrostatic interactions, and MPO chlorinating and oxidizing activity is potentiated upon association with these proteins. Our findings provide new insights into the molecular mechanisms underlying the interaction of MPO with ECM proteins. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Insight into temperature dependence of GTPase activity in human guanylate binding protein-1.

    Directory of Open Access Journals (Sweden)

    Anjana Rani

    Full Text Available Interferon-γ induced human guanylate binding protein-1(hGBP1 belongs to a family of dynamin related large GTPases. Unlike all other GTPases, hGBP1 hydrolyzes GTP to a mixture of GDP and GMP with GMP being the major product at 37°C but GDP became significant when the hydrolysis reaction was carried out at 15°C. The hydrolysis reaction in hGBP1 is believed to involve with a number of catalytic steps. To investigate the effect of temperature in the product formation and on the different catalytic complexes of hGBP1, we carried out temperature dependent GTPase assays, mutational analysis, chemical and thermal denaturation studies. The Arrhenius plot for both GDP and GMP interestingly showed nonlinear behaviour, suggesting that the product formation from the GTP-bound enzyme complex is associated with at least more than one step. The negative activation energy for GDP formation and GTPase assay with external GDP together indicate that GDP formation occurs through the reversible dissociation of GDP-bound enzyme dimer to monomer, which further reversibly dissociates to give the product. Denaturation studies of different catalytic complexes show that unlike other complexes the free energy of GDP-bound hGBP1 decreases significantly at lower temperature. GDP formation is found to be dependent on the free energy of the GDP-bound enzyme complex. The decrease in the free energy of this complex at low temperature compared to at high is the reason for higher GDP formation at low temperature. Thermal denaturation studies also suggest that the difference in the free energy of the GTP-bound enzyme dimer compared to its monomer plays a crucial role in the product formation; higher stability favours GMP but lower favours GDP. Thus, this study provides the first thermodynamic insight into the effect of temperature in the product formation of hGBP1.

  15. Repair of Nerve Cell Membrance Damage by Calcium-Dependent, Membrane-Binding Proteins

    Science.gov (United States)

    2013-09-01

    disease amyloid beta protein forms calcium channels in bilayer membranes: blockade by tromethamine and aluminum , Proc Natl Acad Sci U S A 90 (1993) 567... coagulation cascade in blood and the chaperoning of membrane interactions inside cells.4 Common to these diverse functions is the ability to bind...are quickly resolved restoring the permeability barrier. In vivo, such a transient breakdown of the membrane might constitute a significant initial cost

  16. A Central Nervous System-Dependent Intron-Embedded Gene Encodes a Novel Murine Fyn Binding Protein.

    Science.gov (United States)

    Ben Khalaf, Noureddine; Taha, Safa; Bakhiet, Moiz; Fathallah, M Dahmani

    2016-01-01

    The interplay between the nervous and immune systems is gradually being unraveled. We previously reported in the mouse the novel soluble immune system factor ISRAA, whose activation in the spleen is central nervous system-dependent. We also showed that ISRAA plays a role in modulating anti-infection immunity. Herein, we report the genomic description of the israa locus, along with some insights into the structure-function relationship of the protein. Our findings revealed that israa is nested within intron 6 of the mouse zmiz1 gene. Protein sequence analysis revealed a typical SH2 binding motif (Y102TEV), with Fyn being the most likely binding partner. Docking simulation showed a favorable conformation for the ISRAA-Fyn complex, with a specific binding mode for the binding of the YTEV motif to the SH2 domain. Experimental studies showed that in vitro, recombinant ISRAA is phosphorylated by Fyn at tyrosine 102. Cell transfection and pull-down experiments revealed Fyn as a binding partner of ISRAA in the EL4 mouse T-cell line. Indeed, we demonstrated that ISRAA downregulates T-cell activation and the phosphorylation of an activation tyrosine (Y416) of Src-family kinases in mouse splenocytes. Our observations highlight ISRAA as a novel Fyn binding protein that is likely to be involved in a signaling pathway driven by the nervous system.

  17. Conformational dynamics of L-lysine, L-arginine, L-ornithine binding protein reveals ligand-dependent plasticity.

    Science.gov (United States)

    Silva, Daniel-Adriano; Domínguez-Ramírez, Lenin; Rojo-Domínguez, Arturo; Sosa-Peinado, Alejandro

    2011-07-01

    The molecular basis of multiple ligand binding affinity for amino acids in periplasmic binding proteins (PBPs) and in the homologous domain for class C G-protein coupled receptors is an unsolved question. Here, using unrestrained molecular dynamic simulations, we studied the ligand binding mechanism present in the L-lysine, L-arginine, L-ornithine binding protein. We developed an analysis based on dihedral angles for the description of the conformational changes upon ligand binding. This analysis has an excellent correlation with each of the two main movements described by principal component analysis (PCA) and it's more convenient than RMSD measurements to describe the differences in the conformational ensembles observed. Furthermore, an analysis of hydrogen bonds showed specific interactions for each ligand studied as well as the ligand interaction with the aromatic residues Tyr-14 and Phe-52. Using uncharged histidine tautomers, these interactions are not observed. On the basis of these results, we propose a model in which hydrogen bond interactions place the ligand in the correct orientation to induce a cation-π interaction with Tyr-14 and Phe-52 thereby stabilizing the closed state. Our results also show that this protein adopts slightly different closed conformations to make available specific hydrogen bond interactions for each ligand thus, allowing a single mechanism to attain multiple ligand specificity. These results shed light on the experimental evidence for ligand-dependent conformational plasticity not explained by the previous crystallographic data. Copyright © 2011 Wiley-Liss, Inc.

  18. An abundant DNA binding protein from the hyperthermophilic archaeon Sulfolobus shibatae affects DNA supercoiling in a temperature-dependent fashion.

    Science.gov (United States)

    Xue, H; Guo, R; Wen, Y; Liu, D; Huang, L

    2000-07-01

    The DNA binding protein Ssh10b, a member of the Sac10b family, has been purified from the hyperthermophilic archaeon Sulfolobus shibatae. Ssh10b constitutes about 4% of the cellular protein. Electrophoretic mobility shift assays showed that Ssh10b first bound a double-stranded DNA fragment with an estimated binding size of approximately approximately 12 bp, forming distinct shifts, until the DNA was coated with the protein. Binding of more Ssh10b resulted in the formation of smears of lower mobilities. The migration pattern of the smearing Ssh10b-DNA complexes was affected by temperature, whereas that of complexes associated with the distinct shifts was not. Interestingly, Ssh10b was capable of constraining negative DNA supercoils in a temperature-dependent fashion. While the ability of the protein to constrain supercoils was weak at 25 degrees C, it was enhanced substantially at 45 degrees C or higher temperatures (up to 80 degrees C). Taken together, our data suggest that archaeal proteins of the Sac10b family may affect the topology of chromosomal DNA in thermophilic archaea at their growth temperatures.

  19. Nuclear size is sensitive to NTF2 protein levels in a manner dependent on Ran binding

    Science.gov (United States)

    Vuković, Lidija D.; Jevtić, Predrag; Zhang, Zhaojie; Stohr, Bradley A.; Levy, Daniel L.

    2016-01-01

    ABSTRACT Altered nuclear size is associated with many cancers, and determining whether cancer-associated changes in nuclear size contribute to carcinogenesis necessitates an understanding of mechanisms of nuclear size regulation. Although nuclear import rates generally positively correlate with nuclear size, NTF2 levels negatively affect nuclear size, despite the role of NTF2 (also known as NUTF2) in nuclear recycling of the import factor Ran. We show that binding of Ran to NTF2 is required for NTF2 to inhibit nuclear expansion and import of large cargo molecules in Xenopus laevis egg and embryo extracts, consistent with our observation that NTF2 reduces the diameter of the nuclear pore complex (NPC) in a Ran-binding-dependent manner. Furthermore, we demonstrate that ectopic NTF2 expression in Xenopus embryos and mammalian tissue culture cells alters nuclear size. Finally, we show that increases in nuclear size during melanoma progression correlate with reduced NTF2 expression, and increasing NTF2 levels in melanoma cells is sufficient to reduce nuclear size. These results show a conserved capacity for NTF2 to impact on nuclear size, and we propose that NTF2 might be a new cancer biomarker. PMID:26823604

  20. IGF binding proteins.

    Science.gov (United States)

    Bach, Leon A

    2017-12-18

    Insulin-like growth factor binding proteins (IGFBPs) 1-6 bind IGFs but not insulin with high affinity. They were initially identified as serum carriers and passive inhibitors of IGF actions. However, subsequent studies showed that, although IGFBPs inhibit IGF actions in many circumstances, they may also potentiate these actions. IGFBPs are widely expressed in most tissues, and they are flexible endocrine and autocrine/paracrine regulators of IGF activity, which is essential for this important physiological system. More recently, individual IGFBPs have been shown to have IGF-independent actions. Mechanisms underlying these actions include (i) interaction with non-IGF proteins in compartments including the extracellular space and matrix, the cell surface and intracellularly; (ii) interaction with and modulation of other growth factor pathways including EGF, TGF- and VEGF; and (iii) direct or indirect transcriptional effects following nuclear entry of IGFBPs. Through these IGF-dependent and IGF-independent actions, IGFBPs modulate essential cellular processes including proliferation, survival, migration, senescence, autophagy and angiogenesis. They have been implicated in a range of disorders including malignant, metabolic, neurological and immune diseases. A more complete understanding of their cellular roles may lead to the development of novel IGFBP-based therapeutic opportunities.

  1. Bacillus thuringiensis Cyt2Aa2 binding on lipid/cholesterol bilayer depends on protein concentration and time.

    Science.gov (United States)

    Tharad, Sudarat; Moreno-Cencerrado, Alberto; Üzülmez, Öykü; Promdonkoy, Boonhiang; Toca-Herrera, Jose L

    2017-10-14

    Bacillus thuringiensis produces cytolytic proteins (Cyt) that show toxicity against dipteran insect larvae acting directly on the cell membrane. Up to now, two different models have been proposed to explain the interaction mechanism of the cytolytic protein Cyt2Aa2 on lipid membranes: pore-forming and detergent-like action. Here we report on the interaction of Cyt2Aa2 with lipid/cholesterol bilayers at early stage (far from equilibrium) as a function of protein concentration. Quartz crystal microbalance with dissipation (QCM-D) measurements showed that the rate of protein adsorption increased with concentration, although the mass of the final protein-lipid was similar after two hours. In addition, the dissipation (compliance of the hybrid lipid/protein layer) increased with decreasing protein concentration. Furthermore, atomic force microscopy (AFM) revealed that the structure of the protein-lipid layer was concentration and time dependent. A rigid hybrid homogeneous layer was observed at protein concentrations of 50 μg/ml and 100 μg/ml after 30 min. At lower concentrations, 10 μg/ml and 17.5 μg/ml, protein adsorption on the lipid layer led to the formation of small aggregates. Interestingly, at 25 μg/ml a transition of a hole-like structure into a homogeneous layer was observed. This suggests that 25 μg/ml is a threshold concentration for the binding mechanism of Cyt2Aa2 on to lipid membranes. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. NF-κB-dependent role for cold-inducible RNA binding protein in regulating interleukin 1β.

    Directory of Open Access Journals (Sweden)

    Christian Brochu

    Full Text Available The cold inducible RNA binding protein (CIRBP responds to a wide array of cellular stresses, including short wavelength ultraviolet light (UVC, at the transcriptional and post-translational level. CIRBP can bind the 3'untranslated region of specific transcripts to stabilize them and facilitate their transport to ribosomes for translation. Here we used RNA interference and oligonucleotide microarrays to identify potential downstream targets of CIRBP induced in response to UVC. Twenty eight transcripts were statistically increased in response to UVC and these exhibited a typical UVC response. Only 5 of the 28 UVC-induced transcripts exhibited a CIRBP-dependent pattern of expression. Surprisingly, 3 of the 5 transcripts (IL1B, IL8 and TNFAIP6 encoded proteins important in inflammation with IL-1β apparently contributing to IL8 and TNFAIP6 expression in an autocrine fashion. UVC-induced IL1B expression could be inhibited by pharmacological inhibition of NFκB suggesting that CIRBP was affecting NF-κB signaling as opposed to IL1B mRNA stability directly. Bacterial lipopolysaccharide (LPS was used as an activator of NF-κB to further study the potential link between CIRBP and NFκB. Transfection of siRNAs against CIRBP reduced the extent of the LPS-induced phosphorylation of IκBα, NF-κB DNA binding activity and IL-1β expression. The present work firmly establishes a novel link between CIRBP and NF-κB signaling in response to agents with diverse modes of action. These results have potential implications for disease states associated with inflammation.

  3. The Potato Nucleotide-binding Leucine-rich Repeat (NLR) Immune Receptor Rx1 Is a Pathogen-dependent DNA-deforming Protein.

    Science.gov (United States)

    Fenyk, Stepan; Townsend, Philip D; Dixon, Christopher H; Spies, Gerhard B; de San Eustaquio Campillo, Alba; Slootweg, Erik J; Westerhof, Lotte B; Gawehns, Fleur K K; Knight, Marc R; Sharples, Gary J; Goverse, Aska; Pålsson, Lars-Olof; Takken, Frank L W; Cann, Martin J

    2015-10-09

    Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable cells to respond to pathogen attack. Several NLRs act in the nucleus; however, conserved nuclear targets that support their role in immunity are unknown. Previously, we noted a structural homology between the nucleotide-binding domain of NLRs and DNA replication origin-binding Cdc6/Orc1 proteins. Here we show that the NB-ARC (nucleotide-binding, Apaf-1, R-proteins, and CED-4) domain of the Rx1 NLR of potato binds nucleic acids. Rx1 induces ATP-dependent bending and melting of DNA in vitro, dependent upon a functional P-loop. In situ full-length Rx1 binds nuclear DNA following activation by its cognate pathogen-derived effector protein, the coat protein of potato virus X. In line with its obligatory nucleocytoplasmic distribution, DNA binding was only observed when Rx1 was allowed to freely translocate between both compartments and was activated in the cytoplasm. Immune activation induced by an unrelated NLR-effector pair did not trigger an Rx1-DNA interaction. DNA binding is therefore not merely a consequence of immune activation. These data establish a role for DNA distortion in Rx1 immune signaling and define DNA as a molecular target of an activated NLR. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. The Potato Nucleotide-binding Leucine-rich Repeat (NLR) Immune Receptor Rx1 Is a Pathogen-dependent DNA-deforming Protein*

    Science.gov (United States)

    Fenyk, Stepan; Townsend, Philip D.; Dixon, Christopher H.; Spies, Gerhard B.; de San Eustaquio Campillo, Alba; Slootweg, Erik J.; Westerhof, Lotte B.; Gawehns, Fleur K. K.; Knight, Marc R.; Sharples, Gary J.; Goverse, Aska; Pålsson, Lars-Olof; Takken, Frank L. W.; Cann, Martin J.

    2015-01-01

    Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable cells to respond to pathogen attack. Several NLRs act in the nucleus; however, conserved nuclear targets that support their role in immunity are unknown. Previously, we noted a structural homology between the nucleotide-binding domain of NLRs and DNA replication origin-binding Cdc6/Orc1 proteins. Here we show that the NB-ARC (nucleotide-binding, Apaf-1, R-proteins, and CED-4) domain of the Rx1 NLR of potato binds nucleic acids. Rx1 induces ATP-dependent bending and melting of DNA in vitro, dependent upon a functional P-loop. In situ full-length Rx1 binds nuclear DNA following activation by its cognate pathogen-derived effector protein, the coat protein of potato virus X. In line with its obligatory nucleocytoplasmic distribution, DNA binding was only observed when Rx1 was allowed to freely translocate between both compartments and was activated in the cytoplasm. Immune activation induced by an unrelated NLR-effector pair did not trigger an Rx1-DNA interaction. DNA binding is therefore not merely a consequence of immune activation. These data establish a role for DNA distortion in Rx1 immune signaling and define DNA as a molecular target of an activated NLR. PMID:26306038

  5. RNA-binding protein PSPC1 promotes the differentiation-dependent nuclear export of adipocyte RNAs

    DEFF Research Database (Denmark)

    Wang, Jiexin; Rajbhandari, Prashant; Damianov, Andrey

    2017-01-01

    EBF1. Purification of the paraspeckle complex from adipocytes further showed that PSPC1 associates with the RNA export factor DDX3X in a differentiation-dependent manner. Remarkably, PSPC1 relocates from the nucleus to the cytoplasm during differentiation, coinciding with enhanced export of adipogenic...... RNAs. Mice lacking PSPC1 in fat displayed reduced lipid storage and adipose tissue mass and were resistant to diet-induced obesity and insulin resistance due to a compensatory increase in energy expenditure. These findings highlight a role for PSPC1-dependent RNA maturation in the posttranscriptional...

  6. binding protein (HABP1)

    Indian Academy of Sciences (India)

    Unknown

    of HA in a concentration-dependent manner, suggesting its multiligand affinity amongst carbohydrates. rHABP1 shows differential affinity ... site is seen to correspond to the carbohydrate-binding site in E-selectin, which has similarity in the ... adsorbed on carbon coated copper grid (400 mesh) for. 5 min at room temperature.

  7. Phosphorylation of the PCNA binding domain of the large subunit of replication factor C by Ca2+/calmodulin-dependent protein kinase II inhibits DNA synthesis

    DEFF Research Database (Denmark)

    Maga, G; Mossi, R; Fischer, R

    1997-01-01

    Replication factor C (RF-C) is a heteropentameric protein essential for DNA replication and DNA repair. It is a molecular matchmaker required for loading of the proliferating cell nuclear antigen (PCNA) sliding clamp onto double-strand DNA and for PCNA-dependent DNA synthesis by DNA polymerases...... delta and epsilon. The DNA and PCNA binding domains of the large 140 kDa subunit of human RF-C have been recently cloned [Fotedar, R., Mossi, R., Fitzgerald, P., Rousselle, T., Maga, G., Brickner, H., Messier, H., Khastilba. S., Hübscher, U., & Fotedar, A. (1996) EMBO J. 15, 4423-4433]. Here we show...... that the PCNA binding domain is phosphorylated by the Ca2+/calmodulin-dependent protein kinase II (CaMKII), an enzyme required for cell cycle progression in eukaryotic cells. The DNA binding domain, on the other hand, is not phosphorylated. Phosphorylation by CaMKII reduces the binding of PCNA to RF-C...

  8. Conserved epitope on several human vitamin K-dependent proteins: location of the antigenic site and influence of metal ions on antibody binding

    International Nuclear Information System (INIS)

    Church, W.R.; Messier, T.; Howard, P.R.; Amiral, J.; Meyer, D.; Mann, K.G.

    1988-01-01

    A murine monoclonal antibody (designated H-11) produced by injecting mice with purified human protein C was found to bind several human vitamin K-dependent proteins. Using a solid-phase competitive radioimmunoassay with antibody immobilized onto microtiter plates, binding of 125 I-labeled protein C to the antibody was inhibited by increasing amounts of protein C, prothrombin, and Factors X and VII over a concentration range of 1 x 10 -8 to 1 x 10 -6 M. Chemical treatment of prothrombin with a variety of agents did not destroy the antigenic site recognized by the antibody as measured by immunoblotting of prothrombin or prothrombin derivative immobilized onto nitrocellulose. Immunoblotting of purified vitamin K-dependent polypeptides with the monoclonal antibody following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose indicated that the antigenic site was found on the light chains of protein C and Factor X. The exact location of the antigenic determinant for antibody H-11 was established using synthetic peptides. Comparison of protein sequences of bovine and human vitamin K-dependent proteins suggests that the sequence Phe-Leu-Glu-Glu-Xaa-Arg/Lys is required for antibody binding. Increasing concentrations of Ca 2+ , Mg 2+ , or Mn 2+ partially inhibited binding of 125 I-protein C to the antibody in a solid-phase assay system with half-maximal binding observed at divalent metal ion concentrations of 2, 4, and 0.6 mM, respectively. The antigenic site thus recognized by monoclonal antibody H-11 is located at the amino-terminal region in the highly conserved γ-carboxyglutamic acid-containing domains of several, but not all, vitamin K-dependent proteins

  9. Extratelomeric Binding of the Telomere Binding Protein TRF2 at the PCGF3 Promoter Is G-Quadruplex Motif-Dependent.

    Science.gov (United States)

    Purohit, Gunjan; Mukherjee, Ananda Kishor; Sharma, Shalu; Chowdhury, Shantanu

    2018-04-11

    Telomere repeat binding factor 2 (TRF2) is critical for the protection of chromosome ends. Mounting evidence suggests that TRF2 associates with extratelomeric sites and TRF2 functions may not be limited to telomeres. Here, we show that the PCGF3 promoter harbors a sequence capable of forming the DNA secondary structure G-quadruplex motif, which is required for binding of TRF2 at the PCGF3 promoter. We demonstrate that promoter binding by TRF2 mediates PCGF3 promoter activity, and both the N-terminal and C-terminal domains of TRF2 are necessary for promoter activity. Altogether, this shows for the first time that a telomere binding factor may regulate a component of the polycomb group of proteins.

  10. Coupling of receptor interference and a host-dependent post-binding entry deficiency in a gammaretroviral envelope protein

    Directory of Open Access Journals (Sweden)

    Sørensen Karina

    2010-02-01

    Full Text Available Abstract Background SL3-2 is a unique polytropic murine gammaretroviral isolate that is only able to infect murine cells. We have previously shown that two mutations R212G and T213I located on the surface of the receptor binding domain in a region designated the VR3 loop can alter the species tropism of this envelope protein. This location suggests that the VR3 loop composition has an influence on receptor interaction and thereby affects binding as well as superinfection resistance. In order to investigate this further, we have studied the binding and interference patterns of the SL3-2 envelope and its mutants. Results We find unexpectedly that wild type SL3-2 envelope binds equally well to both permissive and non-permissive cells, indicating a post binding defect when interacting with the human Xpr1. Using replication competent viruses containing envelopes from SL3-2 or its mutants we find that the same amino acid mutations can dramatically alter the interference profile of this polytropic ENV, suggesting that the same amino acid changes that cause the post binding defect also influence interaction with the receptor. Conclusions The envelope protein of SL3-2 MLV shows an entry defect on non-murine cells. This is coupled to a dramatically reduced ability to interfere with entry of other polytropic viruses. Two point mutations in the VR3 loop of the receptor binding domain of this envelope result both in a much increased interference ability and in removing the post-binding defect on non-murine cells, suggesting that both of these phenotypes are a consequence of insufficient interaction between the envelope and the receptor

  11. Hexanucleotide Repeats in ALS/FTD Form Length-Dependent RNA Foci, Sequester RNA Binding Proteins, and Are Neurotoxic

    Directory of Open Access Journals (Sweden)

    Youn-Bok Lee

    2013-12-01

    Full Text Available The GGGGCC (G4C2 intronic repeat expansion within C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS and frontotemporal dementia (FTD. Intranuclear neuronal RNA foci have been observed in ALS and FTD tissues, suggesting that G4C2 RNA may be toxic. Here, we demonstrate that the expression of 38× and 72× G4C2 repeats form intranuclear RNA foci that initiate apoptotic cell death in neuronal cell lines and zebrafish embryos. The foci colocalize with a subset of RNA binding proteins, including SF2, SC35, and hnRNP-H in transfected cells. Only hnRNP-H binds directly to G4C2 repeats following RNA immunoprecipitation, and only hnRNP-H colocalizes with 70% of G4C2 RNA foci detected in C9ORF72 mutant ALS and FTD brain tissues. We show that expanded G4C2 repeats are potently neurotoxic and bind hnRNP-H and other RNA binding proteins. We propose that RNA toxicity and protein sequestration may disrupt RNA processing and contribute to neurodegeneration.

  12. Identification of a phosphorylation-dependent nuclear localization motif in interferon regulatory factor 2 binding protein 2.

    Directory of Open Access Journals (Sweden)

    Allen C T Teng

    Full Text Available Interferon regulatory factor 2 binding protein 2 (IRF2BP2 is a muscle-enriched transcription factor required to activate vascular endothelial growth factor-A (VEGFA expression in muscle. IRF2BP2 is found in the nucleus of cardiac and skeletal muscle cells. During the process of skeletal muscle differentiation, some IRF2BP2 becomes relocated to the cytoplasm, although the functional significance of this relocation and the mechanisms that control nucleocytoplasmic localization of IRF2BP2 are not yet known.Here, by fusing IRF2BP2 to green fluorescent protein and testing a series of deletion and site-directed mutagenesis constructs, we mapped the nuclear localization signal (NLS to an evolutionarily conserved sequence (354ARKRKPSP(361 in IRF2BP2. This sequence corresponds to a classical nuclear localization motif bearing positively charged arginine and lysine residues. Substitution of arginine and lysine with negatively charged aspartic acid residues blocked nuclear localization. However, these residues were not sufficient because nuclear targeting of IRF2BP2 also required phosphorylation of serine 360 (S360. Many large-scale phosphopeptide proteomic studies had reported previously that serine 360 of IRF2BP2 is phosphorylated in numerous human cell types. Alanine substitution at this site abolished IRF2BP2 nuclear localization in C(2C(12 myoblasts and CV1 cells. In contrast, substituting serine 360 with aspartic acid forced nuclear retention and prevented cytoplasmic redistribution in differentiated C(2C(12 muscle cells. As for the effects of these mutations on VEGFA promoter activity, the S360A mutation interfered with VEGFA activation, as expected. Surprisingly, the S360D mutation also interfered with VEGFA activation, suggesting that this mutation, while enforcing nuclear entry, may disrupt an essential activation function of IRF2BP2.Nuclear localization of IRF2BP2 depends on phosphorylation near a conserved NLS. Changes in phosphorylation status

  13. The Rab27a-binding protein, JFC1, regulates androgen-dependent secretion of prostate-specific antigen and prostatic-specific acid phosphatase1

    OpenAIRE

    Johnson, Jennifer L.; Ellis, Beverly A.; Noack, Deborah; Seabra, Miguel C.; Catz, Sergio D.

    2005-01-01

    Two of the major proteins secreted by the prostate epithelium secretory cells are PSA (prostate-specific antigen) and PSAP (prostatic-specific acid phosphatase). The molecules involved in the secretory machinery of PSA and PSAP, and the regulation of this machinery, remain unknown. In the present paper, we provide evidence that JFC1 [synaptotagmin-like protein (slp1)], a Rab27a- and PtdIns(3,4,5)P3-binding protein, regulates the androgen-dependent secretion of PSAP and PSA in human LNCaP pros...

  14. HIF-1-dependent regulation of lifespan in Caenorhabditis elegans by the acyl-CoA-binding protein MAA-1

    DEFF Research Database (Denmark)

    Shamalnasab, Mehrnaz; Dhaoui, Manel; Thondamal, Manjunatha

    2017-01-01

    In yeast, the broadly conserved acyl-CoA-binding protein (ACBP) is a negative regulator of stress resistance and longevity. Here, we have turned to the nematode C. elegans as a model organism in which to determine whether ACBPs play similar roles in multicellular organisms. We systematically...... inactivated each of the seven C. elegans ACBP paralogs and found that one of them, maa-1 (which encodes membrane-associated ACBP 1), is indeed involved in the regulation of longevity. In fact, loss of maa-1 promotes lifespan extension and resistance to different types of stress. Through genetic and gene...... of the proteome. Our work extends to C. elegans the role of ACBP in aging, implicates HIF-1 in the increase of lifespan of maa-1-deficient worms, and sheds light on the anti-aging function of HIF-1. Given that both ACBP and HIF-1 are highly conserved, our results suggest the possible involvement of these proteins...

  15. Septin6 and Septin7 GTP binding proteins regulate AP-3- and ESCRT-dependent multivesicular body biogenesis.

    Directory of Open Access Journals (Sweden)

    Sofia Traikov

    Full Text Available Septins (SEPTs form a family of GTP-binding proteins implicated in cytoskeleton and membrane organization, cell division and host/pathogen interactions. The precise function of many family members remains elusive. We show that SEPT6 and SEPT7 complexes bound to F-actin regulate protein sorting during multivesicular body (MVB biogenesis. These complexes bind AP-3, an adapter complex sorting cargos destined to remain in outer membranes of maturing endosomes, modulate AP-3 membrane interactions and the motility of AP-3-positive endosomes. These SEPT-AP interactions also influence the membrane interaction of ESCRT (endosomal-sorting complex required for transport-I, which selects ubiquitinated cargos for degradation inside MVBs. Whereas our findings demonstrate that SEPT6 and SEPT7 function in the spatial, temporal organization of AP-3- and ESCRT-coated membrane domains, they uncover an unsuspected coordination of these sorting machineries during MVB biogenesis. This requires the E3 ubiquitin ligase LRSAM1, an AP-3 interactor regulating ESCRT-I sorting activity and whose mutations are linked with Charcot-Marie-Tooth neuropathies.

  16. C4b-binding protein inhibits the factor V-dependent but not the factor V-independent cofactor activity of protein S in the activated protein C-mediated inactivation of factor VIIIa

    NARCIS (Netherlands)

    van de Poel, R. H.; Meijers, J. C.; Bouma, B. N.

    2001-01-01

    Activated protein C (APC) is an important inactivator of coagulation factors Va and VIIIa. In the inactivation of factors Va and VIIIa, protein S serves as a cofactor to APC. Protein S can bind to C4b-binding protein (C4BP), and thereby loses its cofactor activity to APC. By modulating free protein

  17. Structure of thrombospondin type 3 repeats in bacterial outer membrane protein A reveals its intra-repeat disulfide bond-dependent calcium-binding capability

    Energy Technology Data Exchange (ETDEWEB)

    Dai, Shuyan; Sun, Cancan; Tan, Kemin; Ye, Sheng; Zhang, Rongguang

    2017-09-01

    Eukaryotic thrombospondin type 3 repeat (TT3R) is an efficient calcium ion (Ca2+) binding motif only found in mammalian thrombospondin family. TT3R has also been found in prokaryotic cellulase Cel5G, which was thought to forfeit the Ca2+-binding capability due to the formation of intra-repeat disulfide bonds, instead of the inter-repeat ones possessed by eukaryotic TT3Rs. In this study, we have identified an enormous number of prokaryotic TT3R-containing proteins belonging to several different protein families, including outer membrane protein A (OmpA), an important structural protein connecting the outer membrane and the periplasmic peptidoglycan layer in gram-negative bacteria. Here, we report the crystal structure of the periplasmic region of OmpA from Capnocytophaga gingivalis, which contains a linker region comprising five consecutive TT3Rs. The structure of OmpA-TT3R exhibits a well-ordered architecture organized around two tightly-coordinated Ca2+ and confirms the presence of abnormal intra-repeat disulfide bonds. Further mutagenesis studies showed that the Ca2+-binding capability of OmpA-TT3R is indeed dependent on the proper formation of intra-repeat disulfide bonds, which help to fix a conserved glycine residue at its proper position for Ca2+ coordination. Additionally, despite lacking inter repeat disulfide bonds, the interfaces between adjacent OmpA-TT3Rs are enhanced by both hydrophobic and conserved aromatic-proline interactions.

  18. Computational study of pH-dependent oligomerization and ligand binding in Alt a 1, a highly allergenic protein with a unique fold.

    Science.gov (United States)

    Garrido-Arandia, María; Bretones, Jorge; Gómez-Casado, Cristina; Cubells, Nuria; Díaz-Perales, Araceli; Pacios, Luis F

    2016-05-01

    Alt a 1 is a highly allergenic protein from Alternaria fungi responsible for several respiratory diseases. Its crystal structure revealed a unique β-barrel fold that defines a new family exclusive to fungi and forms a symmetrical dimer in a butterfly-like shape as well as tetramers. Its biological function is as yet unknown but its localization in cell wall of Alternaria spores and its interactions in the onset of allergy reactions point to a function to transport ligands. However, at odds with binding features in β-barrel proteins, monomeric Alt a 1 seems unable to harbor ligands because the barrel is too narrow. Tetrameric Alt a 1 is able to bind the flavonoid quercetin, yet the stability of the aggregate and the own ligand binding are pH-dependent. At pH 6.5, which Alt a 1 would meet when secreted by spores in bronchial epithelium, tetramer-quercetin complex is stable. At pH 5.5, which Alt a 1 would meet in apoplast when infecting plants, the complex breaks down. By means of a combined computational study that includes docking calculations, empirical pKa estimates, Poisson-Boltzmann electrostatic potentials, and Molecular Dynamics simulations, we identified a putative binding site at the dimeric interface between subunits in tetramer. We propose an explanation on the pH-dependence of both oligomerization states and protein-ligand affinity of Alt a 1 in terms of electrostatic variations associated to distinct protonation states at different pHs. The uniqueness of this singular protein can thus be tracked in the combination of all these features.

  19. Lipid-binding proteins modulate ligand-dependent trans-activation by peroxisome proliferator-activated receptors and localize to the nucleus as well as the cytoplasm

    DEFF Research Database (Denmark)

    Helledie, T; Antonius, M; Sorensen, R V

    2000-01-01

    Peroxisome proliferator-activated receptors (PPARs) are activated by a variety of fatty acids, eicosanoids, and hypolipidemic and insulin-sensitizing drugs. Many of these compounds bind avidly to members of a family of small lipid-binding proteins, the fatty acid-binding proteins (FABPs). Fatty a...

  20. Lipid-binding proteins modulate ligand-dependent trans-activation by peroxisome proliferator-activated receptors and localize to the nucleus as well as the cytoplasm

    DEFF Research Database (Denmark)

    Helledie, T; Antonius, M; Sorensen, R V

    2000-01-01

    Peroxisome proliferator-activated receptors (PPARs) are activated by a variety of fatty acids, eicosanoids, and hypolipidemic and insulin-sensitizing drugs. Many of these compounds bind avidly to members of a family of small lipid-binding proteins, the fatty acid-binding proteins (FABPs). Fatty...

  1. Protein binding of psychotropic agents

    International Nuclear Information System (INIS)

    Hassan, H.A.

    1990-01-01

    Based upon fluorescence measurements, protein binding of some psychotropic agents (chlorpromazine, promethazine, and trifluoperazine) to human IgG and HSA was studied in aqueous cacodylate buffer, PH7. The interaction parameters determined from emission quenching of the proteins. The interaction parameters determined include the equilibrium constant (K), calculated from equations derived by Borazan and coworkers, the number of binding sites (n) available to the monomer molecules on a single protein molecule. The results revealed a high level of affinity, as reflected by high values of K, and the existence of specific binding sites, since a limited number of n values are obtained. 39 tabs.; 37 figs.; 83 refs

  2. Computation of pH-Dependent Binding Free Energies

    Science.gov (United States)

    Kim, M. Olivia; McCammon, J. Andrew

    2015-01-01

    Protein-ligand binding accompanies changes in the surrounding electrostatic environments of the two binding partners and may lead to changes in protonation upon binding. In cases where the complex formation results in a net transfer of protons, the binding process is pH-dependent. However, conventional free energy computations or molecular docking protocols typically employ fixed protonation states for the titratable groups in both binding partners set a priori, which are identical for the free and bound states. In this review, we draw attention to these important yet largely ignored binding-induced protonation changes in protein-ligand association by outlining physical origins and prevalence of the protonation changes upon binding. Following a summary of various theoretical methods for pKa prediction, we discuss the theoretical framework to examine the pH dependence of protein-ligand binding processes. PMID:26202905

  3. binding protein (HABP1)

    Indian Academy of Sciences (India)

    Unknown

    adsorbed on carbon coated copper grid (400 mesh) for. 5 min at room temperature. The grids were subsequently .... and inhibition by GAGs and DMA were determined on polystyrene wells of microtitre plates (Costar, ... for binding inhibition assays was carried out by mixing equal volumes of the conjugate and the inhibitor at ...

  4. Evaluations of in vitro metabolism, drug-drug interactions mediated by reversible and time-dependent inhibition of CYPs, and plasma protein binding of MMB4 DMS.

    Science.gov (United States)

    Hong, S Peter; Lusiak, Bozena D; Burback, Brian L; Johnson, Jerry D

    2013-01-01

    1,1'-Methylenebis[4-[(hydroxyimino)methyl]-pyridinium] (MMB4) dimethanesulfonate (DMS) is a bisquaternary pyridinium aldoxime that reactivates acetylcholinesterase inhibited by organophosphorus nerve agent. Drug metabolism and plasma protein binding for MMB4 DMS were examined using various techniques and a wide range of species. When (14)C-MMB4 DMS was incubated in liver microsomes, 4-pyridine aldoxime (4-PA) and an additional metabolite were detected in all species tested. Identity of the additional metabolite was postulated to be isonicotinic acid (INA) based on liquid chromatography with a tandem mass spectrometry analysis, which was confirmed by comparison with authentic INA. Formation of INA was dependent on species, with the highest level found in monkey liver microsomes. The MMB4 DMS exhibited reversible inhibition in a concentration-dependent manner toward cytochrome P450 1A2 (CYP1A2), CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in human liver microsomes showing the highest inhibition for CYP2D6. Human recombinant CYPs were used to evaluate inhibitory curves more adequately and determine detailed kinetic constants for reversible inhibition and potential time-dependent inhibition (TDI). The MMB4 DMS exhibited reversible inhibition toward human-recombinant CYP2D6 with an inhibition constant (K i) value of 66.6 µmol/L. Based on the k inact/K I values, MMB4 DMS was found to exhibit the most potent TDI toward CYP2D6. The MMB4 DMS at 5 different concentrations was incubated in plasma for 5 hours using an equilibrium dialysis device. For all species tested, there were no concentration-dependent changes in plasma protein binding, ranging from 10% to 17%. These results suggest that MMB4 was not extensively bound to plasma protein, and there were no overt species-related differences in the extent of MMB4 bound to plasma protein.

  5. Licensing of gametogenesis, dependent on RNA binding protein DAZL, as a gateway to sexual differentiation of fetal germ cells

    Science.gov (United States)

    Gill, Mark E.; Hu, Yueh-Chiang; Lin, Yanfeng; Page, David C.

    2011-01-01

    Mammalian oocytes and spermatozoa derive from fetal cells shared by the sexes. These primordial germ cells (PGCs) migrate to the developing somatic gonad, giving rise to oocytes or spermatozoa. These opposing sexual fates are determined not by the PGCs’ own sex chromosome constitution (XX or XY), but by the sexual identity of the fetal gonad that they enter. We asked whether PGCs undergo a developmental transition that enables them to respond to feminizing or masculinizing cues from fetal ovary or testis. We conducted in vivo genetic studies of DAZL, an RNA-binding protein expressed in both ovarian and testicular germ cells. We found that germ cells in C57BL/6 Dazl-deficient fetuses—whether XX or XY—migrate to the gonad but do not develop either male or female features. Instead, they remain in a sexually undifferentiated state similar to that of migrating PGCs. Thus, germ cells in C57BL/6 Dazl-deficient fetuses do not respond to sexual cues from ovary or testis, whereas the earlier processes of germ cell specification and migration are unaffected. We propose that PGCs of both XX and XY fetuses undergo licensing, an active developmental transition that enables the resultant gametogenesis-competent cells to respond to feminizing or masculinizing cues produced by the fetal ovary or testis and hence to embark on oogenesis or spermatogenesis. In C57BL/6 mice, Dazl is required for licensing. Licensing serves as a gateway from the embryonic processes shared between the sexes—germ cell specification and migration—to the sex-specific pathways of oogenesis and spermatogenesis. PMID:21504946

  6. Mechanism of Iron-Dependent Repressor (IdeR Activation and DNA Binding: A Molecular Dynamics and Protein Structure Network Study.

    Directory of Open Access Journals (Sweden)

    Soma Ghosh

    2015-12-01

    Full Text Available Metalloproteins form a major class of enzymes in the living system that are involved in crucial biological functions such as catalysis, redox reactions and as 'switches' in signal transductions. Iron dependent repressor (IdeR is a metal-sensing transcription factor that regulates free iron concentration in Mycobacterium tuberculosis. IdeR is also known to promote bacterial virulence, making it an important target in the field of therapeutics. Mechanistic details of how iron ions modulate IdeR such that it dimerizes and binds to DNA is not understood clearly. In this study, we have performed molecular dynamic simulations and integrated it with protein structure networks to study the influence of iron on IdeR structure and function. A significant structural variation between the metallated and the non-metallated system is observed. Our simulations clearly indicate the importance of iron in stabilizing its monomeric subunit, which in turn promotes dimerization. However, the most striking results are obtained from the simulations of IdeR-DNA complex in the absence of metals, where at the end of 100ns simulations, the protein subunits are seen to rapidly dissociate away from the DNA, thereby forming an excellent resource to investigate the mechanism of DNA binding. We have also investigated the role of iron as an allosteric regulator of IdeR that positively induces IdeR-DNA complex formation. Based on this study, a mechanistic model of IdeR activation and DNA binding has been proposed.

  7. Phenobarbital-dependent protein binding to Barbie box-like sequences in the coding region of cytochrome P450BM-3 gene from Bacillus megaterium.

    Science.gov (United States)

    Gaidamakova, E K; Alpatov, O V; Ischenko, I V; Kovalenko, S P; Lyakhovich, V V

    1996-12-12

    Phenobarbital-dependent protein binding was shown to occur to DNA fragments from the coding region of the cytochrome P450BM-3 gene from Bacillus megaterium. Incubation of the DNA fragments from the coding region of the gene with total cell extract from Bacillus megaterium revealed two DNA regions with protein-binding capacity: +237/+318 and +319/+425 considering 'O' as the start of cytochrome P450BM-3 translation. DNaseI footprint analysis of the fragment +319/+425 with the total cell extract showed that some protein(s) protected DNA stretches from the position +373 up to the position +389 on the transcribed strand and from the position +378 up to the position +398 on the non-transcribed strand. DNaseI footprint analysis of the fragment +237/+318 revealed the protection in the region +262/+277 on the non-transcribed strand. Three regions protected by cell extract protein(s) from DNaseI hydrolysis (+262/+277, +373/+389 and +378/+398) appeared to be strongly homologous to the Barbie box sequence. Barbie-box-like sequences were found in the majority of regulatory regions of phenobarbital-inducible genes whose regulatory sequences had been reported (Fulco et al., 1994). Our results suggest that a functional role of Barbie box sequence takes place not only in regulatory but also in the coding region of the gene. In line with that hypothesis we analyzed all cytochrome P450 genes in respect to the presence of Barbie box-like sequences in their coding parts. At least one cytochrome P450 gene (CYP6A1, phenobarbital-inducible gene from Musca domestica) was shown to contain Barbie box sequence in the coding part of the gene.

  8. Identification of Y-box binding protein 1 as a core regulator of MEK/ERK pathway-dependent gene signatures in colorectal cancer cells.

    Directory of Open Access Journals (Sweden)

    Karsten Jürchott

    2010-12-01

    Full Text Available Transcriptional signatures are an indispensible source of correlative information on disease-related molecular alterations on a genome-wide level. Numerous candidate genes involved in disease and in factors of predictive, as well as of prognostic, value have been deduced from such molecular portraits, e.g. in cancer. However, mechanistic insights into the regulatory principles governing global transcriptional changes are lagging behind extensive compilations of deregulated genes. To identify regulators of transcriptome alterations, we used an integrated approach combining transcriptional profiling of colorectal cancer cell lines treated with inhibitors targeting the receptor tyrosine kinase (RTK/RAS/mitogen-activated protein kinase pathway, computational prediction of regulatory elements in promoters of co-regulated genes, chromatin-based and functional cellular assays. We identified commonly co-regulated, proliferation-associated target genes that respond to the MAPK pathway. We recognized E2F and NFY transcription factor binding sites as prevalent motifs in those pathway-responsive genes and confirmed the predicted regulatory role of Y-box binding protein 1 (YBX1 by reporter gene, gel shift, and chromatin immunoprecipitation assays. We also validated the MAPK-dependent gene signature in colorectal cancers and provided evidence for the association of YBX1 with poor prognosis in colorectal cancer patients. This suggests that MEK/ERK-dependent, YBX1-regulated target genes are involved in executing malignant properties.

  9. Proteomic Identification of Annexins, Calcium-Dependent Membrane Binding Proteins That Mediate Osmotic Stress and Abscisic Acid Signal Transduction in Arabidopsis

    Science.gov (United States)

    Lee, Sumin; Lee, Eun Jung; Yang, Eun Ju; Lee, Ji Eun; Park, Ae Ran; Song, Won Hyun; Park, Ohkmae K.

    2004-01-01

    Comparative proteomic analysis of the Arabidopsis thaliana root microsomal fraction was performed to identify novel components of salt stress signaling. Among the salt-responsive microsomal proteins, two spots that increased upon salt treatment on a two-dimensional gel were identified as the same protein, designated annexin 1 (AnnAt1). Annexins comprise a multigene family of Ca2+-dependent membrane binding proteins and have been extensively studied in animal cells. AnnAt1 is strongly expressed in root but rarely in flower tissue. In this study, the results suggest that salt stress induces translocation from the cytosol to the membrane and potential turnover of existing protein. This process is blocked by EGTA treatment, implying that AnnAt1 functions in stress response are tightly associated with Ca2+. T-DNA insertion mutants of annAt1 and a different isoform, annAt4, displayed hypersensitivity to osmotic stress and abscisic acid (ABA) during germination and early seedling growth. The results collectively suggest that AnnAt1 and AnnAt4 play important roles in osmotic stress and ABA signaling in a Ca2+-dependent manner. PMID:15161963

  10. Rheb Protein Binds CAD (Carbamoyl-phosphate Synthetase 2, Aspartate Transcarbamoylase, and Dihydroorotase) Protein in a GTP- and Effector Domain-dependent Manner and Influences Its Cellular Localization and Carbamoyl-phosphate Synthetase (CPSase) Activity*

    Science.gov (United States)

    Sato, Tatsuhiro; Akasu, Hitomi; Shimono, Wataru; Matsu, Chisa; Fujiwara, Yuki; Shibagaki, Yoshio; Heard, Jeffrey J.; Tamanoi, Fuyuhiko; Hattori, Seisuke

    2015-01-01

    Rheb small GTPases, which consist of Rheb1 and Rheb2 (also known as RhebL1) in mammalian cells, are unique members of the Ras superfamily and play central roles in regulating protein synthesis and cell growth by activating mTOR. To gain further insight into the function of Rheb, we carried out a search for Rheb-binding proteins and found that Rheb binds to CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), a multifunctional enzyme required for the de novo synthesis of pyrimidine nucleotides. CAD binding is more pronounced with Rheb2 than with Rheb1. Rheb binds CAD in a GTP- and effector domain-dependent manner. The region of CAD where Rheb binds is located at the C-terminal region of the carbamoyl-phosphate synthetase domain and not in the dihydroorotase and aspartate transcarbamoylase domains. Rheb stimulated carbamoyl-phosphate synthetase activity of CAD in vitro. In addition, an elevated level of intracellular UTP pyrimidine nucleotide was observed in Tsc2-deficient cells, which was attenuated by knocking down of Rheb. Immunostaining analysis showed that expression of Rheb leads to increased accumulation of CAD on lysosomes. Both a farnesyltransferase inhibitor that blocks membrane association of Rheb and knockdown of Rheb mislocalized CAD. These results establish CAD as a downstream effector of Rheb and suggest a possible role of Rheb in regulating de novo pyrimidine nucleotide synthesis. PMID:25422319

  11. Rheb protein binds CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase) protein in a GTP- and effector domain-dependent manner and influences its cellular localization and carbamoyl-phosphate synthetase (CPSase) activity.

    Science.gov (United States)

    Sato, Tatsuhiro; Akasu, Hitomi; Shimono, Wataru; Matsu, Chisa; Fujiwara, Yuki; Shibagaki, Yoshio; Heard, Jeffrey J; Tamanoi, Fuyuhiko; Hattori, Seisuke

    2015-01-09

    Rheb small GTPases, which consist of Rheb1 and Rheb2 (also known as RhebL1) in mammalian cells, are unique members of the Ras superfamily and play central roles in regulating protein synthesis and cell growth by activating mTOR. To gain further insight into the function of Rheb, we carried out a search for Rheb-binding proteins and found that Rheb binds to CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), a multifunctional enzyme required for the de novo synthesis of pyrimidine nucleotides. CAD binding is more pronounced with Rheb2 than with Rheb1. Rheb binds CAD in a GTP- and effector domain-dependent manner. The region of CAD where Rheb binds is located at the C-terminal region of the carbamoyl-phosphate synthetase domain and not in the dihydroorotase and aspartate transcarbamoylase domains. Rheb stimulated carbamoyl-phosphate synthetase activity of CAD in vitro. In addition, an elevated level of intracellular UTP pyrimidine nucleotide was observed in Tsc2-deficient cells, which was attenuated by knocking down of Rheb. Immunostaining analysis showed that expression of Rheb leads to increased accumulation of CAD on lysosomes. Both a farnesyltransferase inhibitor that blocks membrane association of Rheb and knockdown of Rheb mislocalized CAD. These results establish CAD as a downstream effector of Rheb and suggest a possible role of Rheb in regulating de novo pyrimidine nucleotide synthesis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Guanosine 5'-triphosphate binding protein (G/sub i/) and two additional pertussis toxin substrates associated with muscarinic receptors in rat heart myocytes: characterization and age dependency

    International Nuclear Information System (INIS)

    Moscona-Amir, E.; Henis, Y.I.; Sokolovsky, M.

    1988-01-01

    The coupling of muscarinic receptors with G-proteins was investigated in cultured myocytes prepared from the hearts of newborn rats. The coupling was investigated in both young (5 days after plating) and aged (14 days after plating) cultures, in view of the completely different effects of 5'-guanylyl imidodiphosphate [Gpp(NH)p] on muscarinic agonist binding to homogenates from young vs aged cultures. Pretreatment of cultures from both ages by Bordetella pertussis toxin (IAP) was found to eliminate any Gpp(NH)p effect on carbamylcholine binding. IAP by itself induced a rightward shift in the carbamylcholine competition curve in homogenates from aged cultures, but no such effect was observed in homogenates from young cultures. IAP-catalyzed [ 32 P]ADP-ribosylation of membrane preparations from young and aged cultures revealed major differences between them. Young cultures exhibited a major IAP substrate at 40 kDa, which was also recognized by anti-α/sub i/ antibodies, and two novel IAP substrates at 28 and 42 kDa, which were weakly ADP-ribosylated by the toxin and were not recognized with either anti-α/sub i/ or anti-α 0 antibodies. In aged cultures, only the 40-kDa band (ribosylated to a lower degree) was detected. The parallel age-dependent changes in the three IAP substrates (28, 40, and 42 kDa) and in the interactions of the G-protein(s) with the muscarinic receptors strongly suggest close association between the two phenomena. All of these age-dependent changes in the G-protein related parameters were prevented by phosphatidylcholine-liposome treatment of the aged cultures. The role of the membrane lipid composition in these phenomena is discussed

  13. When is protein binding important?

    Science.gov (United States)

    Heuberger, Jules; Schmidt, Stephan; Derendorf, Hartmut

    2013-09-01

    The present paper is an ode to a classic citation by Benet and Hoener (2002. Clin Pharm Ther 71(3):115-121). The now classic paper had a huge impact on drug development and the way the issue of protein binding is perceived and interpreted. Although the authors very clearly pointed out the limitations and underlying assumptions for their delineations, these are too often overlooked and the classic paper's message is misinterpreted by broadening to cases that were not intended. Some members of the scientific community concluded from the paper that protein binding is not important. This was clearly not intended by the authors, as they finished their paper with a paragraph entitled: "When is protein binding important?" Misinterpretation of the underlying assumptions in the classic work can result in major pitfalls in drug development. Therefore, we revisit the topic of protein binding with the intention of clarifying when clinically relevant changes should be considered during drug development. Copyright © 2013 Wiley Periodicals, Inc.

  14. Multiple protonation equilibria in electrostatics of protein-protein binding.

    Science.gov (United States)

    Piłat, Zofia; Antosiewicz, Jan M

    2008-11-27

    All proteins contain groups capable of exchanging protons with their environment. We present here an approach, based on a rigorous thermodynamic cycle and the partition functions for energy levels characterizing protonation states of the associating proteins and their complex, to compute the electrostatic pH-dependent contribution to the free energy of protein-protein binding. The computed electrostatic binding free energies include the pH of the solution as the variable of state, mutual "polarization" of associating proteins reflected as changes in the distribution of their protonation states upon binding and fluctuations between available protonation states. The only fixed property of both proteins is the conformation; the structure of the monomers is kept in the same conformation as they have in the complex structure. As a reference, we use the electrostatic binding free energies obtained from the traditional Poisson-Boltzmann model, computed for a single macromolecular conformation fixed in a given protonation state, appropriate for given solution conditions. The new approach was tested for 12 protein-protein complexes. It is shown that explicit inclusion of protonation degrees of freedom might lead to a substantially different estimation of the electrostatic contribution to the binding free energy than that based on the traditional Poisson-Boltzmann model. This has important implications for the balancing of different contributions to the energetics of protein-protein binding and other related problems, for example, the choice of protein models for Brownian dynamics simulations of their association. Our procedure can be generalized to include conformational degrees of freedom by combining it with molecular dynamics simulations at constant pH. Unfortunately, in practice, a prohibitive factor is an enormous requirement for computer time and power. However, there may be some hope for solving this problem by combining existing constant pH molecular dynamics

  15. Stimulation of a Cd-binding protein, and inhibition of the vitamin D-dependent calcium-binding protein, by zinc or cadmium in organ-cultured embryonic chick duodenum

    International Nuclear Information System (INIS)

    Corradino, R.A.; Fullmer, C.S.

    1980-01-01

    Embryonic chick duodenum maintained in organ culture responds to 1 α,25-dihydroxy vitamin D 3 in the culture medium by de novo synthesis of a specific calcium-binding protein (CaBP). The addition of Cd 2+ (3-5 x 10 -5 M) or Zn 2+ (10 -5 -10 -4 M) to the medium inhibited CaBP, but stimulated biosynthesis of a Cd-binding protein (CdBP). CdBP in duodenal homogenate supernatants was assessed in two ways: first, by its 109 Cd-binding activity ( 109 CdBA) using a competitive ion exchange procedure; and, second, by the extent of [ 35 S]-cystine incorporation into a specific peak or band after gel filtration or analytical polyacrylamide disc gel electrophoresis, respectively. Regardless of whether cadmium- or zinc-stimulated, the 35 S-labeled CdBP - the only protein significantly labeled under the conditions employed - migrated identically upon gel filtration and electrophoresis, and comigrated with purified chick liver Cd-metallothionein. Neither actinomycin D nor α-amanitin, in concentrations sufficient to severely inhibit CaBP, significantly reduced CdBP production. However, cycloheximide did inhibit either Cd 2+ - or Zn 2+ -stimulated CdBP by about 50% at an inhibitor concentration which abolished CaBP. The inhibitor studies, coupled with the observations of extensive incorporation of [ 35 S]cystine into CdBP, suggest that the metals stimulated biosynthesis by a mechanism operating at the translational level. The organ-cultured duodenum seems well suited for studies of the regulation of CdBP biosynthesis especially since it responds predictably to the steroid hormone, 1α,25-dihydroxy vitamin D 3 , in the induction of another specific protein, CaBP, at the transcriptional level. The biosynthesis of CaBP thus may serve as a convenient control in studies of CdBP production under various experimental conditions

  16. Alcohol induces RNA polymerase III-dependent transcription through c-Jun by co-regulating TATA-binding protein (TBP) and Brf1 expression.

    Science.gov (United States)

    Zhong, Shuping; Machida, Keigo; Tsukamoto, Hide; Johnson, Deborah L

    2011-01-28

    Chronic alcohol consumption is associated with steatohepatitis and cirrhosis, enhancing the risk for hepatocellular carcinoma. RNA polymerase (pol) III transcribes a variety of small, untranslated RNAs, including tRNAs and 5S rRNAs, which determine the biosynthetic capacity of cells. Increased RNA pol III-dependent transcription, observed in transformed cells and human tumors, is required for oncogenic transformation. Given that alcohol consumption increases risk for liver cancer, we examined whether alcohol regulates this class of genes. Ethanol induces RNA pol III-dependent transcription in both HepG2 cells and primary mouse hepatocytes in a manner that requires ethanol metabolism and the activation of JNK1. This regulatory event is mediated, at least in part, through the ability of ethanol to induce expression of the TFIIIB components, Brf1, and the TATA-binding protein (TBP). Induction of TBP, Brf1, and RNA pol III-dependent gene expression is driven by enhanced c-Jun expression. Ethanol promotes a marked increase in the direct recruitment of c-Jun to TBP, Brf1, and tRNA gene promoters. Chronic alcohol administration in mice leads to enhanced expression of TBP, Brf1, tRNA, and 5S rRNA gene transcription in the liver. These alcohol-dependent increases are more pronounced in transgenic animals that express the HCV NS5A protein that display increased incidence of liver tumors. Together, these results identify a new class of genes that are regulated by alcohol through the co-regulation of TFIIIB components and define a central role for c-Jun in this process.

  17. Hepatic overexpression of idol increases circulating protein convertase subtilisin/kexin type 9 in mice and hamsters via dual mechanisms: sterol regulatory element-binding protein 2 and low-density lipoprotein receptor-dependent pathways.

    Science.gov (United States)

    Sasaki, Makoto; Terao, Yoshio; Ayaori, Makoto; Uto-Kondo, Harumi; Iizuka, Maki; Yogo, Makiko; Hagisawa, Kosuke; Takiguchi, Shunichi; Yakushiji, Emi; Nakaya, Kazuhiro; Ogura, Masatsune; Komatsu, Tomohiro; Ikewaki, Katsunori

    2014-06-01

    Low-density lipoprotein receptor (LDLR) is degraded by inducible degrader of LDLR (Idol) and protein convertase subtilisin/kexin type 9 (PCSK9), thereby regulating circulating LDL levels. However, it remains unclear whether, and if so how, these LDLR degraders affect each other. We therefore investigated effects of liver-specific expression of Idol on LDL/PCSK9 metabolism in mice and hamsters. Injection of adenoviral vector expressing Idol (Ad-Idol) induced a liver-specific reduction in LDLR expression which, in turn, increased very-low-density lipoprotein/LDL cholesterol levels in wild-type mice because of delayed LDL catabolism. Interestingly, hepatic Idol overexpression markedly increased plasma PCSK9 levels. In LDLR-deficient mice, plasma PCSK9 levels were already elevated at baseline and unchanged by Idol overexpression, which was comparable with the observation for Ad-Idol-injected wild-type mice, indicating that Idol-induced PCSK9 elevation depended on LDLR. In wild-type mice, but not in LDLR-deficient mice, Ad-Idol enhanced hepatic PCSK9 expression, with activation of sterol regulatory element-binding protein 2 and subsequently increased expression of its target genes. Supporting in vivo findings, Idol transactivated PCSK9/LDLR in sterol regulatory element-binding protein 2/LDLR-dependent manners in vitro. Furthermore, an in vivo kinetic study using (125)I-labeled PCSK9 revealed delayed clearance of circulating PCSK9, which could be another mechanism. Finally, to extend these findings into cholesteryl ester transfer protein-expressing animals, we repeated the above in vivo experiments in hamsters and obtained similar results. A vicious cycle in LDLR degradation might be generated by PCSK9 induced by hepatic Idol overexpression via dual mechanisms: sterol regulatory element-binding protein 2/LDLR. Furthermore, these effects would be independent of cholesteryl ester transfer protein expression. © 2014 American Heart Association, Inc.

  18. Megalin binds and mediates cellular internalization of folate binding protein

    DEFF Research Database (Denmark)

    Birn, Henrik; Zhai, Xiaoyue; Holm, Jan

    2005-01-01

    to express high levels of megalin, is inhibitable by excess unlabeled FBP and by receptor associated protein, a known inhibitor of binding to megalin. Immortalized rat yolk sac cells, representing an established model for studying megalin-mediated uptake, reveal (125)I-labeled FBP uptake which is inhibited...... to bind and mediate cellular uptake of FBP. Surface plasmon resonance analysis shows binding of bovine and human milk FBP to immobilized megalin, but not to low density lipoprotein receptor related protein. Binding of (125)I-labeled folate binding protein (FBP) to sections of kidney proximal tubule, known...

  19. Thermodynamics of Ligand Binding to Acyl-Coenzyme A Binding Protein Studied by Titration Calorimetry

    DEFF Research Database (Denmark)

    Færgeman, Nils Joakim; Sigurskjold, Bent Walther; Kragelund, Birthe B.

    1996-01-01

    Ligand binding to recombinant bovine acyl-CoA binding protein (ACBP) was examined using isothermal microcalorimetry. Microcalorimetric measurements confirm that the binding affinity of acyl-CoA esters for ACBP is strongly dependent on the length of the acyl chain with a clear preference for acyl-...

  20. Thermodynamics of ligand binding to acyl-coenzyme A binding protein studied by titration calorimetry

    DEFF Research Database (Denmark)

    Færgeman, Nils J.; Sigurskjold, B W; Kragelund, B B

    1996-01-01

    Ligand binding to recombinant bovine acyl-CoA binding protein (ACBP) was examined using isothermal microcalorimetry. Microcalorimetric measurements confirm that the binding affinity of acyl-CoA esters for ACBP is strongly dependent on the length of the acyl chain with a clear preference for acyl-...

  1. Adipocyte Fatty Acid Binding Protein Potentiates Toxic Lipids-Induced Endoplasmic Reticulum Stress in Macrophages via Inhibition of Janus Kinase 2-dependent Autophagy.

    Science.gov (United States)

    Hoo, Ruby L C; Shu, Lingling; Cheng, Kenneth K Y; Wu, Xiaoping; Liao, Boya; Wu, Donghai; Zhou, Zhiguang; Xu, Aimin

    2017-01-17

    Lipotoxicity is implicated in the pathogenesis of obesity-related inflammatory complications by promoting macrophage infiltration and activation. Endoplasmic reticulum (ER) stress and adipocyte fatty acid binding protein (A-FABP) play key roles in obesity and mediate inflammatory activity through similar signaling pathways. However, little is known about their interplay in lipid-induced inflammatory responses. Here, we showed that prolonged treatment of palmitic acid (PA) increased ER stress and expression of A-FABP, which was accompanied by reduced autophagic flux in macrophages. Over-expression of A-FABP impaired PA-induced autophagy associating with enhanced ER stress and pro-inflammatory cytokine production, while genetic ablation or pharmacological inhibition of A-FABP reversed the conditions. PA-induced expression of autophagy-related protein (Atg)7 was attenuated in A-FABP over-expressed macrophages, but was elevated in A-FABP-deficient macrophages. Mechanistically, A-FABP potentiated the effects of PA by inhibition of Janus Kinase (JAK)2 activity, thus diminished PA-induced Atg7 expression contributing to impaired autophagy and further augmentation of ER stress. These findings suggest that A-FABP acts as autophagy inhibitor to instigate toxic lipids-induced ER stress through inhibition of JAK2-dependent autophagy, which in turn triggers inflammatory responses in macrophages. A-FABP-JAK2 axis may represent an important pathological pathway contributing to obesity-related inflammatory diseases.

  2. Polymeric competitive protein binding adsorbents for radioassay

    International Nuclear Information System (INIS)

    Adams, R.J.

    1976-01-01

    Serum protein comprising specific binding proteins such as antibodies, B 12 intrinsic factor, thyroxin binding globulin and the like may be copolymerized with globulin constituents of serum by the action of ethylchloroformate to form readily packed insoluble precipitates which, following purification as by washing, are eminently suited for employment as competitive binding protein absorbents in radioassay procedures. 10 claims, no drawings

  3. Actin binding proteins and spermiogenesis

    Science.gov (United States)

    Mruk, Dolores D

    2011-01-01

    Drebrin E, an actin-binding protein lacking intrinsic activity in the regulation of actin dynamics (e.g., polymerization, capping, nucleation, branching, cross-linking, bundling and severing), is known to recruit actin regulatory proteins to a specific cellular site. Herein, we critically evaluate recent findings in the field which illustrate that drebrin E works together with two other actin-binding proteins, namely Arp3 (actin-related protein 3, a component of the Arp2/3 complex that simultaneously controls actin nucleation for polymerization and branching of actin filaments) and Eps8 (epidermal growth factor receptor pathway substrate 8 that controls capping of the barbed ends of actin filaments, as well as actin filament bundling) to regulate the homeostasis of F-actin filament bundles at the ectoplasmic specialization (ES), a testis-specific atypical adherens junction (AJ) in the seminiferous epithelium. This is mediated by the strict temporal and spatial expression of these three actin-binding proteins at the apical and basal ES at the Sertoli cell-spermatid (step 8–19) and Sertoli-Sertoli cell interface, respectively, during the seminiferous epithelial cycle of spermatogenesis. In this Commentary, we put forth a possible model by which drebrin E may be acting as a platform upon which proteins (e.g., Arp3) that are needed to alter the conformation of actin filament bundles at the ES can be recruited to the site, thus facilitating changes in cell shape and cell position in the epithelium during spermiogenesis and spermiation. In short, drebrin E may be acting as a “logistic” distribution center to manage different regulatory proteins at the apical ES, thereby regulating the dynamics of actin filament bundles and modulating the plasticity of the apical ES. This would allow adhesion to be altered continuously throughout the epithelial cycle to accommodate spermatid movement in the seminiferous epithelium during spermiogenesis and spermiation. We also

  4. Human plasminogen binding protein tetranectin

    DEFF Research Database (Denmark)

    Kastrup, J S; Rasmussen, H; Nielsen, B B

    1997-01-01

    The recombinant human plasminogen binding protein tetranectin (TN) and the C-type lectin CRD of this protein (TN3) have been crystallized. TN3 crystallizes in the tetragonal space group P4(2)2(1)2 with cell dimensions a = b = 64.0, c = 75.7 A and with one molecule per asymmetric unit. The crystals...... to at least 2.5 A. A full data set has been collected to 3.0 A. The asymmetric unit contains one monomer of TN. Molecular replacement solutions for TN3 and TN have been obtained using the structure of the C-type lectin CRD of rat mannose-binding protein as search model. The rhombohedral space group indicates...... diffract X-rays to at least 2.0 A resolution. A complete diffraction data set has been collected to 2.7 A resolution. The crystals of TN, obtained by the vapour-diffusion reverse salting-in method at 280 K, are rhombohedral, space group R3, with the hexagonal axes a = b = 89.1, c = 75.8 A, and diffract...

  5. Acyl-CoA-binding protein (ACBP) localizes to the endoplasmic reticulum and Golgi in a ligand-dependent manner in mammalian cells

    DEFF Research Database (Denmark)

    Hansen, Jesper S; Færgeman, Nils J; Kragelund, Birthe B

    2008-01-01

    showed that ACBP targeted to the ER (endoplasmic reticulum) and Golgi in a ligand-binding-dependent manner. A variant Y28F/K32A-FACI-50, which is unable to bind acyl-CoA, did no longer show association with the ER and became segregated from the Golgi, as analysed by intensity correlation calculations....... Depletion of fatty acids from cells by addition of FAFBSA (fatty-acid-free BSA) significantly decreased FACI-50 association with the Golgi, whereas fatty acid overloading increased Golgi association, strongly supporting that ACBP associates with the Golgi in a ligand-dependent manner. FRAP (fluorescence...... recovery after photobleaching) showed that the fatty-acid-induced targeting of FACI-50 to the Golgi resulted in a 5-fold reduction in FACI-50 mobility. We suggest that ACBP is targeted to the ER and Golgi in a ligand-binding-dependent manner in living cells and propose that ACBP may be involved...

  6. The Potato Nucleotide-Binding Leucine-Rich Repeat (NLR) Immune Receptor Rx1 is a Pathogen Dependent DNA-Deforming Protein

    NARCIS (Netherlands)

    Fenyk, S.; Townsend, P.D.; Dixon, C.H.; Spies, G.B.; Campillo, A.S.E.; Slootweg, E.J.; Westerhof, L.B.; Gawehns, F.K.K.; Knight, M.R.; Sharples, G.J.; Goverse, A.; Palsson, L.O.; Takken, F.L.W.; Cann, M.J.

    2015-01-01

    Plant NLR proteins enable cells to respond to pathogen attack. Several NLRs act in the nucleus, however, conserved nuclear targets that support their role in immunity are unknown. Previously we noted a structural homology between the NB domain of NLRs and DNA replication origin-binding Cdc6/Orc1

  7. Acyl-coenzyme A binding protein (ACBP)

    DEFF Research Database (Denmark)

    Kragelund, B B; Knudsen, J; Poulsen, F M

    1999-01-01

    Acyl-coenzyme A binding proteins are known from a large group of eukaryote species and to bind a long chain length acyl-CoA ester with very high affinity. Detailed biochemical mapping of ligand binding properties has been obtained as well as in-depth structural studies on the bovine apo-protein a...

  8. Insulin-like growth factor-binding protein-2 promotes prostate cancer cell growth via IGF-dependent or -independent mechanisms and reduces the efficacy of docetaxel

    Science.gov (United States)

    Uzoh, C C; Holly, J M P; Biernacka, K M; Persad, R A; Bahl, A; Gillatt, D; Perks, C M

    2011-01-01

    Background: The development of androgen independence, chemo-, and radioresistance are critical markers of prostate cancer progression and the predominant reasons for its high mortality. Understanding the resistance to therapy could aid the development of more effective treatments. Aim: The aim of this study is to investigate the effects of insulin-like growth factor-binding protein-2 (IGFBP-2) on prostate cancer cell proliferation and its effects on the response to docetaxel. Methods: DU145 and PC3 cells were treated with IGFBP-2, insulin-like growth factor I (IGF-I) alone or in combination with blockade of the IGF-I receptor or integrin receptors. Cells were also treated with IGFBP-2 short interfering ribonucleic acid with or without a PTEN (phosphatase and tensin homologue deleted on chromosome 10) inhibitor or docetaxel. Tritiated thymidine incorporation was used to measure cell proliferation and Trypan blue cell counting for cell death. Levels of IGFBP-2 mRNA were measured using RT–PCR. Abundance and phosphorylation of proteins were assessed using western immunoblotting. Results: The IGFBP-2 promoted cell growth in both cell lines but with PC3 cells this was in an IGF-dependent manner, whereas with DU145 cells the effect was independent of IGF receptor activation. This IGF-independent effect of IGFBP-2 was mediated by interaction with β-1-containing integrins and a consequent increase in PTEN phosphorylation. We also determined that silencing IGFBP-2 in both cell lines increased the sensitivity of the cells to docetaxel. Conclusion: The IGFBP-2 has a key role in the growth of prostate cancer cells, and silencing IGFBP-2 expression reduced the resistance of these cells to docetaxel. Targeting IGFBP-2 may increase the efficacy of docetaxel. PMID:21487405

  9. End binding proteins are obligatory dimers.

    Directory of Open Access Journals (Sweden)

    Indrani Sen

    Full Text Available End binding (EB proteins are responsible for the recruitment of an array of microtubule plus-end tracking proteins (+TIPs to growing microtubules ends. EBs encompass an N-terminal calponin homology domain that confers microtubule tip tracking activity to the protein. The C-terminal domain of EBs contains a coiled coil that mediates the parallel dimerization of EB monomers. This part of the protein is also responsible for partner binding. While dimerization is not essential for microtubule tip tracking by EBs it is a prerequisite for +TIP partner binding. The concentration of EBs in cells has been estimated to be in the range of hundreds of nanomoles. In contrast, in in vitro single molecule experiments EB concentrations of subnanomoles are employed. From a mechanistic point of view it is important to assess the oligomerization state of EBs at physiologically and experimentally relevant protein concentrations, in particular if the goal of a study is to model the behavior of EB-dependent dynamic +TIP networks. Here we have determined the stability of the EB1 and EB3 dimers using multi-angle light scattering and fluorescence analytical ultracentrifugation. We show that these EBs form stable dimers and do not dissociate even at very low nanomolar concentrations. The dimers remained stable at both room temperature as well as at the physiologically relevant temperature of 37°C. Together, our results reveal that EBs are obligatory dimers, a conclusion that has implications for the mechanistic understanding of these key proteins involved in the orchestration of dynamic protein networks at growing microtubule ends.

  10. UV-induced DNA-binding proteins in human cells

    International Nuclear Information System (INIS)

    Glazer, P.M.; Greggio, N.A.; Metherall, J.E.; Summers, W.C.

    1989-01-01

    To investigate the response of human cells to DNA-damaging agents such as UV irradiation, the authors examined nuclear protein extracts of UV-irradiated HeLa cells for the presence of DNA-binding proteins. Electrophoretically separated proteins were transferred to a nitrocellulose filter that was subsequently immersed in a binding solution containing radioactively labeled DNA probes. Several DNA-binding proteins were induced in HeLa cells after UV irradiation. These included proteins that bind predominantly double-stranded DNA and proteins that bind both double-stranded and single-stranded DNA. The binding proteins were induced in a dose-dependent manner by UV light. Following a dose of 12 J/m 2 , the binding proteins in the nuclear extracts increased over time to a peak in the range of 18 hr after irradiation. Experiments with metabolic inhibitors (cycloheximide and actinomycin D) revealed that de novo synthesis of these proteins is not required for induction of the binding activities, suggesting that the induction is mediated by protein modification

  11. CAP binding proteins associated with the nucleus.

    OpenAIRE

    Patzelt, E; Blaas, D; Kuechler, E

    1983-01-01

    Cap binding proteins of HeLa cells were identified by photo-affinity labelling using the cap analogue gamma-[32P]-[4-(benzoyl-phenyl)methylamido]-7-methylguanosine-5'- triphosphate. Photoreaction with whole cell homogenates resulted in specific labelling of five major polypeptides. The small molecular weight polypeptide appeared to be identical to the 24 000 to 26 000 dalton cap binding protein previously identified in initiation factors. A cap binding protein of 37 000 dalton was found in in...

  12. Retinoid-binding proteins: similar protein architectures bind similar ligands via completely different ways.

    Directory of Open Access Journals (Sweden)

    Yu-Ru Zhang

    Full Text Available BACKGROUND: Retinoids are a class of compounds that are chemically related to vitamin A, which is an essential nutrient that plays a key role in vision, cell growth and differentiation. In vivo, retinoids must bind with specific proteins to perform their necessary functions. Plasma retinol-binding protein (RBP and epididymal retinoic acid binding protein (ERABP carry retinoids in bodily fluids, while cellular retinol-binding proteins (CRBPs and cellular retinoic acid-binding proteins (CRABPs carry retinoids within cells. Interestingly, although all of these transport proteins possess similar structures, the modes of binding for the different retinoid ligands with their carrier proteins are different. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we analyzed the various retinoid transport mechanisms using structure and sequence comparisons, binding site analyses and molecular dynamics simulations. Our results show that in the same family of proteins and subcellular location, the orientation of a retinoid molecule within a binding protein is same, whereas when different families of proteins are considered, the orientation of the bound retinoid is completely different. In addition, none of the amino acid residues involved in ligand binding is conserved between the transport proteins. However, for each specific binding protein, the amino acids involved in the ligand binding are conserved. The results of this study allow us to propose a possible transport model for retinoids. CONCLUSIONS/SIGNIFICANCE: Our results reveal the differences in the binding modes between the different retinoid-binding proteins.

  13. Crystal Structure of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Csn2 Protein Revealed Ca[superscript 2+]-dependent Double-stranded DNA Binding Activity

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Ki Hyun; Kurinov, Igor; Ke, Ailong (Cornell); (NWU)

    2012-05-22

    Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) are widespread in bacteria and archaea. They form a line of RNA-based immunity to eradicate invading bacteriophages and malicious plasmids. A key molecular event during this process is the acquisition of new spacers into the CRISPR loci to guide the selective degradation of the matching foreign genetic elements. Csn2 is a Nmeni subtype-specific cas gene required for new spacer acquisition. Here we characterize the Enterococcus faecalis Csn2 protein as a double-stranded (ds-) DNA-binding protein and report its 2.7 {angstrom} tetrameric ring structure. The inner circle of the Csn2 tetrameric ring is {approx}26 {angstrom} wide and populated with conserved lysine residues poised for nonspecific interactions with ds-DNA. Each Csn2 protomer contains an {alpha}/{beta} domain and an {alpha}-helical domain; significant hinge motion was observed between these two domains. Ca{sup 2+} was located at strategic positions in the oligomerization interface. We further showed that removal of Ca{sup 2+} ions altered the oligomerization state of Csn2, which in turn severely decreased its affinity for ds-DNA. In summary, our results provided the first insight into the function of the Csn2 protein in CRISPR adaptation by revealing that it is a ds-DNA-binding protein functioning at the quaternary structure level and regulated by Ca{sup 2+} ions.

  14. Crystal structure of clustered regularly interspaced short palindromic repeats (CRISPR)-associated Csn2 protein revealed Ca2+-dependent double-stranded DNA binding activity.

    Science.gov (United States)

    Nam, Ki Hyun; Kurinov, Igor; Ke, Ailong

    2011-09-02

    Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) are widespread in bacteria and archaea. They form a line of RNA-based immunity to eradicate invading bacteriophages and malicious plasmids. A key molecular event during this process is the acquisition of new spacers into the CRISPR loci to guide the selective degradation of the matching foreign genetic elements. Csn2 is a Nmeni subtype-specific cas gene required for new spacer acquisition. Here we characterize the Enterococcus faecalis Csn2 protein as a double-stranded (ds-) DNA-binding protein and report its 2.7 Å tetrameric ring structure. The inner circle of the Csn2 tetrameric ring is ∼26 Å wide and populated with conserved lysine residues poised for nonspecific interactions with ds-DNA. Each Csn2 protomer contains an α/β domain and an α-helical domain; significant hinge motion was observed between these two domains. Ca(2+) was located at strategic positions in the oligomerization interface. We further showed that removal of Ca(2+) ions altered the oligomerization state of Csn2, which in turn severely decreased its affinity for ds-DNA. In summary, our results provided the first insight into the function of the Csn2 protein in CRISPR adaptation by revealing that it is a ds-DNA-binding protein functioning at the quaternary structure level and regulated by Ca(2+) ions.

  15. A chimeric protein composed of NuMA fused to the DNA binding domain of LANA is sufficient for the ori-P-dependent DNA replication.

    Science.gov (United States)

    Ohsaki, Eriko; Ueda, Keiji

    2017-01-01

    The Kaposi's sarcoma-associated herpesvirus (KSHV) genome is stably maintained in KSHV-infected PEL cell lines during cell division. We previously showed that accumulation of LANA in the nuclear matrix fraction could be important for the latent DNA replication, and that the functional significance of LANA should be its recruitment of ori-P to the nuclear matrix. Here, we investigated whether the forced localization of the LANA-DNA binding domain (DBD) to the nuclear matrix facilitated ori-P-containing plasmid replication. We demonstrated that chimeric proteins constructed by fusion of LANA DBD with the nuclear mitotic apparatus protein (NuMA), which is one of the components of the nuclear matrix, could bind with ori-P and enhance replication of an ori-P-containing plasmid, compared with that in the presence of DBD alone. These results further suggested that the ori-P recruitment to the nuclear matrix through the binding with DBD is important for latent viral DNA replication. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Identification of calcium-binding proteins in human platelets

    International Nuclear Information System (INIS)

    Brumley, L.M.; Wallace, R.W.

    1986-01-01

    In human platelets, intracellular Ca 2+ is a second messenger for platelet agonists. Two targets for the Ca 2+ signal are calmodulin and the C-,inase; however, other Ca 2+ -binding proteins may also play a role in platelet function. The Western blotting technique of Maruyama et al., which utilizes 45 Ca 2+ to detect Ca 2+ -binding proteins, has been used to identify numerous platelet Ca 2+ -binding proteins ranging in molecular weight from 165K to 15K. The greatest quantity of 45 Ca 2+ was bound to a 165 kilodalton protein which has been identified as thrombospondin based upon its release from thrombin-stimulated platelets and its comigration on SDS gels with purified thrombospondin. Two other major sites for 45 Ca 2+ -binding correspond to proteins of 120K and 108K which are present only in the platelet particulate fraction; they have been identified as glycoproteins IIb and IIIa based upon their labeling by 125 I-concanavalin A. Two proteins with molecular weights of 20K and 15K bound much less 45 Ca 2+ and correspond on SDS gels to calmodulin and subunit B of the calmodulin-dependent phosphatase. A number of other, yet to be identified, Ca 2+ -binding proteins were also detected. These data indicate that human platelets contain numerous Ca 2+ -binding proteins and that Western blotting techniques utilizing 45 Ca 2+ may be useful as an assay system in future attempts to purify platelet Ca 2+ -binding proteins

  17. SONAR Discovers RNA-Binding Proteins from Analysis of Large-Scale Protein-Protein Interactomes.

    Science.gov (United States)

    Brannan, Kristopher W; Jin, Wenhao; Huelga, Stephanie C; Banks, Charles A S; Gilmore, Joshua M; Florens, Laurence; Washburn, Michael P; Van Nostrand, Eric L; Pratt, Gabriel A; Schwinn, Marie K; Daniels, Danette L; Yeo, Gene W

    2016-10-20

    RNA metabolism is controlled by an expanding, yet incomplete, catalog of RNA-binding proteins (RBPs), many of which lack characterized RNA binding domains. Approaches to expand the RBP repertoire to discover non-canonical RBPs are currently needed. Here, HaloTag fusion pull down of 12 nuclear and cytoplasmic RBPs followed by quantitative mass spectrometry (MS) demonstrates that proteins interacting with multiple RBPs in an RNA-dependent manner are enriched for RBPs. This motivated SONAR, a computational approach that predicts RNA binding activity by analyzing large-scale affinity precipitation-MS protein-protein interactomes. Without relying on sequence or structure information, SONAR identifies 1,923 human, 489 fly, and 745 yeast RBPs, including over 100 human candidate RBPs that contain zinc finger domains. Enhanced CLIP confirms RNA binding activity and identifies transcriptome-wide RNA binding sites for SONAR-predicted RBPs, revealing unexpected RNA binding activity for disease-relevant proteins and DNA binding proteins. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. In Situ Protein Binding Assay Using Fc-Fusion Proteins.

    Science.gov (United States)

    Padmanabhan, Nirmala; Siddiqui, Tabrez J

    2017-01-01

    This protocol describes an in situ protein-protein interaction assay between tagged recombinant proteins and cell-surface expressed synaptic proteins. The assay is arguably more sensitive than other traditional protein binding assays such as co-immunoprecipitation and pull-downs and provides a visual readout for binding. This assay has been widely used to determine the dissociation constant of binding of trans-synaptic adhesion proteins. The step-wise description in the protocol should facilitate the adoption of this method in other laboratories.

  19. Discrete persistent-chain model for protein binding on DNA.

    Science.gov (United States)

    Lam, Pui-Man; Zhen, Yi

    2011-04-01

    We describe and solve a discrete persistent-chain model of protein binding on DNA, involving an extra σ(i) at a site i of the DNA. This variable takes the value 1 or 0, depending on whether or not the site is occupied by a protein. In addition, if the site is occupied by a protein, there is an extra energy cost ɛ. For a small force, we obtain analytic expressions for the force-extension curve and the fraction of bound protein on the DNA. For higher forces, the model can be solved numerically to obtain force-extension curves and the average fraction of bound proteins as a function of applied force. Our model can be used to analyze experimental force-extension curves of protein binding on DNA, and hence deduce the number of bound proteins in the case of nonspecific binding. ©2011 American Physical Society

  20. New fluorescent reagents specific for Ca2+-binding proteins

    International Nuclear Information System (INIS)

    Ben-Hail, Danya; Lemelson, Daniela; Israelson, Adrian; Shoshan-Barmatz, Varda

    2012-01-01

    Highlights: ► New reagents specifically inhibit the activity of Ca 2+ -dependent proteins. ► FITC-Ru and EITC-Ru allow for mechanism-independent probing of Ca 2+ -binding proteins. ► Changes in reagents fluorescence allow characterization of protein Ca 2+ -binding properties. -- Abstract: Ca 2+ carries information pivotal to cell life and death via its interactions with specific binding sites in a protein. We previously developed a novel photoreactive reagent, azido ruthenium (AzRu), which strongly inhibits Ca 2+ -dependent activities. Here, we synthesized new fluorescent ruthenium-based reagents containing FITC or EITC, FITC-Ru and EITC-Ru. These reagents were purified, characterized and found to specifically interact with and markedly inhibit Ca 2+ -dependent activities but not the activity of Ca 2+ -independent reactions. In contrast to many reagents that serve as probes for Ca 2+ , FITC-Ru and EITC-Ru are the first fluorescent divalent cation analogs to be synthesized and characterized that specifically bind to Ca 2+ -binding proteins and inhibit their activity. Such reagents will assist in characterizing Ca 2+ -binding proteins, thereby facilitating better understanding of the function of Ca 2+ as a key bio-regulator.

  1. Radiation damage to DNA-binding proteins

    International Nuclear Information System (INIS)

    Culard, G.; Eon, S.; DeVuyst, G.; Charlier, M.; Spotheim-Maurizot, M.

    2003-01-01

    The DNA-binding properties of proteins are strongly affected upon irradiation. The tetrameric lactose repressor (a dimer of dimers) losses its ability to bind operator DNA as soon as at least two damages per protomer of each dimer occur. The monomeric MC1 protein losses its ability to bind DNA in two steps : i) at low doses only the specific binding is abolished, whereas the non-specific one is still possible; ii) at high doses all binding vanishes. Moreover, the DNA bending induced by MC1 binding is less pronounced for a protein that underwent the low dose irradiation. When the entire DNA-protein complexes are irradiated, the observed disruption of the complexes is mainly due to the damage of the proteins and not to that of DNA. The doses necessary for complex disruption are higher than those inactivating the free protein. This difference, larger for MC1 than for lactose repressor, is due to the protection of the protein by the bound DNA. The oxidation of the protein side chains that are accessible to the radiation-induced hydroxyl radicals seems to represent the inactivating damage

  2. CaMELS: In silico prediction of calmodulin binding proteins and their binding sites.

    Science.gov (United States)

    Abbasi, Wajid Arshad; Asif, Amina; Andleeb, Saiqa; Minhas, Fayyaz Ul Amir Afsar

    2017-09-01

    Due to Ca 2+ -dependent binding and the sequence diversity of Calmodulin (CaM) binding proteins, identifying CaM interactions and binding sites in the wet-lab is tedious and costly. Therefore, computational methods for this purpose are crucial to the design of such wet-lab experiments. We present an algorithm suite called CaMELS (CalModulin intEraction Learning System) for predicting proteins that interact with CaM as well as their binding sites using sequence information alone. CaMELS offers state of the art accuracy for both CaM interaction and binding site prediction and can aid biologists in studying CaM binding proteins. For CaM interaction prediction, CaMELS uses protein sequence features coupled with a large-margin classifier. CaMELS models the binding site prediction problem using multiple instance machine learning with a custom optimization algorithm which allows more effective learning over imprecisely annotated CaM-binding sites during training. CaMELS has been extensively benchmarked using a variety of data sets, mutagenic studies, proteome-wide Gene Ontology enrichment analyses and protein structures. Our experiments indicate that CaMELS outperforms simple motif-based search and other existing methods for interaction and binding site prediction. We have also found that the whole sequence of a protein, rather than just its binding site, is important for predicting its interaction with CaM. Using the machine learning model in CaMELS, we have identified important features of protein sequences for CaM interaction prediction as well as characteristic amino acid sub-sequences and their relative position for identifying CaM binding sites. Python code for training and evaluating CaMELS together with a webserver implementation is available at the URL: http://faculty.pieas.edu.pk/fayyaz/software.html#camels. © 2017 Wiley Periodicals, Inc.

  3. Salt modulates the stability and lipid binding affinity of the adipocyte lipid-binding proteins

    Science.gov (United States)

    Schoeffler, Allyn J.; Ruiz, Carmen R.; Joubert, Allison M.; Yang, Xuemei; LiCata, Vince J.

    2003-01-01

    Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its Delta G of unfolding by 3-5 kcal/mol. At 37 degrees C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilinonaphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP.

  4. Are many Z-DNA binding proteins actually phospholipid-binding proteins?

    OpenAIRE

    Krishna, P; Kennedy, B P; Waisman, D M; van de Sande, J H; McGhee, J D

    1990-01-01

    We used a Z-DNA affinity column to isolate a collection of Z-DNA binding proteins from a high salt extract of Escherichia coli. We identified one of the major Z-DNA binding proteins of this fraction, not as a protein involved in gene regulation or genetic recombination, but rather as an outer membrane porin protein. We then showed that several other known phospholipid-binding proteins (bovine lung annexins and human serum lipoproteins) also bind much more tightly to Z-DNA than to B-DNA. In al...

  5. Fatty Acid Binding Proteins in Prostate Cancer

    National Research Council Canada - National Science Library

    Jett, Marti

    2000-01-01

    We have shown that there is a distinct pattern of fatty acid binding protein (FAEP) expression in prostate cancer vs normal cells and that finding has be confirmed in patient samples of biopsy specimens...

  6. Ubiquitin-binding proteins: similar, but different

    DEFF Research Database (Denmark)

    Andersen, Katrine M; Hofmann, Kay; Hartmann-Petersen, Rasmus

    2005-01-01

    and phosphatases, specific sets of ubiquitinating/deubiquitinating enzymes control the degree of ubiquitination. A large number of ubiquitin-binding proteins act at different steps in the downstream pathways, followed by the ubiquitinated protein. Different families of ubiquitin-binding proteins have been...... described. UBA (ubiquitin-associated) domain-containing proteins is the largest family and includes members involved in different cell processes. The smaller groups of UIM (ubiquitin-interacting motif), GAT [GGA (Golgi-associated gamma-adaptin homologous) and Tom1 (target of Myb 1)], CUE (coupling...

  7. Telomere-binding proteins of Arabidopsis thaliana.

    Science.gov (United States)

    Zentgraf, U

    1995-02-01

    The nucleoprotein structure of Arabidopsis thaliana telomeres was investigated. A protein specifically binding to telomeric sequences was characterized by gel mobility shift assays with synthetic oligonucleotides consisting of four 7 bp telomeric repeats of Arabidopsis (TTTAGGG) and crude nuclear protein extracts of Arabidopsis leaves. These DNA-protein binding studies revealed that the binding affinity of this telomere-binding protein to the G-rich single-strand as well as to the double-stranded telomeric DNA is much higher than to the C-rich single-strand. The molecular mass of the protein was identified by SDS-PAGE to be 67 kDa. The isoelectric points were determined to be 5.0, 4.85 and 4.7, respectively, indicating that either one protein with different modifications or three slightly different proteins have been isolated. An RNA component, possibly serving as a template for reverse transcription of a plant telomerase, does not mediate the DNA-protein contact because the DNA-protein interactions were not RNAse-sensitive.

  8. RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins

    Directory of Open Access Journals (Sweden)

    Elisa E. Figueroa-Angulo

    2015-11-01

    Full Text Available Iron homeostasis is highly regulated in vertebrates through a regulatory system mediated by RNA-protein interactions between the iron regulatory proteins (IRPs that interact with an iron responsive element (IRE located in certain mRNAs, dubbed the IRE-IRP regulatory system. Trichomonas vaginalis, the causal agent of trichomoniasis, presents high iron dependency to regulate its growth, metabolism, and virulence properties. Although T. vaginalis lacks IRPs or proteins with aconitase activity, possesses gene expression mechanisms of iron regulation at the transcriptional and posttranscriptional levels. However, only one gene with iron regulation at the transcriptional level has been described. Recently, our research group described an iron posttranscriptional regulatory mechanism in the T. vaginalis tvcp4 and tvcp12 cysteine proteinase mRNAs. The tvcp4 and tvcp12 mRNAs have a stem-loop structure in the 5'-coding region or in the 3'-UTR, respectively that interacts with T. vaginalis multifunctional proteins HSP70, α-Actinin, and Actin under iron starvation condition, causing translation inhibition or mRNA stabilization similar to the previously characterized IRE-IRP system in eukaryotes. Herein, we summarize recent progress and shed some light on atypical RNA-binding proteins that may participate in the iron posttranscriptional regulation in T. vaginalis.

  9. Drosophila DNA-Binding Proteins in Polycomb Repression

    Directory of Open Access Journals (Sweden)

    Maksim Erokhin

    2018-01-01

    Full Text Available The formation of individual gene expression patterns in different cell types is required during differentiation and development of multicellular organisms. Polycomb group (PcG proteins are key epigenetic regulators responsible for gene repression, and dysregulation of their activities leads to developmental abnormalities and diseases. PcG proteins were first identified in Drosophila, which still remains the most convenient system for studying PcG-dependent repression. In the Drosophila genome, these proteins bind to DNA regions called Polycomb response elements (PREs. A major role in the recruitment of PcG proteins to PREs is played by DNA-binding factors, several of which have been characterized in detail. However, current knowledge is insufficient for comprehensively describing the mechanism of this process. In this review, we summarize and discuss the available data on the role of DNA-binding proteins in PcG recruitment to chromatin.

  10. Measurement of plasma protein and lipoprotein binding of pyrethroids.

    Science.gov (United States)

    Sethi, Pankaj K; Muralidhara, S; Bruckner, James V; White, Catherine A

    2014-01-01

    A simple, reliable procedure was developed to measure binding of pyrethroid insecticides to total proteins and lipoproteins of rat and human plasma. The extent of binding of (14)C-labeled deltamethrin (DLM), cis-permethrin (CIS) and trans-permethrin (TRANS) was quantified by a 3-step organic solvent extraction technique. Rat and human plasma samples, containing NaF to inhibit esterases, were spiked with a range of concentrations of each radiolabeled pyrethroid. Protein binding reached equilibrium within ~1h of incubation at 37°C. The samples were extracted in turn with: isooctane to collect the unbound fraction; 2-octanol to extract the lipoprotein-bound fraction; and acetonitrile to obtain the protein-bound fraction. Absolute recoveries of DLM, CIS and TRANS ranged from 86 to 95%. Adherence of these very lipophilic chemicals to glass and plastic was minimized by using silanized glass vials and LoBind® plastic pipettes. The method's ability to distinguish lipoprotein from protein binding was confirmed by experiments with diazepam and cyclosporine, drugs that bind selectively to albumin and lipoproteins, respectively. This procedure was effectively utilized for studies of the species-dependence of plasma protein and lipoprotein binding of three pyrethroids for inclusion in physiologically-based pharmacokinetic models of pyrethroids for use in health risk assessments of the insecticides in children and adults. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Haptenation: Chemical Reactivity and Protein Binding

    Directory of Open Access Journals (Sweden)

    Itai Chipinda

    2011-01-01

    Full Text Available Low molecular weight chemical (LMW allergens are commonly referred to as haptens. Haptens must complex with proteins to be recognized by the immune system. The majority of occupationally related haptens are reactive, electrophilic chemicals, or are metabolized to reactive metabolites that form covalent bonds with nucleophilic centers on proteins. Nonelectrophilic protein binding may occur through disulfide exchange, coordinate covalent binding onto metal ions on metalloproteins or of metal allergens, themselves, to the major histocompatibility complex. Recent chemical reactivity kinetic studies suggest that the rate of protein binding is a major determinant of allergenic potency; however, electrophilic strength does not seem to predict the ability of a hapten to skew the response between Th1 and Th2. Modern proteomic mass spectrometry methods that allow detailed delineation of potential differences in protein binding sites may be valuable in predicting if a chemical will stimulate an immediate or delayed hypersensitivity. Chemical aspects related to both reactivity and protein-specific binding are discussed.

  12. Loss of the zona pellucida-binding protein 2 (Zpbp2) gene in mice impacts airway hypersensitivity and lung lipid metabolism in a sex-dependent fashion.

    Science.gov (United States)

    Kanagaratham, Cynthia; Chiwara, Victoria; Ho, Bianca; Moussette, Sanny; Youssef, Mina; Venuto, David; Jeannotte, Lucie; Bourque, Guillaume; de Sanctis, Juan Bautista; Radzioch, Danuta; Naumova, Anna K

    2018-04-01

    The human chromosomal region 17q12-q21 is one of the best replicated genome-wide association study loci for childhood asthma. The associated SNPs span a large genomic interval that includes several protein-coding genes. Here, we tested the hypothesis that the zona pellucida-binding protein 2 (ZPBP2) gene residing in this region contributes to asthma pathogenesis using a mouse model. We tested the lung phenotypes of knock-out (KO) mice that carry a deletion of the Zpbp2 gene. The deletion attenuated airway hypersensitivity (AHR) in female, but not male, mice in the absence of allergic sensitization. Analysis of the lipid profiles of their lungs showed that female, but not male, KO mice had significantly lower levels of sphingosine-1-phosphate (S1P), very long-chain ceramides (VLCCs), and higher levels of long-chain ceramides compared to wild-type controls. Furthermore, in females, lung resistance following methacholine challenge correlated with lung S1P levels (Pearson correlation coefficient 0.57) suggesting a link between reduced AHR in KO females, Zpbp2 deletion, and S1P level regulation. In livers, spleens and blood plasma, however, VLCC, S1P, and sphingosine levels were reduced in both KO females and males. We also find that the Zpbp2 deletion was associated with gain of methylation in the adjacent DNA regions. Thus, we demonstrate that the mouse ortholog of ZPBP2 has a role in controlling AHR in female mice. Our data also suggest that Zpbp2 may act through regulation of ceramide metabolism. These findings highlight the importance of phospholipid metabolism for sexual dimorphism in AHR.

  13. Computational search for aflatoxin binding proteins

    Science.gov (United States)

    Wang, Ying; Liu, Jinfeng; Zhang, Lujia; He, Xiao; Zhang, John Z. H.

    2017-10-01

    Aflatoxin is one of the mycotoxins that contaminate various food products. Among various aflatoxin types (B1, B2, G1, G2 and M1), aflatoxin B1 is the most important and the most toxic one. In this study, through computational screening, we found that several proteins may bind specifically with different type of aflatoxins. Combination of theoretical methods including target fishing, molecular docking, molecular dynamics (MD) simulation, MM/PBSA calculation were utilized to search for new aflatoxin B1 binding proteins. A recently developed method for calculating entropic contribution to binding free energy called interaction entropy (IE) was employed to compute the binding free energy between the protein and aflatoxin B1. Through comprehensive comparison, three proteins, namely, trihydroxynaphthalene reductase, GSK-3b, and Pim-1 were eventually selected as potent aflatoxin B1 binding proteins. GSK-3b and Pim-1 are drug targets of cancers or neurological diseases. GSK-3b is the strongest binder for aflatoxin B1.

  14. Binding mechanisms of intrinsically disordered proteins: theory, simulation, and experiment

    Directory of Open Access Journals (Sweden)

    Luca Mollica

    2016-09-01

    Full Text Available In recent years, protein science has been revolutionized by the discovery of intrinsically disordered proteins (IDPs. In contrast to the classical paradigm that a given protein sequence corresponds to a defined structure and an associated function, we now know that proteins can be functional in the absence of a stable three-dimensional structure. In many cases, disordered proteins or protein regions become structured, at least locally, upon interacting with their physiological partners. Many, sometimes conflicting, hypotheses have been put forward regarding the interaction mechanisms of IDPs and the potential advantages of disorder for protein-protein interactions. Whether disorder may increase, as proposed e.g. in the fly-casting hypothesis, or decrease binding rates, increase or decrease binding specificity, or what role pre-formed structure might play in interactions involving IDPs (conformational selection vs. induced fit, are subjects of intense debate. Experimentally, these questions remain difficult to address. Here, we review experimental studies of binding mechanisms of IDPs using NMR spectroscopy and transient kinetic techniques, as well as the underlying theoretical concepts and numerical methods that can be applied to describe these interactions at the atomic level. The available literature suggests that the kinetic and thermodynamic parameters characterizing interactions involving IDPs can vary widely and that there may be no single common mechanism that can explain the different binding modes observed experimentally. Rather, disordered proteins appear to make combined use of features such as pre-formed structure and flexibility, depending on the individual system and the functional context.

  15. Co-suppression of sterol-regulatory element binding protein ...

    African Journals Online (AJOL)

    Administrator

    2011-06-22

    Jun 22, 2011 ... In Arabidopsis,. At5g35220 gene being sterol regulatory element-binding protein site 2, protease and metalloendopeptidase activity were required for chloroplast development and play a role in regulation of endodermal plastid size and number that are involved in ethylene-dependent gravitropism of light-.

  16. Binding and measuring natural rubber latex proteins on glove powder.

    Science.gov (United States)

    Tomazic-Jezic, Vesna J; Lucas, Anne D; Sanchez, Beatriz A

    2004-01-01

    does not depend only on the initial protein levels in the raw NRL. More likely, other physical or chemical factors introduced during the manufacturing process, as well as the properties of the donning powder itself, may influence protein binding. Moreover, we demonstrated that the ELISA inhibition assay could be successfully modified for quantitation of proteins adsorbed on the glove powder, together with water-extractable proteins.

  17. Quantitative analysis of EGR proteins binding to DNA: assessing additivity in both the binding site and the protein

    Directory of Open Access Journals (Sweden)

    Stormo Gary D

    2005-07-01

    Full Text Available Abstract Background Recognition codes for protein-DNA interactions typically assume that the interacting positions contribute additively to the binding energy. While this is known to not be precisely true, an additive model over the DNA positions can be a good approximation, at least for some proteins. Much less information is available about whether the protein positions contribute additively to the interaction. Results Using EGR zinc finger proteins, we measure the binding affinity of six different variants of the protein to each of six different variants of the consensus binding site. Both the protein and binding site variants include single and double mutations that allow us to assess how well additive models can account for the data. For each protein and DNA alone we find that additive models are good approximations, but over the combined set of data there are context effects that limit their accuracy. However, a small modification to the purely additive model, with only three additional parameters, improves the fit significantly. Conclusion The additive model holds very well for every DNA site and every protein included in this study, but clear context dependence in the interactions was detected. A simple modification to the independent model provides a better fit to the complete data.

  18. Echinococcus granulosus fatty acid binding proteins subcellular localization.

    Science.gov (United States)

    Alvite, Gabriela; Esteves, Adriana

    2016-05-01

    Two fatty acid binding proteins, EgFABP1 and EgFABP2, were isolated from the parasitic platyhelminth Echinococcus granulosus. These proteins bind fatty acids and have particular relevance in flatworms since de novo fatty acids synthesis is absent. Therefore platyhelminthes depend on the capture and intracellular distribution of host's lipids and fatty acid binding proteins could participate in lipid distribution. To elucidate EgFABP's roles, we investigated their intracellular distribution in the larval stage by a proteomic approach. Our results demonstrated the presence of EgFABP1 isoforms in cytosolic, nuclear, mitochondrial and microsomal fractions, suggesting that these molecules could be involved in several cellular processes. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. AUXIN BINDING PROTEIN1: the outsider.

    Science.gov (United States)

    Sauer, Michael; Kleine-Vehn, Jürgen

    2011-06-01

    AUXIN BINDING PROTEIN1 (ABP1) is one of the first characterized proteins that bind auxin and has been implied as a receptor for a number of auxin responses. Early studies characterized its auxin binding properties and focused on rapid electrophysiological and cell expansion responses, while subsequent work indicated a role in cell cycle and cell division control. Very recently, ABP1 has been ascribed a role in modulating endocytic events at the plasma membrane and RHO OF PLANTS-mediated cytoskeletal rearrangements during asymmetric cell expansion. The exact molecular function of ABP1 is still unresolved, but its main activity apparently lies in influencing events at the plasma membrane. This review aims to connect the novel findings with the more classical literature on ABP1 and to point out the many open questions that still separate us from a comprehensive model of ABP1 action, almost 40 years after the first reports of its existence.

  20. ALG-2, a multifunctional calcium binding protein?

    DEFF Research Database (Denmark)

    Tarabykina, Svetlana; Mollerup, Jens; Winding Gojkovic, P.

    2004-01-01

    ALG-2 was originally discovered as a pro-apoptotic protein in a genetic screen. Due to its ability to bind calcium with high affinity it was postulated to provide a link between the known effect of calcium in programmed cell death and the molecular death execution machinery. This review article d...

  1. CAG trinucleotide RNA repeats interact with RNA-binding proteins

    Energy Technology Data Exchange (ETDEWEB)

    McLaughlin, B.A.; Eberwine, J.; Spencer, C. [Univ. of Pennsylvania, Philadelphia, PA (United States)

    1996-09-01

    Genes associated with several neurological diseases are characterized by the presence of an abnormally long trinucleotide repeat sequence. By way of example, Huntington`s disease (HD), is characterized by selective neuronal degeneration associated with the expansion of a polyglutamine-encoding CAG tract. Normally, this CAG tract is comprised of 11-34 repeats, but in HD it is expanded to >37 repeats in affected individuals. The mechanism by which CAG repeats cause neuronal degeneration is unknown, but it has been speculated that the expansion primarily causes abnormal protein functioning, which in turn causes HD pathology. Other mechanisms, however, have not been ruled out. Interactions between RNA and RNA-binding proteins have previously been shown to play a role in the expression of several eukaryotic genes. Herein, we report the association of cytoplasmic proteins with normal length and extended CAG repeats, using gel shift and LJV crosslinking assays. Cytoplasmic protein extracts from several rat brain regions, including the striatum and cortex, sites of neuronal degeneration in HD, contain a 63-kD RNA-binding protein that specifically interacts with these CAG-repeat sequences. These protein-RNA interactions are dependent on the length of the CAG repeat, with longer repeats binding substantially more protein. Two CAG repeat-binding proteins are present in human cortex and striatum; one comigrates with the rat protein at 63 kD, while the other migrates at 49 kD. These data suggest mechanisms by which RNA-binding proteins may be involved in the pathological course of trinucleotide repeat-associated neurological diseases. 47 refs., 5 figs.

  2. Free enthalpies of replacing water molecules in protein binding pockets.

    Science.gov (United States)

    Riniker, Sereina; Barandun, Luzi J; Diederich, François; Krämer, Oliver; Steffen, Andreas; van Gunsteren, Wilfred F

    2012-12-01

    Water molecules in the binding pocket of a protein and their role in ligand binding have increasingly raised interest in recent years. Displacement of such water molecules by ligand atoms can be either favourable or unfavourable for ligand binding depending on the change in free enthalpy. In this study, we investigate the displacement of water molecules by an apolar probe in the binding pocket of two proteins, cyclin-dependent kinase 2 and tRNA-guanine transglycosylase, using the method of enveloping distribution sampling (EDS) to obtain free enthalpy differences. In both cases, a ligand core is placed inside the respective pocket and the remaining water molecules are converted to apolar probes, both individually and in pairs. The free enthalpy difference between a water molecule and a CH(3) group at the same location in the pocket in comparison to their presence in bulk solution calculated from EDS molecular dynamics simulations corresponds to the binding free enthalpy of CH(3) at this location. From the free enthalpy difference and the enthalpy difference, the entropic contribution of the displacement can be obtained too. The overlay of the resulting occupancy volumes of the water molecules with crystal structures of analogous ligands shows qualitative correlation between experimentally measured inhibition constants and the calculated free enthalpy differences. Thus, such an EDS analysis of the water molecules in the binding pocket may give valuable insight for potency optimization in drug design.

  3. Thermal unfolding of a Ca- and Lanthanide-binding protein

    Energy Technology Data Exchange (ETDEWEB)

    Fahmy, Karim [Helmholtz-Zentrum Dresden-Rossendorf e.V., Dresden (Germany). Biophysics; Goettfert, M. [Technische Univ. Dresden (Germany); Knoeppel, J.

    2017-06-01

    The MIIA (metal ion-induced autocleavage)-domain of the protein Vic001052 from the pathogen Vibrio coralliilyticus, comprises 173 amino acids and exhibits Ca-dependent autoproteolytic activity. It shows homology to nodulation proteins which are secreted by Rhizobiacea into plant host cells where they exert Ca-dependent functions. We have studied the structural and energetic aspects of metal protein interactions of the MIIA domain which appear attractive for engineering metal-binding synthetic peptides. Using a non-cleavable MIIA domain construct, we detected very similar structural changes upon binding to Ca{sup 2+} and Eu{sup 3+}. The thermal denaturation of the Ca-bound state was studied by circular dichroism spectroscopy. The metal-bound folded state unfolds reversibly into an unstructured metal-free state similar to the metal-free state at room temperature.

  4. Complementarity of structure ensembles in protein-protein binding.

    Science.gov (United States)

    Grünberg, Raik; Leckner, Johan; Nilges, Michael

    2004-12-01

    Protein-protein association is often accompanied by changes in receptor and ligand structure. This interplay between protein flexibility and protein-protein recognition is currently the largest obstacle both to our understanding of and to the reliable prediction of protein complexes. We performed two sets of molecular dynamics simulations for the unbound receptor and ligand structures of 17 protein complexes and applied shape-driven rigid body docking to all combinations of representative snapshots. The crossdocking of structure ensembles increased the likelihood of finding near-native solutions. The free ensembles appeared to contain multiple complementary conformations. These were in general not related to the bound structure. We suggest that protein-protein binding follows a three-step mechanism of diffusion, free conformer selection, and refolding. This model combines previously conflicting ideas and is in better agreement with the current data on interaction forces, time scales, and kinetics.

  5. Protein Binding Capacity of Different Forages Tannin

    Science.gov (United States)

    Yusiati, L. M.; Kurniawati, A.; Hanim, C.; Anas, M. A.

    2018-02-01

    Eight forages of tannin sources(Leucaena leucocephala, Arachis hypogaea, Mimosa pudica, Morus alba L, Swietenia mahagoni, Manihot esculenta, Gliricidia sepium, and Bauhinia purpurea)were evaluated their tannin content and protein binding capacity. The protein binding capacity of tannin were determined using precipitation of bovine serum albumin (BSA). Swietenia mahagonihas higest total tannin level and condensed tannin (CT) compared with other forages (Ptannin (HT) level (Ptannin content of Swietenia mahagoni were 11.928±0.04 mg/100 mg and 9.241±0.02mg/100mg dry matter (DM) of leaves. The hydrolysable tannin content of Leucaena leucocephala was 5.338±0.03 mg/100 mg DM of leaves. Binding capacity was highest in Swietenia mahagoni and Leucaena leucocephala compared to the other forages (Ptannin in Leucaena leucocephala and Swietenia mahagoniwere1.181±0.44 and 1.217±0.60mg/mg dry matter of leaves. The present study reports that Swietenia mahagoni has highest of tannin content and Leucaena leucocephala and Swietenia mahagoni capacity of protein binding.

  6. A structural classification of substrate-binding proteins

    NARCIS (Netherlands)

    Berntsson, Ronnie P. -A.; Smits, Sander H. J.; Schmitt, Lutz; Slotboom, Dirk-Jan; Poolman, Bert

    2010-01-01

    Substrate-binding proteins (SBP) are associated with a wide variety of protein complexes. The proteins are part of ATP-binding cassette transporters for substrate uptake, ion gradient driven transporters, DNA-binding proteins, as well as channels and receptors from both pro-and eukaryotes. A wealth

  7. Calcium-dependent binding of Myc to calmodulin

    Science.gov (United States)

    Raffeiner, Philipp; Schraffl, Andrea; Schwarz, Thomas; Röck, Ruth; Ledolter, Karin; Hartl, Markus; Konrat, Robert; Stefan, Eduard; Bister, Klaus

    2017-01-01

    The bHLH-LZ (basic region/helix-loop-helix/leucine zipper) oncoprotein Myc and the bHLH-LZ protein Max form a binary transcription factor complex controlling fundamental cellular processes. Deregulated Myc expression leads to neoplastic transformation and is a hallmark of most human cancers. The dynamics of Myc transcription factor activity are post-translationally coordinated by defined protein-protein interactions. Here, we present evidence for a second messenger controlled physical interaction between the Ca2+ sensor calmodulin (CaM) and all Myc variants (v-Myc, c-Myc, N-Myc, and L-Myc). The predominantly cytoplasmic Myc:CaM interaction is Ca2+-dependent, and the binding site maps to the conserved bHLH domain of Myc. Ca2+-loaded CaM binds the monomeric and intrinsically disordered Myc protein with high affinity, whereas Myc:Max heterodimers show less, and Max homodimers no affinity for CaM. NMR spectroscopic analyses using alternating mixtures of 15N-labeled and unlabeled preparations of CaM and a monomeric Myc fragment containing the bHLH-LZ domain corroborate the biochemical results on the Myc:CaM interaction and confirm the interaction site mapping. In electrophoretic mobility shift assays, addition of CaM does not affect high-affinity DNA-binding of Myc:Max heterodimers. However, cell-based reporter analyses and cell transformation assays suggest that increasing CaM levels enhance Myc transcriptional and oncogenic activities. Our results point to a possible involvement of Ca2+ sensing CaM in the fine-tuning of Myc function. PMID:27926480

  8. XBP1 (X-Box-Binding Protein-1)-Dependent O-GlcNAcylation Is Neuroprotective in Ischemic Stroke in Young Mice and Its Impairment in Aged Mice Is Rescued by Thiamet-G.

    Science.gov (United States)

    Jiang, Meng; Yu, Shu; Yu, Zhui; Sheng, Huaxin; Li, Ying; Liu, Shuai; Warner, David S; Paschen, Wulf; Yang, Wei

    2017-06-01

    Impaired protein homeostasis induced by endoplasmic reticulum dysfunction is a key feature of a variety of age-related brain diseases including stroke. To restore endoplasmic reticulum function impaired by stress, the unfolded protein response is activated. A key unfolded protein response prosurvival pathway is controlled by the endoplasmic reticulum stress sensor (inositol-requiring enzyme-1), XBP1 (downstream X-box-binding protein-1), and O-GlcNAc (O-linked β-N-acetylglucosamine) modification of proteins (O-GlcNAcylation). Stroke impairs endoplasmic reticulum function, which activates unfolded protein response. The rationale of this study was to explore the potentials of the IRE1/XBP1/O-GlcNAc axis as a target for neuroprotection in ischemic stroke. Mice with Xbp1 loss and gain of function in neurons were generated. Stroke was induced by transient or permanent occlusion of the middle cerebral artery in young and aged mice. Thiamet-G was used to increase O-GlcNAcylation. Deletion of Xbp1 worsened outcome after transient and permanent middle cerebral artery occlusion. After stroke, O-GlcNAcylation was activated in neurons of the stroke penumbra in young mice, which was largely Xbp1 dependent. This activation of O-GlcNAcylation was impaired in aged mice. Pharmacological increase of O-GlcNAcylation before or after stroke improved outcome in both young and aged mice. Our study indicates a critical role for the IRE1/XBP1 unfolded protein response branch in stroke outcome. O-GlcNAcylation is a prosurvival pathway that is activated in the stroke penumbra in young mice but impaired in aged mice. Boosting prosurvival pathways to counterbalance the age-related decline in the brain's self-healing capacity could be a promising strategy to improve ischemic stroke outcome in aged brains. © 2017 American Heart Association, Inc.

  9. Dependence of protein binding capacity of dimethylamino-γ-butyric-acid (DMGABA)-immobilized porous membrane on composition of solvent used for DMGABA immobilization

    International Nuclear Information System (INIS)

    Iwanade, Akio; Umeno, Daisuke; Saito, Kyoichi; Sugo, Takanobu

    2013-01-01

    Dimethylamino-γ-butyric acid (DMGABA) as an ampholite was reacted with the epoxy group of the poly-glycidyl methacrylate chain grafted onto the pore surface of a porous hollow-fiber polyethylene membrane by radiation-induced graft polymerization. DMGABA was dissolved in a mixture of dioxane and water at various dioxane volume fractions, defined by dividing the dioxane volume by the total volume. The equilibrium binding capacity (EBC) of the DMGABA-immobilized porous hollow-fiber membrane for lysozyme was evaluated in the permeation mode. The EBC was varied from a 1/50-fold monolayer binding capacity to a 10-fold monolayer binding capacity by controlling the composition of the solvent used for DMGABA immobilization and the molar conversion of the epoxy group into the DMGABA group. - Highlights: ► A DMGABA membrane was immobilized by irradiation induced graft polymerization. ► The DMGABA was immobilized in a mixture of dioxane and water of various compositions. ► Lysozyme adsorptivity of DMGABA-immobilized membranes evaluated in the permeation mode. ► The composition of the DMGABA immobilized solvent can control adsorptivity

  10. Amino Acid Change in the Carbohydrate Response Element Binding Protein is associated with lower triglycerides and myocardial infarction incidence depending on level of adherence to the Mediterranean diet in the PREDIMED trial

    Science.gov (United States)

    A variant (rs3812316, C771G, and Gln241His) in the MLXIPL (Max-like protein X interacting protein-like) gene encoding the carbohydrate response element binding protein has been associated with lower triglycerides. However, its association with cardiovascular diseases and gene-diet interactions modul...

  11. Polyamine binding to proteins in oat and Petunia protoplasts

    Science.gov (United States)

    Mizrahi, Y.; Applewhite, P. B.; Galston, A. W.

    1989-01-01

    Previous work (A Apelbaum et al. [1988] Plant Physiol 88: 996-998) has demonstrated binding of labeled spermidine (Spd) to a developmentally regulated 18 kilodalton protein in tobacco tissue cultures derived from thin surface layer explants. To assess the general importance of such Spd-protein complexes, we attempted bulk isolation from protoplasts of Petunia and oat (Avena sativa). In Petunia, as in tobacco, fed radioactive Spd is bound to protein, but in oat, Spd is first converted to 1,3,-diaminopropane (DAP), probably by polyamine oxidase action. In oat, binding of DAP to protein depends on age of donor leaf and conditions of illumination and temperature, and the extraction of the DAP-protein complex depends upon buffer and pH. The yield of the DAP-protein complex was maximized by extraction of frozen-thawed protoplasts with a pH 8.8 carbonate buffer containing SDS. Its molecular size, based on Sephacryl column fractionation of ammonium sulfate precipitated material, exceeded 45 kilodaltons. Bound Spd or DAP can be released from their complexes by the action of Pronase, but not DNAse, RNAse, or strong salt solutions, indicating covalent attachment to protein.

  12. Binding of radionuclides to proteins in fish

    International Nuclear Information System (INIS)

    Suzuki, Yuzuru

    1981-01-01

    Radioisotope tracer experiments on binding of radionuclides to proteins in fish were carried out in order to gain further information on biochemical behavior of radionuclides in marine fish. The radionuclides, which were bound to proteins in fish through sea water and food, were extracted with a Trisacetate buffer solution and separated by gel filtration on Sephadex G-75. Most of 137 Cs in the fish liver were bound only to a peptide with a molecular weight of 1,100 - 1,300. The most remarkable feature of 60 Co in the profiles of the gel filtration was the presence of two clear radioactivity pearks and the radioactivity appeared to transfer from a low molecular weight protein to a high molecular weight protein in the case of the uptake, and the reverse phenomenon was observed in the case of the excretion. Therefore, this suggested that these proteins had each inherent turnover rate for 60 Co. The profiles of the gel filtration of 65 Zn varied widely among species of fish, tissues or organs even in the same fish and pathways of the uptake. 125 I was bound to a relatively low molecular weight substance in cultured eel, however, the binding of 125 I to a protein with higher molecular weight was observed in the eel head including thyroid gland marked through food, and this protein was estimated to be thyroglobulin with molecular weight of 670,000. Although 95 Nb, 144 Ce- 144 Pr and 106 Ru- 106 Rh probably have no biological function in fish, it was apparently found to be organically bound in tissues or organs of the marine fish. (author)

  13. Maximizing binding capacity for protein A chromatography.

    Science.gov (United States)

    Ghose, Sanchayita; Zhang, Jennifer; Conley, Lynn; Caple, Ryan; Williams, Kevin P; Cecchini, Douglas

    2014-01-01

    Advances in cell culture expression levels in the last two decades have resulted in monoclonal antibody titers of ≥10 g/L to be purified downstream. A high capacity capture step is crucial to prevent purification from being the bottleneck in the manufacturing process. Despite its high cost and other disadvantages, Protein A chromatography still remains the optimal choice for antibody capture due to the excellent selectivity provided by this step. A dual flow loading strategy was used in conjunction with a new generation high capacity Protein A resin to maximize binding capacity without significantly increasing processing time. Optimum conditions were established using a simple empirical Design of Experiment (DOE) based model and verified with a wide panel of antibodies. Dynamic binding capacities of >65 g/L could be achieved under these new conditions, significantly higher by more than one and half times the values that have been typically achieved with Protein A in the past. Furthermore, comparable process performance and product quality was demonstrated for the Protein A step at the increased loading. © 2014 American Institute of Chemical Engineers.

  14. Growth hormone-binding proteins in high-speed cytosols of multiple tissues of the rabbit.

    Science.gov (United States)

    Herington, A C; Ymer, S; Roupas, P; Stevenson, J

    1986-04-11

    Soluble, specific binding protein(s) for growth hormone (GH) have been identified and partially characterized in high-speed cytosolic preparations from a number of rabbit tissues. The binding of 125I-labelled human GH to proteins in liver, heart, adipose tissue, skeletal muscle and kidney cytosols was dependent on time and cytosolic protein concentration. By Scatchard analysis, the binding affinities (KA: (2-7) X 10(9) M-1) were somewhat higher than those generally reported for membrane GH receptors. The binding proteins had a greater specificity for somatotrophic hormones than lactogenic hormones, although the kidney appeared to have, in addition, a lactogen-binding protein. By gel filtration, the Mr of the cytosolic GH-binding protein was approximately 100 000 in all tissues. None of the binding proteins was detectable by the poly(ethylene glycol) precipitation method used widely for soluble hormone receptors. The cytosolic GH-binding proteins also cross-reacted with a monoclonal antibody to the rabbit liver membrane GH receptor. These results indicate the ubiquitous presence of apparently naturally soluble GH-binding proteins in the cytosolic fractions of several tissues in the rabbit. Of great interest is their presence in muscle, where GH receptors or binding proteins have not previously been detected, despite muscle being recognized as a classical GH target tissue.

  15. DNA and RNA Quadruplex-Binding Proteins

    Directory of Open Access Journals (Sweden)

    Václav Brázda

    2014-09-01

    Full Text Available Four-stranded DNA structures were structurally characterized in vitro by NMR, X-ray and Circular Dichroism spectroscopy in detail. Among the different types of quadruplexes (i-Motifs, minor groove quadruplexes, G-quadruplexes, etc., the best described are G-quadruplexes which are featured by Hoogsteen base-paring. Sequences with the potential to form quadruplexes are widely present in genome of all organisms. They are found often in repetitive sequences such as telomeric ones, and also in promoter regions and 5' non-coding sequences. Recently, many proteins with binding affinity to G-quadruplexes have been identified. One of the initially portrayed G-rich regions, the human telomeric sequence (TTAGGGn, is recognized by many proteins which can modulate telomerase activity. Sequences with the potential to form G-quadruplexes are often located in promoter regions of various oncogenes. The NHE III1 region of the c-MYC promoter has been shown to interact with nucleolin protein as well as other G-quadruplex-binding proteins. A number of G-rich sequences are also present in promoter region of estrogen receptor alpha. In addition to DNA quadruplexes, RNA quadruplexes, which are critical in translational regulation, have also been predicted and observed. For example, the RNA quadruplex formation in telomere-repeat-containing RNA is involved in interaction with TRF2 (telomere repeat binding factor 2 and plays key role in telomere regulation. All these fundamental examples suggest the importance of quadruplex structures in cell processes and their understanding may provide better insight into aging and disease development.

  16. Properties of Human Embryonic Stem Cells and Their Differentiated Derivatives Depend on Nonhistone DNA-Binding HMGB1 and HMGB2 Proteins

    Czech Academy of Sciences Publication Activity Database

    Bagherpoor, Alireza Jian; Doležalová, D.; Bárta, T.; Kučírek, Martin; Sani, Soodabeh Abbasi; Esner, M.; Bosakova, M.K.; Vinařský, V.; Peškova, L.; Hampl, A.; Štros, Michal

    2017-01-01

    Roč. 26, č. 5 (2017), s. 328-340 ISSN 1547-3287 R&D Projects: GA ČR GA15-01354S Grant - others:GA ČR(CZ) GA15-23033S Institutional support: RVO:68081707 Keywords : group box 1 * chromatin protein * expression Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemistry and molecular biology Impact factor: 3.562, year: 2016

  17. Cobalamin and its binding protein in rat milk

    DEFF Research Database (Denmark)

    Raaberg, Lasse; Nexø, Ebba; Poulsen, Steen Seier

    1989-01-01

    Cobalamin and its binding protein, haptocorrin, are present in rat milk throughout the lactation period. The concentration of cobalamin is approximately 0.3-times the concentration of the unsaturated binding protein. The concentration of the unsaturated cobalamin-binding protein varies between 18...... nmol l-1 and 16 nmol l-1. The binding protein has a Stokes radius of 2.49 nm when saturated with cobalamin and 2.61 nm when unsaturated. It binds cobalamin over a broad range of pH and is able to bind cobinamide also. With immunohistochemistry, we find haptocorrin immunoreactivity in the mammary glands...

  18. Observation of Protein Structural Vibrational Mode Sensitivity to Ligand Binding

    Science.gov (United States)

    Niessen, Katherine; Xu, Mengyang; Snell, Edward; Markelz, Andrea

    2014-03-01

    We report the first measurements of the dependence of large-scale protein intramolecular vibrational modes on ligand binding. These collective vibrational modes in the terahertz (THz) frequency range (5-100 cm-1) are of great interest due to their predicted relation to protein function. Our technique, Crystals Anisotropy Terahertz Microscopy (CATM), allows for room temperature, table-top measurements of the optically active intramolecular modes. CATM measurements have revealed surprisingly narrowband features. CATM measurements are performed on single crystals of chicken egg-white lysozyme (CEWL) as well as CEWL bound to tri-N-acetylglucosamine (CEWL-3NAG) inhibitor. We find narrow band resonances that dramatically shift with binding. Quasiharmonic calculations are performed on CEWL and CEWL-3NAG proteins with CHARMM using normal mode analysis. The expected CATM response of the crystals is then calculated by summing over all protein orientations within the unit cell. We will compare the CATM measurements with the calculated results and discuss the changes which arise with protein-ligand binding. This work is supported by NSF grant MRI 2 grant DBI2959989.

  19. Viral Proteins That Bind Double-Stranded RNA: Countermeasures Against Host Antiviral Responses

    OpenAIRE

    Krug, Robert M.

    2014-01-01

    Several animal viruses encode proteins that bind double-stranded RNA (dsRNA) to counteract host dsRNA-dependent antiviral responses. This article discusses the structure and function of the dsRNA-binding proteins of influenza A virus and Ebola viruses (EBOVs).

  20. The binding site for regulatory 14-3-3 protein in plant plasma membrane H+-ATPase: Involvement of a region promoting phosphorylation-independent interaction in addition to the phosphorylation-dependent C-terminal end

    DEFF Research Database (Denmark)

    Fuglsang, Anja T; Borch, Jonas; Bych, Katrine

    2003-01-01

    14-3-3 proteins constitute a family of well conserved proteins interacting with a large number of phosphorylated binding partners in eukaryotic cells. The plant plasma membrane H+-ATPase is an unusual target in that a unique phosphothreonine motif (946YpTV, where pT represents phosphothreonine......, Thr-924 is important for interaction with 14-3-3 protein even when Thr-947 is phosphorylated. We suggest that the role of phosphorylation, which is accentuated by fusicoccin, is to stabilize protein-protein interaction between 14-3-3 protein and several residues of the H+-ATPase C-terminal domain....

  1. Measuring Binding Affinity of Protein-Ligand Interaction Using Spectrophotometry: Binding of Neutral Red to Riboflavin-Binding Protein

    Science.gov (United States)

    Chenprakhon, Pirom; Sucharitakul, Jeerus; Panijpan, Bhinyo; Chaiyen, Pimchai

    2010-01-01

    The dissociation constant, K[subscript d], of the binding of riboflavin-binding protein (RP) with neutral red (NR) can be determined by titrating RP to a fixed concentration of NR. Upon adding RP to the NR solution, the maximum absorption peak of NR shifts to 545 nm from 450 nm for the free NR. The change of the absorption can be used to determine…

  2. What Happened to the IGF Binding Proteins?

    Science.gov (United States)

    Bach, Leon A

    2018-02-01

    Insulinlike growth factor (IGF) binding proteins (IGFBPs) 1 to 6 are high-affinity regulators of IGF activity. They generally inhibit IGF actions by preventing binding to the IGF-I receptor but can also enhance their actions under some conditions. Posttranslational modifications such as glycosylation and phosphorylation modulate IGFBP properties, and IGFBP proteolysis results in IGF release. IGFBPs have more recently been shown to have IGF-independent actions. A number of mechanisms are involved, including modulation of other growth factor pathways, nuclear localization and transcriptional regulation, interaction with the sphingolipid pathway, and binding to non-IGF biomolecules in the extracellular space and matrix, on the cell surface and intracellularly. IGFBPs modulate important biological processes, including cell proliferation, survival, migration, senescence, autophagy, and angiogenesis. Their actions have been implicated in growth, metabolism, cancer, stem cell maintenance and differentiation, and immune regulation. Recent studies have shown that epigenetic mechanisms are involved in the regulation of IGFBP abundance. A more complete understanding of IGFBP biology is necessary to further define their cellular roles and determine their therapeutic potential. Copyright © 2018 Endocrine Society.

  3. Surface selective binding of nanoclay particles to polyampholyte protein chains.

    Science.gov (United States)

    Pawar, Nisha; Bohidar, H B

    2009-07-28

    Binding of nanoclay (Laponite) to gelatin-A and gelatin-B (both polyampholytes) molecules was investigated at room temperature (25 degrees C) both experimentally and theoretically. The stoichiometric binding ratio between gelatin and Laponite was found to be strongly dependent on the solution ionic strength. Large soluble complexes were formed at higher ionic strengths of the solution, a result supported by data obtained from light scattering, viscosity, and zeta potential measurements. The binding problem was theoretically modeled by choosing a suitable two-body screened Coulomb potential, U(R(+)) = (q(-)/2epsilon)[(Q(-)/R(-))e(-kR(-))-(Q(+)/R(+))e(-kR(+))], where the protein dipole has charges Q(+) and Q(-) that are located at distances R(+) and R(-) from the point Laponite charge q(-) and the dispersion liquid has dielectric constant (epsilon). U(R(+)) accounted for electrostatic interactions between a dipole (protein molecule) and an effective charge (Laponite particle) located at an angular position theta. Gelatin-A and Laponite association was facilitated by a strong attractive interaction potential that led to preferential binding of the biopolymer chains to negatively charged face of Laponite particles. In the case of gelatin-B selective surf ace patch binding dominated the process where the positively charged rim and negatively charged face of the particles were selectively bound to the oppositely charged segments of the biopolymer. The equilibrium separation (R(e)) between the protein and nanoclay particle revealed monovalent salt concentration dependence given by R(e) approximately [NaCl](alpha) where alpha = 0.6+/-0.2 for gelatin-A and alpha = 0.4+/-0.2 for gelatin-B systems. The equilibrium separations were approximately 30% less compared to the gelatin-A system implying preferential short-range ordering of the gelatin-B-nanoclay pair in the solvent.

  4. Surfactant protein D binds to human immunodeficiency virus (HIV) envelope protein gp120 and inhibits HIV replication

    DEFF Research Database (Denmark)

    Meschi, Joseph; Crouch, Erika C; Skolnik, Paul

    2005-01-01

    The envelope protein (gp120) of human immunodeficiency virus (HIV) contains highly conserved mannosylated oligosaccharides. These glycoconjugates contribute to resistance to antibody neutralization, and binding to cell surface lectins on macrophages and dendritic cells. Mannose-binding lectin (MBL......) binds to gp120 and plays a role in defence against the virus. In this study it is demonstrated that surfactant protein D (SP-D) binds to gp120 and inhibits HIV infectivity at significantly lower concentrations than MBL. The binding of SP-D was mediated by its calcium-dependent carbohydrate...... defence against HIV. A chimeric protein containing the N-terminal and collagen domains of SP-D linked to the neck and carbohydrate-recognition domains of MBL (called SP-D/MBL(neck+CRD)) had greater ability to bind to gp120 and inhibit virus replication than either SP-D or MBL. The enhanced binding of SP...

  5. Structural and binding studies of SAP-1 protein with heparin.

    Science.gov (United States)

    Yadav, Vikash K; Mandal, Rahul S; Puniya, Bhanwar L; Kumar, Rahul; Dey, Sharmistha; Singh, Sarman; Yadav, Savita

    2015-03-01

    SAP-1 is a low molecular weight cysteine protease inhibitor (CPI) which belongs to type-2 cystatins family. SAP-1 protein purified from human seminal plasma (HuSP) has been shown to inhibit cysteine and serine proteases and exhibit interesting biological properties, including high temperature and pH stability. Heparin is a naturally occurring glycosaminoglycan (with varied chain length) which interacts with a number of proteins and regulates multiple steps in different biological processes. As an anticoagulant, heparin enhances inhibition of thrombin by the serpin antithrombin III. Therefore, we have employed surface plasmon resonance (SPR) to improve our understanding of the binding interaction between heparin and SAP-1 (protease inhibitor). SPR data suggest that SAP-1 binds to heparin with a significant affinity (KD = 158 nm). SPR solution competition studies using heparin oligosaccharides showed that the binding of SAP-1 to heparin is dependent on chain length. Large oligosaccharides show strong binding affinity for SAP-1. Further to get insight into the structural aspect of interactions between SAP-1 and heparin, we used modelled structure of the SAP-1 and docked with heparin and heparin-derived polysaccharides. The results suggest that a positively charged residue lysine plays important role in these interactions. Such information should improve our understanding of how heparin, present in the reproductive tract, regulates cystatins activity. © 2014 John Wiley & Sons A/S.

  6. Glycan masking of Plasmodium vivax Duffy Binding Protein for probing protein binding function and vaccine development.

    Directory of Open Access Journals (Sweden)

    Sowmya Sampath

    Full Text Available Glycan masking is an emerging vaccine design strategy to focus antibody responses to specific epitopes, but it has mostly been evaluated on the already heavily glycosylated HIV gp120 envelope glycoprotein. Here this approach was used to investigate the binding interaction of Plasmodium vivax Duffy Binding Protein (PvDBP and the Duffy Antigen Receptor for Chemokines (DARC and to evaluate if glycan-masked PvDBPII immunogens would focus the antibody response on key interaction surfaces. Four variants of PVDBPII were generated and probed for function and immunogenicity. Whereas two PvDBPII glycosylation variants with increased glycan surface coverage distant from predicted interaction sites had equivalent binding activity to wild-type protein, one of them elicited slightly better DARC-binding-inhibitory activity than wild-type immunogen. Conversely, the addition of an N-glycosylation site adjacent to a predicted PvDBP interaction site both abolished its interaction with DARC and resulted in weaker inhibitory antibody responses. PvDBP is composed of three subdomains and is thought to function as a dimer; a meta-analysis of published PvDBP mutants and the new DBPII glycosylation variants indicates that critical DARC binding residues are concentrated at the dimer interface and along a relatively flat surface spanning portions of two subdomains. Our findings suggest that DARC-binding-inhibitory antibody epitope(s lie close to the predicted DARC interaction site, and that addition of N-glycan sites distant from this site may augment inhibitory antibodies. Thus, glycan resurfacing is an attractive and feasible tool to investigate protein structure-function, and glycan-masked PvDBPII immunogens might contribute to P. vivax vaccine development.

  7. Insulin-like growth factor binding protein 3 has opposing actions on malignant and nonmalignant breast epithelial cells that are each reversible and dependent upon cholesterol-stabilized integrin receptor complexes.

    Science.gov (United States)

    Burrows, C; Holly, J M P; Laurence, N J; Vernon, E G; Carter, J V; Clark, M A; McIntosh, J; McCaig, C; Winters, Z E; Perks, C M

    2006-07-01

    IGF-binding protein (IGFBP)-3 is generally considered to have actions that counterbalance those of IGFs and is therefore being developed as a cancer treatment. In breast tumors, however, high levels are associated with aggressive tumors and poor prognosis. Consistent with this we have demonstrated that although IGFBP-3 and a non-IGF-binding fragment (serine phosphorylation domain peptide) reduced attachment and enhanced apoptosis of Hs578T breast cancer cells cultured on collagen or laminin, it promoted their attachment and survival on fibronectin, which is abundant in the matrix of aggressive tumors. We have now examined the factors that determine whether IGFBP-3 has positive or negative actions on breast epithelial cells. IGFBP-3 also promoted survival of Hs578T cells in the presence of an antibody to the beta1-integrin subunit or when cholesterol-stabilized complexes were disrupted. These actions were blocked by IGF-I or a MAPK inhibitor. Serine phosphorylation domain peptide had similar actions on MCF-7 cells that were again reversed on fibronectin or with disruption of cholesterol-stabilized complexes and blocked by the beta1-integrin antibody. In contrast, IGFBP-3 promoted growth and survival for nonmalignant MCF-10A cells, but these effects were again reversed on fibronectin and blocked by the beta1 antibody or a MAPK inhibitor or by disruption of cholesterol-stabilized complexes. On Hs578T cells, IGFBP-3 bound to caveolin-1 and beta1-integrins, enhancing their aggregation, the recruitment of focal adhesion kinase, and the activation of MAPK. In summary, with three breast epithelial cell lines, IGFBP-3 had positive or negative effects on growth and survival dependent upon the status of cholesterol-stabilized integrin receptor complexes.

  8. Ice-Binding Proteins in Plants

    Directory of Open Access Journals (Sweden)

    Melissa Bredow

    2017-12-01

    Full Text Available Sub-zero temperatures put plants at risk of damage associated with the formation of ice crystals in the apoplast. Some freeze-tolerant plants mitigate this risk by expressing ice-binding proteins (IBPs, that adsorb to ice crystals and modify their growth. IBPs are found across several biological kingdoms, with their ice-binding activity and function uniquely suited to the lifestyle they have evolved to protect, be it in fishes, insects or plants. While IBPs from freeze-avoidant species significantly depress the freezing point, plant IBPs typically have a reduced ability to lower the freezing temperature. Nevertheless, they have a superior ability to inhibit the recrystallization of formed ice. This latter activity prevents ice crystals from growing larger at temperatures close to melting. Attempts to engineer frost-hardy plants by the controlled transfer of IBPs from freeze-avoiding fish and insects have been largely unsuccessful. In contrast, the expression of recombinant IBP sequences from freeze-tolerant plants significantly reduced electrolyte leakage and enhanced freezing survival in freeze-sensitive plants. These promising results have spurred additional investigations into plant IBP localization and post-translational modifications, as well as a re-evaluation of IBPs as part of the anti-stress and anti-pathogen axis of freeze-tolerant plants. Here we present an overview of plant freezing stress and adaptation mechanisms and discuss the potential utility of IBPs for the generation of freeze-tolerant crops.

  9. Apolipoprotein B is a calcium binding protein

    Energy Technology Data Exchange (ETDEWEB)

    Dashti, N.; Lee, D.M.; Mok, T.

    1986-05-29

    Human hepatocarcinoma Hep G2 cells were grown in culture medium containing (/sup 45/Ca/sup 2 +/). The secreted lipoproteins of d < 1.063 g/ml and d 1.063-1.21 g/ml were isolated from the culture media and analyzed by 3.3% and 7% SDS-polyacrylamide gel electrophoresis. Radioactivity profiles of (/sup 45/Ca) from the gels showed that the peak of radioactivity corresponded to the apolipoprotein B band. The molar ratio of the incorporated (/sup 45/Ca/sup 2 +/) and apolipoprotein B was close to unity. No radioactivity was found associated with any other secreted apolipoproteins. To confirm these findings, apolipoprotein B-containing lipoproteins were precipitated with anti-apolipoprotein B and high density lipoproteins were precipitated with anti-apolipoprotein A-I. Only the former precipitate was radioactive. These results suggest that apolipoprotein B is a calcium binding protein.

  10. Guanylate kinase domains of the MAGUK family scaffold proteins as specific phospho-protein-binding modules.

    Science.gov (United States)

    Zhu, Jinwei; Shang, Yuan; Xia, Caihao; Wang, Wenning; Wen, Wenyu; Zhang, Mingjie

    2011-11-25

    Membrane-associated guanylate kinases (MAGUKs) are a large family of scaffold proteins that play essential roles in tissue developments, cell-cell communications, cell polarity control, and cellular signal transductions. Despite extensive studies over the past two decades, the functions of the signature guanylate kinase domain (GK) of MAGUKs are poorly understood. Here we show that the GK domain of DLG1/SAP97 binds to asymmetric cell division regulatory protein LGN in a phosphorylation-dependent manner. The structure of the DLG1 SH3-GK tandem in complex with a phospho-LGN peptide reveals that the GMP-binding site of GK has evolved into a specific pSer/pThr-binding pocket. Residues both N- and C-terminal to the pSer are also critical for the specific binding of the phospho-LGN peptide to GK. We further demonstrate that the previously reported GK domain-mediated interactions of DLGs with other targets, such as GKAP/DLGAP1/SAPAP1 and SPAR, are also phosphorylation dependent. Finally, we provide evidence that other MAGUK GKs also function as phospho-peptide-binding modules. The discovery of the phosphorylation-dependent MAGUK GK/target interactions indicates that MAGUK scaffold-mediated signalling complex organizations are dynamically regulated.

  11. Dominant Alcohol-Protein Interaction via Hydration-Enabled Enthalpy-Driven Binding Mechanism.

    Science.gov (United States)

    Chong, Yuan; Kleinhammes, Alfred; Tang, Pei; Xu, Yan; Wu, Yue

    2015-04-30

    Water plays an important role in weak associations of small drug molecules with proteins. Intense focus has been on binding-induced structural changes in the water network surrounding protein binding sites, especially their contributions to binding thermodynamics. However, water is also tightly coupled to protein conformations and dynamics, and so far little is known about the influence of water-protein interactions on ligand binding. Alcohols are a type of low-affinity drugs, and it remains unclear how water affects alcohol-protein interactions. Here, we present alcohol adsorption isotherms under controlled protein hydration using in situ NMR detection. As functions of hydration level, Gibbs free energy, enthalpy, and entropy of binding were determined from the temperature dependence of isotherms. Two types of alcohol binding were found. The dominant type is low-affinity nonspecific binding, which is strongly dependent on temperature and the level of hydration. At low hydration levels, this nonspecific binding only occurs above a threshold of alcohol vapor pressure. An increased hydration level reduces this threshold, with it finally disappearing at a hydration level of h ≈ 0.2 (g water/g protein), gradually shifting alcohol binding from an entropy-driven to an enthalpy-driven process. Water at charged and polar groups on the protein surface was found to be particularly important in enabling this binding. Although further increase in hydration has smaller effects on the changes of binding enthalpy and entropy, it results in a significant negative change in Gibbs free energy due to unmatched enthalpy-entropy compensation. These results show the crucial role of water-protein interplay in alcohol binding.

  12. Dominant Alcohol-Protein Interaction via Hydration-Enabled Enthalpy-Driven Binding Mechanism

    Science.gov (United States)

    Chong, Yuan; Kleinhammes, Alfred; Tang, Pei; Xu, Yan; Wu, Yue

    2015-01-01

    Water plays an important role in weak associations of small drug molecules with proteins. Intense focus has been on binding-induced structural changes in the water network surrounding protein binding sites, especially their contributions to binding thermodynamics. However, water is also tightly coupled to protein conformations and dynamics, and so far little is known about the influence of water-protein interactions on ligand binding. Alcohols are a type of low-affinity drugs, and it remains unclear how water affects alcohol-protein interactions. Here, we present alcohol adsorption isotherms under controlled protein hydration using in-situ NMR detection. As functions of hydration level, Gibbs free energy, enthalpy, and entropy of binding were determined from the temperature dependence of isotherms. Two types of alcohol binding were found. The dominant type is low-affinity nonspecific binding, which is strongly dependent on temperature and the level of hydration. At low hydration levels, this nonspecific binding only occurs above a threshold of alcohol vapor pressure. An increased hydration level reduces this threshold, with it finally disappearing at a hydration level of h~0.2 (g water/g protein), gradually shifting alcohol binding from an entropy-driven to an enthalpy-driven process. Water at charged and polar groups on the protein surface was found to be particularly important in enabling this binding. Although further increase in hydration has smaller effects on the changes of binding enthalpy and entropy, it results in significant negative change in Gibbs free energy due to unmatched enthalpy-entropy compensation. These results show the crucial role of water-protein interplay in alcohol binding. PMID:25856773

  13. Helical propensity in an intrinsically disordered protein accelerates ligand binding

    DEFF Research Database (Denmark)

    Iesmantavicius, Vytautas; Dogan, Jakob; Jemth, Per

    2014-01-01

    domain of the activator for thyroid hormone and retinoid receptors (ACTR) is intrinsically disordered and folds upon binding to the nuclear coactivator binding domain (NCBD) of the CREB binding protein. A number of mutants was designed that selectively perturbs the amount of secondary structure......Many intrinsically disordered proteins fold upon binding to other macromolecules. The secondary structure present in the well-ordered complex is often formed transiently in the unbound state. The consequence of such transient structure for the binding process is, however, not clear. The activation...... the notion of preformed secondary structure as an important determinant for molecular recognition in intrinsically disordered proteins....

  14. Identification of Lipid Binding Modulators Using the Protein-Lipid Overlay Assay.

    Science.gov (United States)

    Tang, Tuo-Xian; Xiong, Wen; Finkielstein, Carla V; Capelluto, Daniel G S

    2017-01-01

    The protein-lipid overlay assay is an inexpensive, easy-to-implement, and high-throughput methodology that employs nitrocellulose membranes to immobilize lipids in order to rapid screen and identify protein-lipid interactions. In this chapter, we show how this methodology can identify potential modulators of protein-lipid interactions by screening water-soluble lipid competitors or even the introduction of pH changes during the binding assay to identify pH-dependent lipid binding events.

  15. Detection of secondary binding sites in proteins using fragment screening.

    Science.gov (United States)

    Ludlow, R Frederick; Verdonk, Marcel L; Saini, Harpreet K; Tickle, Ian J; Jhoti, Harren

    2015-12-29

    Proteins need to be tightly regulated as they control biological processes in most normal cellular functions. The precise mechanisms of regulation are rarely completely understood but can involve binding of endogenous ligands and/or partner proteins at specific locations on a protein that can modulate function. Often, these additional secondary binding sites appear separate to the primary binding site, which, for example for an enzyme, may bind a substrate. In previous work, we have uncovered several examples in which secondary binding sites were discovered on proteins using fragment screening approaches. In each case, we were able to establish that the newly identified secondary binding site was biologically relevant as it was able to modulate function by the binding of a small molecule. In this study, we investigate how often secondary binding sites are located on proteins by analyzing 24 protein targets for which we have performed a fragment screen using X-ray crystallography. Our analysis shows that, surprisingly, the majority of proteins contain secondary binding sites based on their ability to bind fragments. Furthermore, sequence analysis of these previously unknown sites indicate high conservation, which suggests that they may have a biological function, perhaps via an allosteric mechanism. Comparing the physicochemical properties of the secondary sites with known primary ligand binding sites also shows broad similarities indicating that many of the secondary sites may be druggable in nature with small molecules that could provide new opportunities to modulate potential therapeutic targets.

  16. Roles of RNA-Binding Proteins in DNA Damage Response

    Directory of Open Access Journals (Sweden)

    Mihoko Kai

    2016-02-01

    Full Text Available Living cells experience DNA damage as a result of replication errors and oxidative metabolism, exposure to environmental agents (e.g., ultraviolet light, ionizing radiation (IR, and radiation therapies and chemotherapies for cancer treatments. Accumulation of DNA damage can lead to multiple diseases such as neurodegenerative disorders, cancers, immune deficiencies, infertility, and also aging. Cells have evolved elaborate mechanisms to deal with DNA damage. Networks of DNA damage response (DDR pathways are coordinated to detect and repair DNA damage, regulate cell cycle and transcription, and determine the cell fate. Upstream factors of DNA damage checkpoints and repair, “sensor” proteins, detect DNA damage and send the signals to downstream factors in order to maintain genomic integrity. Unexpectedly, we have discovered that an RNA-processing factor is involved in DNA repair processes. We have identified a gene that contributes to glioblastoma multiforme (GBM’s treatment resistance and recurrence. This gene, RBM14, is known to function in transcription and RNA splicing. RBM14 is also required for maintaining the stem-like state of GBM spheres, and it controls the DNA-PK-dependent non-homologous end-joining (NHEJ pathway by interacting with KU80. RBM14 is a RNA-binding protein (RBP with low complexity domains, called intrinsically disordered proteins (IDPs, and it also physically interacts with PARP1. Furthermore, RBM14 is recruited to DNA double-strand breaks (DSBs in a poly(ADP-ribose (PAR-dependent manner (unpublished data. DNA-dependent PARP1 (poly-(ADP ribose polymerase 1 makes key contributions in the DNA damage response (DDR network. RBM14 therefore plays an important role in a PARP-dependent DSB repair process. Most recently, it was shown that the other RBPs with intrinsically disordered domains are recruited to DNA damage sites in a PAR-dependent manner, and that these RBPs form liquid compartments (also known as

  17. Rapid identification of DNA-binding proteins by mass spectrometry

    DEFF Research Database (Denmark)

    Nordhoff, E; Krogsdam, A M; Jorgensen, H F

    1999-01-01

    We report a protocol for the rapid identification of DNA-binding proteins. Immobilized DNA probes harboring a specific sequence motif are incubated with cell or nuclear extract. Proteins are analyzed directly off the solid support by matrix-assisted laser desorption/ionization time-of-flight mass...... was validated by the identification of known prokaryotic and eukaryotic DNA-binding proteins, and its use provided evidence that poly(ADP-ribose) polymerase exhibits DNA sequence-specific binding to DNA....

  18. Generation of Soluble Advanced Glycation End Products Receptor (sRAGE)-Binding Ligands during Extensive Heat Treatment of Whey Protein/Lactose Mixtures Is Dependent on Glycation and Aggregation

    NARCIS (Netherlands)

    Liu, Fahui; Teodorowicz, Gosia; Wichers, Harry J.; Boekel, van Tiny; Hettinga, Kasper A.

    2016-01-01

    Heating of protein- and sugar-containing materials is considered the primary factor affecting the formation of advanced glycation end products (AGEs). This study aimed to investigate the influence of heating conditions, digestion, and aggregation on the binding capacity of AGEs to the soluble AGE

  19. In vitro binding of germanium to proteins of rice shoots

    International Nuclear Information System (INIS)

    Matsumoto, Hideaki; Takahashi, Eiichi

    1976-01-01

    The possibility of in vitro binding between proteins of rice shoots and germanium (Ge) was investigated. The proteins in mixtures of aqueous extracts of rice shoots and radioactive germanium ( 68 GeO 2 ) were fractionated. The binding of radioactivity to the proteins was observed even after 5 successive fractionation steps from the original mixtures. At the final fractionation step using polyacrylamide gel electrophoresis, a constant proportionality between protein concentration and associated radioactivity was found in most samples although not all. These results indicate that the binding of 68 Ge to proteins is not due to the simple adsorption by proteins. (auth.)

  20. CXCL4 is a novel nickel-binding protein and augments nickel allergy.

    Science.gov (United States)

    Kuroishi, T; Bando, K; Tanaka, Y; Shishido, K; Kinbara, M; Ogawa, T; Muramoto, K; Endo, Y; Sugawara, S

    2017-08-01

    Nickel (Ni) is the most frequent metal allergen and induces a TH 1 -dependent type-IV allergy. Although Ni 2+ is considered to bind to endogenous proteins, it currently remains unclear whether these Ni-binding proteins are involved in Ni allergy in vivo. We previously reported the adjuvant effects of lipopolysaccharide (LPS) in a Ni allergy mouse model. As LPS induces a number of inflammatory mediators, we hypothesized that Ni-binding protein(s) are also induced by LPS. The objective of this study was to purify and identify Ni-binding protein(s) from serum taken from LPS-injected mice (referred as LPS serum) and examined the augmenting effects of these Ni-binding protein(s) on Ni allergy in an in vivo model. BALB/cA mice were sensitized with an i.p. injection of NiCl 2 and LPS. Ten days after sensitization, mice were challenged with NiCl 2 by an i.d. injection into ear pinnae. Ni-binding protein(s) were purified by Ni-affinity column chromatography and gel filtration. Lipopolysaccharide serum, but not serum taken from saline-injected mice, augmented ear swelling induced by Ni-allergic inflammation. Ni-binding, but not non-binding fraction, purified from LPS serum augmented Ni-allergic inflammation. Mass spectrometry and Western blotting detected CXCL4 in the active fraction. A batch analysis with Ni-sepharose and a surface plasmon resonance analysis revealed direct binding between CXCL4 and Ni 2+ . Recombinant CXCL4 augmented Ni-allergic inflammation and exerted adjuvant effects at the sensitization phase. These results indicate that CXCL4 is a novel Ni-binding protein that augments Ni allergy at the elicitation and sensitization phases. This is the first study to demonstrate that the Ni-binding protein augments Ni allergy in vivo. © 2017 John Wiley & Sons Ltd.

  1. Development of radioimmunoassay for prolactin binding protein

    International Nuclear Information System (INIS)

    Raikar, R.S.; Sheth, A.R.

    1982-01-01

    Using a homogenous prolactin binding protein (PBP) preparations from rat seminal vesicle secretion, a sensitive and specific radioimmunoassay (RIA) for PBP has been developed. The assay was highly specific and showed no cross-reaction with other protein hormones from various species. The antiserum had an affinity constant (Ka) of 2.66 x 10 10 M -1 . The assay sensitivity was in the range of 0.5-1.0 ng of pure PBP per assay tube and the intra- and inter-assay coefficients of variations were 6-8% and 12-14.5% respectively. The overall recovery of PBP to the rat seminal vesicle secretion was 96.8%. Using this RIA, PBP levels in various biological fluids and reproductive tissues were measured. Azoospermic human semen contained significantly higher levels of PBP than normospermic semen. The seminal vesicle of rat exhibited the highest concentration of PBP. Administration of antiserum to PBP to mature male rats resulted in a significant reduction in the weight of ventral prostrate and serum prolactin levels were significantly elevated in these animals suggesting that the antibody raised against the PBP was capable of blocking prolactin receptors. (author)

  2. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    International Nuclear Information System (INIS)

    Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya; Apicella, Michael A.; Ramaswamy, S.

    2014-01-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states

  3. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    Energy Technology Data Exchange (ETDEWEB)

    Gangi Setty, Thanuja [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Cho, Christine [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Govindappa, Sowmya [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Apicella, Michael A. [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Ramaswamy, S., E-mail: ramas@instem.res.in [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India)

    2014-07-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

  4. Distribution in rat tissues of modulator-binding protein of particulate nature

    International Nuclear Information System (INIS)

    Sobue, K.; Muramoto, Y.; Kakiuchi, S.; Yamazaki, R.

    1979-01-01

    Studies on Ca 2+ -activatable cyclic nucleotide phosphodiesterase led to the discovery of a protein modulator that is required for the activation of this enzyme by Ca 2+ . Later, this protein has been shown to cause the Ca 2+ -dependent activation of several enzymes that include phosphodiesterase, adenylate cyclase, a protein kinase from muscles, phosphorylase b kinase, actomyosin ATPase, membranous ATPase from erythrocytes and nerve synapses. Thus, modulator protein appears to be an intracellular mediator of actions of Ca 2+ . The present work shows the distribution of this particulate modulator-binding component in rat tissues. This paper also describes the labeling of modulator protein with tritium without deteriorating its biological activities and application of this 3 H-modulator protein to the determination of the Ca ++ dependent binding of modulator protein with membranous protein. This technique proves to be useful in studying enzymes or proteins whose functions are regulated by Ca ++ /modulator protein system. (Auth.)

  5. The BRCT domain is a phospho-protein binding domain.

    Science.gov (United States)

    Yu, Xiaochun; Chini, Claudia Christiano Silva; He, Miao; Mer, Georges; Chen, Junjie

    2003-10-24

    The carboxyl-terminal domain (BRCT) of the Breast Cancer Gene 1 (BRCA1) protein is an evolutionarily conserved module that exists in a large number of proteins from prokaryotes to eukaryotes. Although most BRCT domain-containing proteins participate in DNA-damage checkpoint or DNA-repair pathways, or both, the function of the BRCT domain is not fully understood. We show that the BRCA1 BRCT domain directly interacts with phosphorylated BRCA1-Associated Carboxyl-terminal Helicase (BACH1). This specific interaction between BRCA1 and phosphorylated BACH1 is cell cycle regulated and is required for DNA damage-induced checkpoint control during the transition from G2 to M phase of the cell cycle. Further, we show that two other BRCT domains interact with their respective physiological partners in a phosphorylation-dependent manner. Thirteen additional BRCT domains also preferentially bind phospho-peptides rather than nonphosphorylated control peptides. These data imply that the BRCT domain is a phospho-protein binding domain involved in cell cycle control.

  6. Partial characterization of GTP-binding proteins in Neurospora

    International Nuclear Information System (INIS)

    Hasunuma, K.; Miyamoto-Shinohara, Y.; Furukawa, K.

    1987-01-01

    Six fractions of GTP-binding proteins separated by gel filtration of a mycelial extract containing membrane components of Neurospora crassa were partially characterized. [ 35 S]GTP gamma S bound to GTP-binding protein was assayed by repeated treatments with a Norit solution and centrifugation. The binding of [ 35 S]GTP gamma S to GTP-binding proteins was competitively prevented in the presence of 0.1 to 1 mM GTP but not in the presence of ATP. These GTP-binding proteins fractionated by the gel column had Km values of 20, 7, 4, 4, 80 and 2 nM. All six fractions of these GTP-binding proteins showed the capacity to be ADP-ribosylated by pertussis toxin

  7. SFS, a Novel Fibronectin-Binding Protein from Streptococcus equi, Inhibits the Binding between Fibronectin and Collagen

    Science.gov (United States)

    Lindmark, Hans; Guss, Bengt

    1999-01-01

    The obligate parasitic bacterium Streptococcus equi subsp. equi is the causative agent of strangles, a serious disease of the upper respiratory tract in horses. In this study we have, using shotgun phage display, cloned from S. equi subsp. equi and characterized a gene, called sfs, encoding a protein termed SFS, representing a new type of fibronectin (Fn)-binding protein. The sfs gene was found to be present in all 50 isolates of S. equi subsp. equi tested and in 41 of 48 S. equi subsp. zooepidemicus isolates tested. The sfs gene is down-regulated during growth in vitro compared to fnz, a previously characterized gene encoding an Fn-binding protein from S. equi subsp. zooepidemicus. Sequence comparisons revealed no similarities to previously characterized Fn-binding proteins, but high scores were obtained against collagen. Besides similarity due to the high content of glycine, serine, and proline residues present in both proteins, there was a nine-residue motif present both in collagen and in the Fn-binding domain of SFS. By searching the Oklahoma S. pyogenes database, we found that this motif is also present in a potential cell surface protein from S. pyogenes. Protein SFS was found to inhibit the binding between Fn and collagen in a concentration-dependent way. PMID:10225899

  8. Effects of single-stranded DNA binding proteins on primer extension by telomerase.

    Science.gov (United States)

    Cohen, Shlomit; Jacob, Eyal; Manor, Haim

    2004-08-12

    We present a biochemical analysis of the effects of three single-stranded DNA binding proteins on extension of oligonucleotide primers by the Tetrahymena telomerase. One of them, a human protein designated translin, which was shown to specifically bind the G-rich Tetrahymena and human telomeric repeats, slightly stimulated the primer extension reactions at molar ratios of translin/primer of primers, rather than by a direct interaction of this protein with telomerase. A second protein, the general human single-stranded DNA binding protein Replication Protein A (RPA), similarly affected the primer extension by telomerase, even though its mode of binding to DNA differs from that of translin. A third protein, the E. coli single-stranded DNA binding protein (SSB), whose binding to DNA is highly cooperative, caused more substantial stimulation and inhibition at the lower and the higher molar ratios of SSB/primer, respectively. Both telomere-specific and general single-stranded DNA binding proteins are found in living cells in telomeric complexes. Based on our data, we propose that these proteins may exert either stimulatory or inhibitory effects on intracellular telomerases, depending on their local concentrations. Copyright 2004 Elsevier B.V.

  9. Collagen-binding proteins of Streptococcus mutans and related streptococci.

    Science.gov (United States)

    Avilés-Reyes, A; Miller, J H; Lemos, J A; Abranches, J

    2017-04-01

    The ability of Streptococcus mutans to interact with collagen through the expression of collagen-binding proteins (CBPs) bestows this oral pathogen with an alternative to the sucrose-dependent mechanism of colonization classically attributed to caries development. Based on the abundance and distribution of collagen throughout the human body, stringent adherence to this molecule grants S. mutans with the opportunity to establish infection at different host sites. Surface proteins, such as SpaP, WapA, Cnm and Cbm, have been shown to bind collagen in vitro, and it has been suggested that these molecules play a role in colonization of oral and extra-oral tissues. However, robust collagen binding is not achieved by all strains of S. mutans, particularly those that lack Cnm or Cbm. These observations merit careful dissection of the contribution from these different CBPs towards tissue colonization and virulence. In this review, we will discuss the current understanding of mechanisms used by S. mutans and related streptococci to colonize collagenous tissues, and the possible contribution of CBPs to infections in different sites of the host. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  10. The Collagen Binding Proteins of Streptococcus mutans and Related Streptococci

    Science.gov (United States)

    Avilés-Reyes, Alejandro; Miller, James H.; Lemos, José A.; Abranches, Jacqueline

    2016-01-01

    Summary The ability of Streptococcus mutans to interact with collagen through the expression of collagen-binding proteins (CBPs) bestows this oral pathogen with an alternative to the sucrose-dependent mechanism of colonization classically attributed to caries development. Based on the abundance and distribution of collagen throughout the human body, stringent adherence to this molecule grants S. mutans with the opportunity to establish infection at different host sites. Surface proteins, such as SpaP, WapA, Cnm and Cbm, have been shown to bind collagen in vitro, and it has been suggested that these molecules play a role in colonization of oral and extra-oral tissues. However, robust collagen binding is not achieved by all strains of S. mutans, particularly those that lack Cnm or Cbm. These observations merit careful dissection of the contribution from these different CBPs towards tissue colonization and virulence. In this review, we will discuss the current understanding of mechanisms utilized by S. mutans and related streptococci to colonize collagenous tissues, and the possible contribution of CBPs to infections in different sites of the host. PMID:26991416

  11. Enhanced expression of a calcium-dependent protein kinase from ...

    Indian Academy of Sciences (India)

    Among the downstream targets of calcium in plants, calcium-dependent protein kinases (CDPKs) form an interesting class of kinases which are activated by calcium binding. They have been implicated in a diverse array of responses to hormonal and environmental stimuli. In order to dissect the role of CDPKs in the moss ...

  12. Enhanced expression of a calcium-dependent protein kinase

    Indian Academy of Sciences (India)

    Among the downstream targets of calcium in plants, calcium-dependent protein kinases (CDPKs) form an interesting class of kinases which are activated by calcium binding. They have been implicated in a diverse array of responses to hormonal and environmental stimuli. In order to dissect the role of CDPKs in the moss ...

  13. Translation initiation mediated by nuclear cap-binding protein complex.

    Science.gov (United States)

    Ryu, Incheol; Kim, Yoon Ki

    2017-04-01

    In mammals, cap-dependent translation of mRNAs is initiated by two distinct mechanisms: cap-binding complex (CBC; a heterodimer of CBP80 and 20)-dependent translation (CT) and eIF4E-dependent translation (ET). Both translation initiation mechanisms share common features in driving cap- dependent translation; nevertheless, they can be distinguished from each other based on their molecular features and biological roles. CT is largely associated with mRNA surveillance such as nonsense-mediated mRNA decay (NMD), whereas ET is predominantly involved in the bulk of protein synthesis. However, several recent studies have demonstrated that CT and ET have similar roles in protein synthesis and mRNA surveillance. In a subset of mRNAs, CT preferentially drives the cap-dependent translation, as ET does, and ET is responsible for mRNA surveillance, as CT does. In this review, we summarize and compare the molecular features of CT and ET with a focus on the emerging roles of CT in translation. [BMB Reports 2017; 50(4): 186-193].

  14. Actin binding proteins, spermatid transport and spermiation*

    Science.gov (United States)

    Qian, Xiaojing; Mruk, Dolores D.; Cheng, Yan-Ho; Tang, Elizabeth I.; Han, Daishu; Lee, Will M.; Wong, Elissa W. P.; Cheng, C. Yan

    2014-01-01

    The transport of germ cells across the seminiferous epithelium is composed of a series of cellular events during the epithelial cycle essential to the completion of spermatogenesis. Without the timely transport of spermatids during spermiogenesis, spermatozoa that are transformed from step 19 spermatids in the rat testis fail to reach the luminal edge of the apical compartment and enter the tubule lumen at spermiation, thereby entering the epididymis for further maturation. Step 19 spermatids and/or sperms that remain in the epithelium will be removed by the Sertoli cell via phagocytosis to form phagosomes and be degraded by lysosomes, leading to subfertility and/or infertility. However, the biology of spermatid transport, in particular the final events that lead to spermiation remain elusive. Based on recent data in the field, we critically evaluate the biology of spermiation herein by focusing on the actin binding proteins (ABPs) that regulate the organization of actin microfilaments at the Sertoli-spermatid interface, which is crucial for spermatid transport during this event. The hypothesis we put forth herein also highlights some specific areas of research that can be pursued by investigators in the years to come. PMID:24735648

  15. Interleukin-18 and interleukin-18 Binding Protein

    Directory of Open Access Journals (Sweden)

    Charles eDinarello

    2013-10-01

    Full Text Available Interleukin-18 (IL 18 is a member of the IL 1 family of cytokines. Increasing reports have expanded the role of IL 18 in mediating inflammation in animal models of disease using IL 18 deficient mice, neutralization of IL 18 or deficiency in the IL 18 receptor alpha chain. Similar to IL 1β, IL 18 is synthesized as an inactive precursor requiering processing by caspase 1 into an active cytokine but unlike IL 1β, the IL 18 precursor is constitutively present in nearly all cells in healthy humans and animals. The activity of IL 18 is balanced by the presence of a high-affinity naturally occuring IL 18 binding protein (IL 18BP. In humans, disease increased disease severity can be associated with an imbalance of IL 18 to IL 18BP such that the levels of free IL 18 are elevated in the circulation. A role for IL 18 has been implicated in several autoimmune diseases, myocardial function, emphysema, metabolic syndromes, psoriasis, inflammatory bowel disease, hemophagocytic syndromes, macrophage activation syndrome, sepsis and acute kidney injury, although in some diseases, IL 18 is protective. IL 18 plays a major role in the production of interferon-g from natural killer cells. The IL 18BP has been used safely in humans and clinical trials of IL 18BP as well as neutralizing anti-IL 18 antibodies are in clinical trials. This review updates the biology of IL 18 as well as its role in human disease

  16. IGF Binding Protein-5 Induces Cell Senescence

    Directory of Open Access Journals (Sweden)

    Fumihiro Sanada

    2018-02-01

    Full Text Available Cellular senescence is the complex process of deterioration that drives the aging of an organism, resulting in the progressive loss of organ function and eventually phenotypic aging. Senescent cells undergo irreversible growth arrest, usually by inducing telomere shortening. Alternatively, senescence may also occur prematurely in response to various stress stimuli, such as oxidative stress, DNA damage, or activated oncogenes. Recently, it has been shown that IGF binding protein-5 (IGFBP-5 with the induction of the tumor suppressor p53 is upregulated during cellular senescence. This mechanism mediates interleukin-6/gp130-induced premature senescence in human fibroblasts, irradiation-induced premature senescence in human endothelial cells (ECs, and replicative senescence in human ECs independent of insulin-like growth factor I (IGF-I and IGF-II. Additionally, a link between IGFBP-5, hyper-coagulation, and inflammation, which occur with age, has been implicated. Thus, IGFBP-5 seems to play decisive roles in controlling cell senescence and cell inflammation. In this review, we describe the accumulating evidence for this role of IGFBP-5 including our new finding.

  17. Immobilized purified folate-binding protein: binding characteristics and use for quantifying folate in erythrocytes

    International Nuclear Information System (INIS)

    Hansen, S.I.; Holm, J.; Nexo, E.

    1987-01-01

    Purified folate-binding protein from cow's milk was immobilized on monodisperse polymer particles (Dynospheres) activated by rho-toluenesulfonyl chloride. Leakage from the spheres was less than 0.1%, and the binding properties were similar to those of the soluble protein with regard to dissociation, pH optimum for binding pteroylglutamic acid, and specificity for binding various folate derivatives. We used the immobilized folate-binding protein as binding protein in an isotope-dilution assay for quantifying folate in erythrocytes. The detection limit was 50 nmol/L and the CV over a six-month period was 2.3% (means = 1.25 mumol/L, n = 15). The reference interval, for folate measured in erythrocytes of 43 blood donors, was 0.4-1.5 mumol/L

  18. Specificity of the second binding protein of the peptide ABC-transporter (Dpp) of Lactococcus lactis IL1403

    NARCIS (Netherlands)

    Sanz, Y; Toldra, F; Renault, P; Poolman, B

    2003-01-01

    The genome sequence of Lactococcus lactis IL1403 revealed the presence of a putative peptide-binding protein-dependent ABC-transporter (Dpp). The genes for two peptide-binding proteins (dppA and dppP) precede the membrane components, which include two transmembrane protein genes (dppB and dppC) and

  19. Ligand Binding Domain Protein in Tetracycline-Inducible Expression

    African Journals Online (AJOL)

    Purpose: To investigate tetracycline-inducible expression system for producing clinically usable, highquality liver X receptor ligand-binding domain recombinant protein. Methods: In this study, we have expressed and purified the recombinant liver X receptor β-ligand binding domain proteins in E. coli using a tetracycline ...

  20. SCOWLP classification: Structural comparison and analysis of protein binding regions

    Directory of Open Access Journals (Sweden)

    Anders Gerd

    2008-01-01

    Full Text Available Abstract Background Detailed information about protein interactions is critical for our understanding of the principles governing protein recognition mechanisms. The structures of many proteins have been experimentally determined in complex with different ligands bound either in the same or different binding regions. Thus, the structural interactome requires the development of tools to classify protein binding regions. A proper classification may provide a general view of the regions that a protein uses to bind others and also facilitate a detailed comparative analysis of the interacting information for specific protein binding regions at atomic level. Such classification might be of potential use for deciphering protein interaction networks, understanding protein function, rational engineering and design. Description Protein binding regions (PBRs might be ideally described as well-defined separated regions that share no interacting residues one another. However, PBRs are often irregular, discontinuous and can share a wide range of interacting residues among them. The criteria to define an individual binding region can be often arbitrary and may differ from other binding regions within a protein family. Therefore, the rational behind protein interface classification should aim to fulfil the requirements of the analysis to be performed. We extract detailed interaction information of protein domains, peptides and interfacial solvent from the SCOWLP database and we classify the PBRs of each domain family. For this purpose, we define a similarity index based on the overlapping of interacting residues mapped in pair-wise structural alignments. We perform our classification with agglomerative hierarchical clustering using the complete-linkage method. Our classification is calculated at different similarity cut-offs to allow flexibility in the analysis of PBRs, feature especially interesting for those protein families with conflictive binding regions

  1. Accurate prediction of peptide binding sites on protein surfaces.

    Directory of Open Access Journals (Sweden)

    Evangelia Petsalaki

    2009-03-01

    Full Text Available Many important protein-protein interactions are mediated by the binding of a short peptide stretch in one protein to a large globular segment in another. Recent efforts have provided hundreds of examples of new peptides binding to proteins for which a three-dimensional structure is available (either known experimentally or readily modeled but where no structure of the protein-peptide complex is known. To address this gap, we present an approach that can accurately predict peptide binding sites on protein surfaces. For peptides known to bind a particular protein, the method predicts binding sites with great accuracy, and the specificity of the approach means that it can also be used to predict whether or not a putative or predicted peptide partner will bind. We used known protein-peptide complexes to derive preferences, in the form of spatial position specific scoring matrices, which describe the binding-site environment in globular proteins for each type of amino acid in bound peptides. We then scan the surface of a putative binding protein for sites for each of the amino acids present in a peptide partner and search for combinations of high-scoring amino acid sites that satisfy constraints deduced from the peptide sequence. The method performed well in a benchmark and largely agreed with experimental data mapping binding sites for several recently discovered interactions mediated by peptides, including RG-rich proteins with SMN domains, Epstein-Barr virus LMP1 with TRADD domains, DBC1 with Sir2, and the Ago hook with Argonaute PIWI domain. The method, and associated statistics, is an excellent tool for predicting and studying binding sites for newly discovered peptides mediating critical events in biology.

  2. Genes encoding calmodulin-binding proteins in the Arabidopsis genome

    Science.gov (United States)

    Reddy, Vaka S.; Ali, Gul S.; Reddy, Anireddy S N.

    2002-01-01

    Analysis of the recently completed Arabidopsis genome sequence indicates that approximately 31% of the predicted genes could not be assigned to functional categories, as they do not show any sequence similarity with proteins of known function from other organisms. Calmodulin (CaM), a ubiquitous and multifunctional Ca(2+) sensor, interacts with a wide variety of cellular proteins and modulates their activity/function in regulating diverse cellular processes. However, the primary amino acid sequence of the CaM-binding domain in different CaM-binding proteins (CBPs) is not conserved. One way to identify most of the CBPs in the Arabidopsis genome is by protein-protein interaction-based screening of expression libraries with CaM. Here, using a mixture of radiolabeled CaM isoforms from Arabidopsis, we screened several expression libraries prepared from flower meristem, seedlings, or tissues treated with hormones, an elicitor, or a pathogen. Sequence analysis of 77 positive clones that interact with CaM in a Ca(2+)-dependent manner revealed 20 CBPs, including 14 previously unknown CBPs. In addition, by searching the Arabidopsis genome sequence with the newly identified and known plant or animal CBPs, we identified a total of 27 CBPs. Among these, 16 CBPs are represented by families with 2-20 members in each family. Gene expression analysis revealed that CBPs and CBP paralogs are expressed differentially. Our data suggest that Arabidopsis has a large number of CBPs including several plant-specific ones. Although CaM is highly conserved between plants and animals, only a few CBPs are common to both plants and animals. Analysis of Arabidopsis CBPs revealed the presence of a variety of interesting domains. Our analyses identified several hypothetical proteins in the Arabidopsis genome as CaM targets, suggesting their involvement in Ca(2+)-mediated signaling networks.

  3. Antigenic and structural conservation of herpesvirus DNA-binding proteins.

    Science.gov (United States)

    Littler, E; Yeo, J; Killington, R A; Purifoy, D J; Powell, K L

    1981-10-01

    Previously, we have shown a common antigen of several herpesviruses (pseudorabies virus, equine abortion virus and bovine mammillitis virus) to be antigenically related to the major DNA-binding proteins of herpes simplex virus types 1 and 2. In this study we have purified the cross-reacting polypeptide from cells infected with pseudorabies virus, equine abortion virus and bovine mammillitis virus and shown the cross-reacting protein to be a major DNA-binding protein for each virus. Tryptic peptide analysis of the cross-reacting DNA-binding proteins of all five viruses has shown structural similarities. The proteins thus were shown to share common antigenic sites, to have similar biological properties and to have a highly conserved amino acid sequence. This unexpected similarity between proteins from diverse herpes viruses suggests an essential and fundamental role of the major DNA-binding protein in herpes virus replication.

  4. LRRK2 kinase activity is dependent on LRRK2 GTP binding capacity but independent of LRRK2 GTP binding.

    Directory of Open Access Journals (Sweden)

    Jean-Marc Taymans

    Full Text Available Leucine rich repeat kinase 2 (LRRK2 is a Parkinson's disease (PD gene that encodes a large multidomain protein including both a GTPase and a kinase domain. GTPases often regulate kinases within signal transduction cascades, where GTPases act as molecular switches cycling between a GTP bound "on" state and a GDP bound "off" state. It has been proposed that LRRK2 kinase activity may be increased upon GTP binding at the LRRK2 Ras of complex proteins (ROC GTPase domain. Here we extensively test this hypothesis by measuring LRRK2 phosphorylation activity under influence of GDP, GTP or non-hydrolyzable GTP analogues GTPγS or GMPPCP. We show that autophosphorylation and lrrktide phosphorylation activity of recombinant LRRK2 protein is unaltered by guanine nucleotides, when co-incubated with LRRK2 during phosphorylation reactions. Also phosphorylation activity of LRRK2 is unchanged when the LRRK2 guanine nucleotide binding pocket is previously saturated with various nucleotides, in contrast to the greatly reduced activity measured for the guanine nucleotide binding site mutant T1348N. Interestingly, when nucleotides were incubated with cell lysates prior to purification of LRRK2, kinase activity was slightly enhanced by GTPγS or GMPPCP compared to GDP, pointing to an upstream guanine nucleotide binding protein that may activate LRRK2 in a GTP-dependent manner. Using metabolic labeling, we also found that cellular phosphorylation of LRRK2 was not significantly modulated by nucleotides, although labeling is significantly reduced by guanine nucleotide binding site mutants. We conclude that while kinase activity of LRRK2 requires an intact ROC-GTPase domain, it is independent of GDP or GTP binding to ROC.

  5. Sialic Acid Binding Properties of Soluble Coronavirus Spike (S1) Proteins: Differences between Infectious Bronchitis Virus and Transmissible Gastroenteritis Virus

    Science.gov (United States)

    Shahwan, Katarina; Hesse, Martina; Mork, Ann-Kathrin; Herrler, Georg; Winter, Christine

    2013-01-01

    The spike proteins of a number of coronaviruses are able to bind to sialic acids present on the cell surface. The importance of this sialic acid binding ability during infection is, however, quite different. We compared the spike protein of transmissible gastroenteritis virus (TGEV) and the spike protein of infectious bronchitis virus (IBV). Whereas sialic acid is the only receptor determinant known so far for IBV, TGEV requires interaction with its receptor aminopeptidase N to initiate infection of cells. Binding tests with soluble spike proteins carrying an IgG Fc-tag revealed pronounced differences between these two viral proteins. Binding of the IBV spike protein to host cells was in all experiments sialic acid dependent, whereas the soluble TGEV spike showed binding to APN but had no detectable sialic acid binding activity. Our results underline the different ways in which binding to sialoglycoconjugates is mediated by coronavirus spike proteins. PMID:23896748

  6. Sialic Acid Binding Properties of Soluble Coronavirus Spike (S1 Proteins: Differences between Infectious Bronchitis Virus and Transmissible Gastroenteritis Virus

    Directory of Open Access Journals (Sweden)

    Christine Winter

    2013-07-01

    Full Text Available The spike proteins of a number of coronaviruses are able to bind to sialic acids present on the cell surface. The importance of this sialic acid binding ability during infection is, however, quite different. We compared the spike protein of transmissible gastroenteritis virus (TGEV and the spike protein of infectious bronchitis virus (IBV. Whereas sialic acid is the only receptor determinant known so far for IBV, TGEV requires interaction with its receptor aminopeptidase N to initiate infection of cells. Binding tests with soluble spike proteins carrying an IgG Fc-tag revealed pronounced differences between these two viral proteins. Binding of the IBV spike protein to host cells was in all experiments sialic acid dependent, whereas the soluble TGEV spike showed binding to APN but had no detectable sialic acid binding activity. Our results underline the different ways in which binding to sialoglycoconjugates is mediated by coronavirus spike proteins.

  7. Carbene footprinting accurately maps binding sites in protein-ligand and protein-protein interactions

    Science.gov (United States)

    Manzi, Lucio; Barrow, Andrew S.; Scott, Daniel; Layfield, Robert; Wright, Timothy G.; Moses, John E.; Oldham, Neil J.

    2016-11-01

    Specific interactions between proteins and their binding partners are fundamental to life processes. The ability to detect protein complexes, and map their sites of binding, is crucial to understanding basic biology at the molecular level. Methods that employ sensitive analytical techniques such as mass spectrometry have the potential to provide valuable insights with very little material and on short time scales. Here we present a differential protein footprinting technique employing an efficient photo-activated probe for use with mass spectrometry. Using this methodology the location of a carbohydrate substrate was accurately mapped to the binding cleft of lysozyme, and in a more complex example, the interactions between a 100 kDa, multi-domain deubiquitinating enzyme, USP5 and a diubiquitin substrate were located to different functional domains. The much improved properties of this probe make carbene footprinting a viable method for rapid and accurate identification of protein binding sites utilizing benign, near-UV photoactivation.

  8. A brave new world of RNA-binding proteins.

    Science.gov (United States)

    Hentze, Matthias W; Castello, Alfredo; Schwarzl, Thomas; Preiss, Thomas

    2018-01-17

    RNA-binding proteins (RBPs) are typically thought of as proteins that bind RNA through one or multiple globular RNA-binding domains (RBDs) and change the fate or function of the bound RNAs. Several hundred such RBPs have been discovered and investigated over the years. Recent proteome-wide studies have more than doubled the number of proteins implicated in RNA binding and uncovered hundreds of additional RBPs lacking conventional RBDs. In this Review, we discuss these new RBPs and the emerging understanding of their unexpected modes of RNA binding, which can be mediated by intrinsically disordered regions, protein-protein interaction interfaces and enzymatic cores, among others. We also discuss the RNA targets and molecular and cellular functions of the new RBPs, as well as the possibility that some RBPs may be regulated by RNA rather than regulate RNA.

  9. Rate Constants and Mechanisms of Protein-Ligand Binding.

    Science.gov (United States)

    Pang, Xiaodong; Zhou, Huan-Xiang

    2017-05-22

    Whereas protein-ligand binding affinities have long-established prominence, binding rate constants and binding mechanisms have gained increasing attention in recent years. Both new computational methods and new experimental techniques have been developed to characterize the latter properties. It is now realized that binding mechanisms, like binding rate constants, can and should be quantitatively determined. In this review, we summarize studies and synthesize ideas on several topics in the hope of providing a coherent picture of and physical insight into binding kinetics. The topics include microscopic formulation of the kinetic problem and its reduction to simple rate equations; computation of binding rate constants; quantitative determination of binding mechanisms; and elucidation of physical factors that control binding rate constants and mechanisms.

  10. ICBP90, a Novel Methyl K9 H3 Binding Protein Linking Protein Ubiquitination with Heterochromatin Formation▿

    Science.gov (United States)

    Karagianni, Panagiota; Amazit, Larbi; Qin, Jun; Wong, Jiemin

    2008-01-01

    Methylation of histone H3 on lysine 9 is critical for diverse biological processes including transcriptional repression, heterochromatin formation, and X inactivation. The biological effects of histone methylation are thought to be mediated by effector proteins that recognize and bind to specific patterns of methylation. Using an unbiased in vitro biochemical approach, we have identified ICBP90, a transcription and cell cycle regulator, as a novel methyl K9 H3-specific binding protein. ICBP90 and its murine homologue Np95 are enriched in pericentric heterochromatin of interphase nuclei, and this localization is dependent on H3K9 methylation. Specific binding of ICBP90 to methyl K9 H3 depends on two functional domains, a PHD (plant homeodomain) finger that defines the binding specificity and an SRA (SET- and RING-associated) domain that promotes binding activity. Furthermore, we present evidence that ICBP90 is required for proper heterochromatin formation in mammalian cells. PMID:17967883

  11. ICBP90, a novel methyl K9 H3 binding protein linking protein ubiquitination with heterochromatin formation.

    Science.gov (United States)

    Karagianni, Panagiota; Amazit, Larbi; Qin, Jun; Wong, Jiemin

    2008-01-01

    Methylation of histone H3 on lysine 9 is critical for diverse biological processes including transcriptional repression, heterochromatin formation, and X inactivation. The biological effects of histone methylation are thought to be mediated by effector proteins that recognize and bind to specific patterns of methylation. Using an unbiased in vitro biochemical approach, we have identified ICBP90, a transcription and cell cycle regulator, as a novel methyl K9 H3-specific binding protein. ICBP90 and its murine homologue Np95 are enriched in pericentric heterochromatin of interphase nuclei, and this localization is dependent on H3K9 methylation. Specific binding of ICBP90 to methyl K9 H3 depends on two functional domains, a PHD (plant homeodomain) finger that defines the binding specificity and an SRA (SET- and RING-associated) domain that promotes binding activity. Furthermore, we present evidence that ICBP90 is required for proper heterochromatin formation in mammalian cells.

  12. The Cobalamin-binding Protein in Zebrafish is an Intermediate Between the Three Cobalamin-binding Proteins in Human

    DEFF Research Database (Denmark)

    Greibe, Eva Holm; Fedosov, Sergey; Nexø, Ebba

    2012-01-01

    In humans, three soluble extracellular cobalamin-binding proteins; transcobalamin (TC), intrinsic factor (IF), and haptocorrin (HC), are involved in the uptake and transport of cobalamin. In this study, we investigate a cobalamin-binding protein from zebrafish (Danio rerio) and summarize current...

  13. The Movable Type Method Applied to Protein-Ligand Binding.

    Science.gov (United States)

    Zheng, Zheng; Ucisik, Melek N; Merz, Kenneth M

    2013-12-10

    Accurately computing the free energy for biological processes like protein folding or protein-ligand association remains a challenging problem. Both describing the complex intermolecular forces involved and sampling the requisite configuration space make understanding these processes innately difficult. Herein, we address the sampling problem using a novel methodology we term "movable type". Conceptually it can be understood by analogy with the evolution of printing and, hence, the name movable type. For example, a common approach to the study of protein-ligand complexation involves taking a database of intact drug-like molecules and exhaustively docking them into a binding pocket. This is reminiscent of early woodblock printing where each page had to be laboriously created prior to printing a book. However, printing evolved to an approach where a database of symbols (letters, numerals, etc.) was created and then assembled using a movable type system, which allowed for the creation of all possible combinations of symbols on a given page, thereby, revolutionizing the dissemination of knowledge. Our movable type (MT) method involves the identification of all atom pairs seen in protein-ligand complexes and then creating two databases: one with their associated pairwise distant dependent energies and another associated with the probability of how these pairs can combine in terms of bonds, angles, dihedrals and non-bonded interactions. Combining these two databases coupled with the principles of statistical mechanics allows us to accurately estimate binding free energies as well as the pose of a ligand in a receptor. This method, by its mathematical construction, samples all of configuration space of a selected region (the protein active site here) in one shot without resorting to brute force sampling schemes involving Monte Carlo, genetic algorithms or molecular dynamics simulations making the methodology extremely efficient. Importantly, this method explores the free

  14. Calcium-binding proteins from human platelets

    International Nuclear Information System (INIS)

    Gogstad, G.O.; Krutnes, M.B.; Solum, N.O.

    1983-01-01

    Calcium-binding platelet proteins were examined by crossed immunoelectrophoresis of solubilized platelets against antibodies to whole platelets followed by incubation of the immunoplates with 45 Ca 2 + and autoradiography. When the immunoplates had been pretreated with EDTA at pH 9.0 in order to remove divalent cations, three immunoprecipitates were markedly labelled with 45 Ca 2 + . These corresponded to the glycoprotein IIb-IIIa complex, glycoprotein Ia and a presently unidentified antigen termed G18. These antigens were membrane-bound and surface-oriented. When an excess of EDTA was introduced in the incubation media the results revealed that the glycoprotein IIb-IIIa complex and antigen G18, but not glycoprotein Ia, contained sites with a stronger affinity for calcium than has EDTA at pH 7.4 Immunoprecipitates of the separate glycoproteins IIb and IIIa both bound calcium in the same manner as the glycoprotein IIb-IIIa complex. As another approach, platelet-rich plasma was incubated with 45 Ca 2 + prior to crossed immunoelectrophoresis of the solubilized platelets. A single immunoprecipitate was wekly labelled. This did not correspond to any of the immunoprecipitates which were visible after staining with Coomassie blue. The labelling of this antigen was markedly increased when the platelt-rich plasma had been preincubated with EDTA and in this case a weak labelling of the glycoprotein IIB-IIIa precipitate also became apparent. No increased incorporation of calcium occured in any of these immunoprecipitates when the platelets were aggregated with ADP in the presence of 45 Ca 2 + . (orig.)

  15. Identification of Cisplatin-Binding Proteins Using Agarose Conjugates of Platinum Compounds

    Science.gov (United States)

    Karasawa, Takatoshi; Sibrian-Vazquez, Martha; Strongin, Robert M.; Steyger, Peter S.

    2013-01-01

    Cisplatin is widely used as an antineoplastic drug, but its ototoxic and nephrotoxic side-effects, as well as the inherent or acquired resistance of some cancers to cisplatin, remain significant clinical problems. Cisplatin's selectivity in killing rapidly proliferating cancer cells is largely dependent on covalent binding to DNA via cisplatin's chloride sites that had been aquated. We hypothesized that cisplatin's toxicity in slowly proliferating or terminally differentiated cells is primarily due to drug-protein interactions, instead of drug-DNA binding. To identify proteins that bind to cisplatin, we synthesized two different platinum-agarose conjugates, one with two amino groups and another with two chlorides attached to platinum that are available for protein binding, and conducted pull-down assays using cochlear and kidney cells. Mass spectrometric analysis on protein bands after gel electrophoresis and Coomassie blue staining identified several proteins, including myosin IIA, glucose-regulated protein 94 (GRP94), heat shock protein 90 (HSP90), calreticulin, valosin containing protein (VCP), and ribosomal protein L5, as cisplatin-binding proteins. Future studies on the interaction of these proteins with cisplatin will elucidate whether these drug-protein interactions are involved in ototoxicity and nephrotoxicity, or contribute to tumor sensitivity or resistance to cisplatin treatment. PMID:23755301

  16. Identification of cisplatin-binding proteins using agarose conjugates of platinum compounds.

    Directory of Open Access Journals (Sweden)

    Takatoshi Karasawa

    Full Text Available Cisplatin is widely used as an antineoplastic drug, but its ototoxic and nephrotoxic side-effects, as well as the inherent or acquired resistance of some cancers to cisplatin, remain significant clinical problems. Cisplatin's selectivity in killing rapidly proliferating cancer cells is largely dependent on covalent binding to DNA via cisplatin's chloride sites that had been aquated. We hypothesized that cisplatin's toxicity in slowly proliferating or terminally differentiated cells is primarily due to drug-protein interactions, instead of drug-DNA binding. To identify proteins that bind to cisplatin, we synthesized two different platinum-agarose conjugates, one with two amino groups and another with two chlorides attached to platinum that are available for protein binding, and conducted pull-down assays using cochlear and kidney cells. Mass spectrometric analysis on protein bands after gel electrophoresis and Coomassie blue staining identified several proteins, including myosin IIA, glucose-regulated protein 94 (GRP94, heat shock protein 90 (HSP90, calreticulin, valosin containing protein (VCP, and ribosomal protein L5, as cisplatin-binding proteins. Future studies on the interaction of these proteins with cisplatin will elucidate whether these drug-protein interactions are involved in ototoxicity and nephrotoxicity, or contribute to tumor sensitivity or resistance to cisplatin treatment.

  17. Mechanosensitive kinetic preference of actin-binding protein to actin filament.

    Science.gov (United States)

    Inoue, Yasuhiro; Adachi, Taiji

    2016-04-01

    The kinetic preference of actin-binding proteins to actin filaments is altered by external forces on the filament. Such an altered kinetic preference is largely responsible for remodeling the actin cytoskeletal structure in response to intracellular forces. During remodeling, actin-binding proteins and actin filaments interact under isothermal conditions, because the cells are homeostatic. In such a temperature homeostatic state, we can rigorously and thermodynamically link the chemical potential of actin-binding proteins to stresses on the actin filaments. From this relationship, we can construct a physical model that explains the force-dependent kinetic preference of actin-binding proteins to actin filaments. To confirm the model, we have analyzed the mechanosensitive alternation of the kinetic preference of Arp2/3 and cofilin to actin filaments. We show that this model captures the qualitative responses of these actin-binding proteins to the forces, as observed experimentally. Moreover, our theoretical results demonstrate that, depending on the structural parameters of the binding region, actin-binding proteins can show different kinetic responses even to the same mechanical signal tension, in which the double-helix nature of the actin filament also plays a critical role in a stretch-twist coupling of the filament.

  18. Differential roles for Fos and Jun in DNA-binding: redox-dependent and independent functions.

    Science.gov (United States)

    Ng, L; Forrest, D; Curran, T

    1993-01-01

    The Fos and Jun family of transcription factors contain an invariant sequence motif lysine-cysteine-arginine (KCR) in the highly conserved DNA-binding region. Reduction of the cysteine residue is necessary to facilitate DNA-binding. Here, we examined the potential dual roles of the flanking lysine and arginine residues in influencing the redox reactivity of the cysteine and the DNA-binding activity of Fos and Jun. Two sets of Fos and Jun mutants were generated: the KCR and KSR series representing proteins capable of redox-dependent and redox-independent DNA-binding activity, respectively. Mutation of the lysine in Fos-Jun heterodimers had no obvious effect on the redox reactivity of the cysteine, suggesting that lysine is not essential in this respect. However, mutation of the arginine but not lysine, in both the KCR and the KSR series abolished DNA-binding activity. Thus, the arginine but not the lysine residue in the KCR motif is critical for both redox-dependent and redox-independent functions in DNA-binding. Surprisingly, the triple substitution, ISI, exhibited high levels of DNA-binding activity. This demonstrates that the effects of amino acid substitutions can be highly dependent on context and that non-basic amino acids can function efficiently in DNA-binding. Analysis of combinations of wild-type and mutant Fos and Jun proteins indicated that Fos was dominant in dictating the DNA-binding ability of Fos-Jun heterodimers. This suggests that the lysine and arginine residues in the KCR region of Fos are not equivalent to those in Jun and that they interact with DNA differently. Images PMID:8290340

  19. Further biochemical characterization of Mycobacterium leprae laminin-binding proteins

    Directory of Open Access Journals (Sweden)

    M.A.M. Marques

    2001-04-01

    Full Text Available It has been demonstrated that the alpha2 chain of laminin-2 present on the surface of Schwann cells is involved in the process of attachment of Mycobacterium leprae to these cells. Searching for M. leprae laminin-binding molecules, in a previous study we isolated and characterized the cationic proteins histone-like protein (Hlp and ribosomal proteins S4 and S5 as potential adhesins involved in M. leprae-Schwann cell interaction. Hlp was shown to bind alpha2-laminins and to greatly enhance the attachment of mycobacteria to ST88-14 Schwann cells. In the present study, we investigated the laminin-binding capacity of the ribosomal proteins S4 and S5. The genes coding for these proteins were PCR amplified and their recombinant products were shown to bind alpha2-laminins in overlay assays. However, when tested in ELISA-based assays and in adhesion assays with ST88-14 cells, in contrast to Hlp, S4 and S5 failed to bind laminin and act as adhesins. The laminin-binding property and adhesin capacity of two basic host-derived proteins were also tested, and only histones, but not cytochrome c, were able to increase bacterial attachment to ST88-14 cells. Our data suggest that the alanine/lysine-rich sequences shared by Hlp and eukaryotic H1 histones might be involved in the binding of these cationic proteins to laminin.

  20. Predicting binding within disordered protein regions to structurally characterised peptide-binding domains.

    Directory of Open Access Journals (Sweden)

    Waqasuddin Khan

    Full Text Available Disordered regions of proteins often bind to structured domains, mediating interactions within and between proteins. However, it is difficult to identify a priori the short disordered regions involved in binding. We set out to determine if docking such peptide regions to peptide binding domains would assist in these predictions.We assembled a redundancy reduced dataset of SLiM (Short Linear Motif containing proteins from the ELM database. We selected 84 sequences which had an associated PDB structures showing the SLiM bound to a protein receptor, where the SLiM was found within a 50 residue region of the protein sequence which was predicted to be disordered. First, we investigated the Vina docking scores of overlapping tripeptides from the 50 residue SLiM containing disordered regions of the protein sequence to the corresponding PDB domain. We found only weak discrimination of docking scores between peptides involved in binding and adjacent non-binding peptides in this context (AUC 0.58.Next, we trained a bidirectional recurrent neural network (BRNN using as input the protein sequence, predicted secondary structure, Vina docking score and predicted disorder score. The results were very promising (AUC 0.72 showing that multiple sources of information can be combined to produce results which are clearly superior to any single source.We conclude that the Vina docking score alone has only modest power to define the location of a peptide within a larger protein region known to contain it. However, combining this information with other knowledge (using machine learning methods clearly improves the identification of peptide binding regions within a protein sequence. This approach combining docking with machine learning is primarily a predictor of binding to peptide-binding sites, and is not intended as a predictor of specificity of binding to particular receptors.

  1. Guardian of Genetic Messenger-RNA-Binding Proteins

    Directory of Open Access Journals (Sweden)

    Antje Anji

    2016-01-01

    Full Text Available RNA in cells is always associated with RNA-binding proteins that regulate all aspects of RNA metabolism including RNA splicing, export from the nucleus, RNA localization, mRNA turn-over as well as translation. Given their diverse functions, cells express a variety of RNA-binding proteins, which play important roles in the pathologies of a number of diseases. In this review we focus on the effect of alcohol on different RNA-binding proteins and their possible contribution to alcohol-related disorders, and discuss the role of these proteins in the development of neurological diseases and cancer. We further discuss the conventional methods and newer techniques that are employed to identify RNA-binding proteins.

  2. Binding Properties of General Odorant Binding Proteins from the Oriental Fruit Moth, Grapholita molesta (Busck (Lepidoptera: Tortricidae.

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    Guangwei Li

    Full Text Available The oriental fruit moth Grapholita molesta is a host-switching pest species. The adults highly depend on olfactory cues in locating optimal host plants and oviposition sites. Odorant binding proteins (OBPs are thought to be responsible for recognizing and transporting hydrophobic odorants across the aqueous sensillum lymph to stimulate the odorant receptors (ORs within the antennal sensilla and activate the olfactory signal transduction pathway. Exploring the physiological function of these OBPs could facilitate understanding insect chemical communications.Two antennae-specific general OBPs (GOBPs of G. molesta were expressed and purified in vitro. The binding affinities of G. molesta GOBP1 and 2 (GmolGOBP1 and 2 for sex pheromone components and host plant volatiles were measured by fluorescence ligand-binding assays. The distribution of GmolGOBP1 and 2 in the antennal sensillum were defined by whole mount fluorescence immunohistochemistry (WM-FIHC experiments. The binding sites of GmolGOBP2 were predicted using homology modeling, molecular docking and site-directed mutagenesis. Both GmolGOBP1 and 2 are housing in sensilla basiconica and with no differences in male and female antennae. Recombinant GmolGOBP1 (rGmolGOBP1 exhibited broad binding properties towards host plant volatiles and sex pheromone components; rGmolGOBP2 could not effectively bind host plant volatiles but showed specific binding affinity with a minor sex pheromone component dodecanol. We chose GmolGOBP2 and dodecanol for further homology modeling, molecular docking, and site-directed mutagenesis. Binding affinities of mutants demonstrated that Thr9 was the key binding site and confirmed dodecanol bonding to protein involves a hydrogen bond. Combined with the pH effect on binding affinities of rGmolGOBP2, ligand binding and release of GmolGOBP2 were related to a pH-dependent conformational transition.Two rGmolGOBPs exhibit different binding characteristics for tested ligands. r

  3. Binding Properties of General Odorant Binding Proteins from the Oriental Fruit Moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae).

    Science.gov (United States)

    Li, Guangwei; Chen, Xiulin; Li, Boliao; Zhang, Guohui; Li, Yiping; Wu, Junxiang

    2016-01-01

    The oriental fruit moth Grapholita molesta is a host-switching pest species. The adults highly depend on olfactory cues in locating optimal host plants and oviposition sites. Odorant binding proteins (OBPs) are thought to be responsible for recognizing and transporting hydrophobic odorants across the aqueous sensillum lymph to stimulate the odorant receptors (ORs) within the antennal sensilla and activate the olfactory signal transduction pathway. Exploring the physiological function of these OBPs could facilitate understanding insect chemical communications. Two antennae-specific general OBPs (GOBPs) of G. molesta were expressed and purified in vitro. The binding affinities of G. molesta GOBP1 and 2 (GmolGOBP1 and 2) for sex pheromone components and host plant volatiles were measured by fluorescence ligand-binding assays. The distribution of GmolGOBP1 and 2 in the antennal sensillum were defined by whole mount fluorescence immunohistochemistry (WM-FIHC) experiments. The binding sites of GmolGOBP2 were predicted using homology modeling, molecular docking and site-directed mutagenesis. Both GmolGOBP1 and 2 are housing in sensilla basiconica and with no differences in male and female antennae. Recombinant GmolGOBP1 (rGmolGOBP1) exhibited broad binding properties towards host plant volatiles and sex pheromone components; rGmolGOBP2 could not effectively bind host plant volatiles but showed specific binding affinity with a minor sex pheromone component dodecanol. We chose GmolGOBP2 and dodecanol for further homology modeling, molecular docking, and site-directed mutagenesis. Binding affinities of mutants demonstrated that Thr9 was the key binding site and confirmed dodecanol bonding to protein involves a hydrogen bond. Combined with the pH effect on binding affinities of rGmolGOBP2, ligand binding and release of GmolGOBP2 were related to a pH-dependent conformational transition. Two rGmolGOBPs exhibit different binding characteristics for tested ligands. rGmolGOBP1 has

  4. Binding Properties of General Odorant Binding Proteins from the Oriental Fruit Moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae)

    Science.gov (United States)

    Li, Guangwei; Chen, Xiulin; Li, Boliao; Zhang, Guohui; Li, Yiping; Wu, Junxiang

    2016-01-01

    Background The oriental fruit moth Grapholita molesta is a host-switching pest species. The adults highly depend on olfactory cues in locating optimal host plants and oviposition sites. Odorant binding proteins (OBPs) are thought to be responsible for recognizing and transporting hydrophobic odorants across the aqueous sensillum lymph to stimulate the odorant receptors (ORs) within the antennal sensilla and activate the olfactory signal transduction pathway. Exploring the physiological function of these OBPs could facilitate understanding insect chemical communications. Methodology/Principal Finding Two antennae-specific general OBPs (GOBPs) of G. molesta were expressed and purified in vitro. The binding affinities of G. molesta GOBP1 and 2 (GmolGOBP1 and 2) for sex pheromone components and host plant volatiles were measured by fluorescence ligand-binding assays. The distribution of GmolGOBP1 and 2 in the antennal sensillum were defined by whole mount fluorescence immunohistochemistry (WM-FIHC) experiments. The binding sites of GmolGOBP2 were predicted using homology modeling, molecular docking and site-directed mutagenesis. Both GmolGOBP1 and 2 are housing in sensilla basiconica and with no differences in male and female antennae. Recombinant GmolGOBP1 (rGmolGOBP1) exhibited broad binding properties towards host plant volatiles and sex pheromone components; rGmolGOBP2 could not effectively bind host plant volatiles but showed specific binding affinity with a minor sex pheromone component dodecanol. We chose GmolGOBP2 and dodecanol for further homology modeling, molecular docking, and site-directed mutagenesis. Binding affinities of mutants demonstrated that Thr9 was the key binding site and confirmed dodecanol bonding to protein involves a hydrogen bond. Combined with the pH effect on binding affinities of rGmolGOBP2, ligand binding and release of GmolGOBP2 were related to a pH-dependent conformational transition. Conclusion Two rGmolGOBPs exhibit different

  5. Characterization of a cocaine binding protein in human placenta

    International Nuclear Information System (INIS)

    Ahmed, M.S.; Zhou, D.H.; Maulik, D.; Eldefrawi, M.E.

    1990-01-01

    [ 3 H]-Cocaine binding sites are identified in human placental villus tissue plasma membranes. These binding sites are associated with a protein and show saturable and specific binding of [ 3 H]-cocaine with a high affinity site of 170 fmole/mg protein. The binding is lost with pretreatment with trypsin or heat. The membrane bound protein is solubilized with the detergent 3-(3-cholamidopropyl)dimethyl-ammonio-1-propane sulphonate (CHAPS) with retention of its saturable and specific binding of [ 3 H]-cocaine. The detergent-protein complex migrates on a sepharose CL-6B gel chromatography column as a protein with an apparent molecular weight of 75,900. The protein has an S 20,w value of 5.1. The binding of this protein to norcocaine, pseudococaine, nomifensine, imipramine, desipramine, amphetamine and dopamine indicates that it shares some, but not all, the properties of the brain cocaine receptor. The physiologic significance of this protein in human placenta is currently unclear

  6. Predicting nucleic acid binding interfaces from structural models of proteins.

    Science.gov (United States)

    Dror, Iris; Shazman, Shula; Mukherjee, Srayanta; Zhang, Yang; Glaser, Fabian; Mandel-Gutfreund, Yael

    2012-02-01

    The function of DNA- and RNA-binding proteins can be inferred from the characterization and accurate prediction of their binding interfaces. However, the main pitfall of various structure-based methods for predicting nucleic acid binding function is that they are all limited to a relatively small number of proteins for which high-resolution three-dimensional structures are available. In this study, we developed a pipeline for extracting functional electrostatic patches from surfaces of protein structural models, obtained using the I-TASSER protein structure predictor. The largest positive patches are extracted from the protein surface using the patchfinder algorithm. We show that functional electrostatic patches extracted from an ensemble of structural models highly overlap the patches extracted from high-resolution structures. Furthermore, by testing our pipeline on a set of 55 known nucleic acid binding proteins for which I-TASSER produces high-quality models, we show that the method accurately identifies the nucleic acids binding interface on structural models of proteins. Employing a combined patch approach we show that patches extracted from an ensemble of models better predicts the real nucleic acid binding interfaces compared with patches extracted from independent models. Overall, these results suggest that combining information from a collection of low-resolution structural models could be a valuable approach for functional annotation. We suggest that our method will be further applicable for predicting other functional surfaces of proteins with unknown structure. Copyright © 2011 Wiley Periodicals, Inc.

  7. New binding mode to TNF-alpha revealed by ubiquitin-based artificial binding protein.

    Directory of Open Access Journals (Sweden)

    Andreas Hoffmann

    Full Text Available A variety of approaches have been employed to generate binding proteins from non-antibody scaffolds. Utilizing a beta-sheet of the human ubiquitin for paratope creation we obtained binding proteins against tumor necrosis factor (TNF-alpha. The bioactive form of this validated pharmacological target protein is a non-covalently linked homo-trimer. This structural feature leads to the observation of a certain heterogeneity concerning the binding mode of TNF-alpha binding molecules, for instance in terms of monomer/trimer specificity. We analyzed a ubiquitin-based TNF-alpha binder, selected by ribosome display, with a particular focus on its mode of interaction. Using enzyme-linked immunosorbent assays, specific binding to TNF-alpha with nanomolar affinity was observed. In isothermal titration calorimetry we obtained comparable results regarding the affinity and detected an exothermic reaction with one ubiquitin-derived binding molecule binding one TNF-alpha trimer. Using NMR spectroscopy and other analytical methods the 1:3 stoichiometry could be confirmed. Detailed binding analysis showed that the interaction is affected by the detergent Tween-20. Previously, this phenomenon was reported only for one other type of alternative scaffold-derived binding proteins--designed ankyrin repeat proteins--without further investigation. As demonstrated by size exclusion chromatography and NMR spectroscopy, the presence of the detergent increases the association rate significantly. Since the special architecture of TNF-alpha is known to be modulated by detergents, the access to the recognized epitope is indicated to be restricted by conformational transitions within the target protein. Our results suggest that the ubiquitin-derived binding protein targets a new epitope on TNF-alpha, which differs from the epitopes recognized by TNF-alpha neutralizing antibodies.

  8. Analysis of the ligand binding properties of recombinant bovine liver-type fatty acid binding protein

    DEFF Research Database (Denmark)

    Rolf, B; Oudenampsen-Krüger, E; Börchers, T

    1995-01-01

    The coding part of the cDNA for bovine liver-type fatty acid binding protein (L-FABP) has been amplified by RT-PCR, cloned and used for the construction of an Escherichia coli (E. coli) expression system. The recombinant protein made up to 25% of the soluble E. coli proteins and could be isolated...

  9. The protein inhibitor of nNOS (PIN/DLC1/LC8) binding does not inhibit the NADPH-dependent heme reduction in nNOS, a key step in NO synthesis.

    Science.gov (United States)

    Parhad, Swapnil S; Jaiswal, Deepa; Ray, Krishanu; Mazumdar, Shyamalava

    2016-03-25

    The neuronal nitric oxide synthase (nNOS) is an essential enzyme involved in the synthesis of nitric oxide (NO), a potent neurotransmitter. Although previous studies have indicated that the dynein light chain 1 (DLC1) binding to nNOS could inhibit the NO synthesis, the claim is challenged by contradicting reports. Thus, the mechanism of nNOS regulation remained unclear. nNOS has a heme-bearing, Cytochrome P450 core, and the functional enzyme is a dimer. The electron flow from NADPH to Flavin, and finally to the heme of the paired nNOS subunit within a dimer, is facilitated upon calmodulin (CaM) binding. Here, we show that DLC1 binding to nNOS-CaM complex does not affect the electron transport from the reductase to the oxygenase domain. Therefore, it cannot inhibit the rate of NADPH-dependent heme reduction in nNOS, which results in l-Arginine oxidation. Also, the NO release activity does not decrease with increasing DLC1 concentration in the reaction mix, which further confirmed that DLC1 does not inhibit nNOS activity. These findings suggest that the DLC1 binding may have other implications for the nNOS function in the cell. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Conformational Dynamics of the Receptor Protein Galactose/Glucose Binding Protein

    Science.gov (United States)

    Messina, Troy; Talaga, David

    2006-03-01

    We have performed time-correlated single photon counting (TCSPC) anisotropy and Stokes Shift measurements on bulk solutions of galactose/glucose binding protein. Site-directed mutagenesis was used to provide a single cysteine amino acid near the sugar-binding center of the protein (glutamine 26 to cysteine -- Q26C). The cysteine was covalently labeled with the environmentally-sensitive fluorophore acrylodan, and a long-lived ruthenium complex was covalently attached to the N-terminus to provide a fluorescent reference. The TCSPC data were analyzed using global convolute-and-compare fitting routines over the entire glucose titration and temperature range to provide minimal reduced chi-squared values and the highest time resolution possible. Using a standard ligand-binding model, the resulting distributions show that the closed (ligand-bound) conformation exists even at zero glucose concentration. At 20^oC, the relative abundance of this conformation is as high as 40%. The temperature dependence of this conformational study will be discussed and related to the ligand-binding free energy surface.

  11. The protein inhibitor of nNOS (PIN/DLC1/LC8) binding does not inhibit the NADPH-dependent heme reduction in nNOS, a key step in NO synthesis

    International Nuclear Information System (INIS)

    Parhad, Swapnil S.; Jaiswal, Deepa; Ray, Krishanu; Mazumdar, Shyamalava

    2016-01-01

    The neuronal nitric oxide synthase (nNOS) is an essential enzyme involved in the synthesis of nitric oxide (NO), a potent neurotransmitter. Although previous studies have indicated that the dynein light chain 1 (DLC1) binding to nNOS could inhibit the NO synthesis, the claim is challenged by contradicting reports. Thus, the mechanism of nNOS regulation remained unclear. nNOS has a heme-bearing, Cytochrome P450 core, and the functional enzyme is a dimer. The electron flow from NADPH to Flavin, and finally to the heme of the paired nNOS subunit within a dimer, is facilitated upon calmodulin (CaM) binding. Here, we show that DLC1 binding to nNOS-CaM complex does not affect the electron transport from the reductase to the oxygenase domain. Therefore, it cannot inhibit the rate of NADPH-dependent heme reduction in nNOS, which results in L-Arginine oxidation. Also, the NO release activity does not decrease with increasing DLC1 concentration in the reaction mix, which further confirmed that DLC1 does not inhibit nNOS activity. These findings suggest that the DLC1 binding may have other implications for the nNOS function in the cell. - Highlights: • The effect of interaction of nNOS with DLC1 has been debatable with contradicting reports in literature. • Purified DLC1 has no effect on electron transport between reductase and oxygenase domain of purified nNOS-CaM. • The NO release activity of nNOS was not altered by DLC1, supporting that DLC1 does not inhibit the enzyme. • These findings suggest that the DLC1 binding may have other implications for the nNOS function in the cell.

  12. The protein inhibitor of nNOS (PIN/DLC1/LC8) binding does not inhibit the NADPH-dependent heme reduction in nNOS, a key step in NO synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Parhad, Swapnil S. [Tata Institute of Fundamental Research (TIFR), Homi Bhabha Road, Mumbai 400 005 (India); Jaiswal, Deepa [Tata Institute of Fundamental Research (TIFR), Homi Bhabha Road, Mumbai 400 005 (India); TIFR Centre for Interdisciplinary Sciences, 21 Brundavan Colony, Narsingi, Hyderabad 500075 (India); Ray, Krishanu, E-mail: krishanu@tifr.res.in [Tata Institute of Fundamental Research (TIFR), Homi Bhabha Road, Mumbai 400 005 (India); Mazumdar, Shyamalava, E-mail: shyamal@tifr.res.in [Tata Institute of Fundamental Research (TIFR), Homi Bhabha Road, Mumbai 400 005 (India)

    2016-03-25

    The neuronal nitric oxide synthase (nNOS) is an essential enzyme involved in the synthesis of nitric oxide (NO), a potent neurotransmitter. Although previous studies have indicated that the dynein light chain 1 (DLC1) binding to nNOS could inhibit the NO synthesis, the claim is challenged by contradicting reports. Thus, the mechanism of nNOS regulation remained unclear. nNOS has a heme-bearing, Cytochrome P450 core, and the functional enzyme is a dimer. The electron flow from NADPH to Flavin, and finally to the heme of the paired nNOS subunit within a dimer, is facilitated upon calmodulin (CaM) binding. Here, we show that DLC1 binding to nNOS-CaM complex does not affect the electron transport from the reductase to the oxygenase domain. Therefore, it cannot inhibit the rate of NADPH-dependent heme reduction in nNOS, which results in L-Arginine oxidation. Also, the NO release activity does not decrease with increasing DLC1 concentration in the reaction mix, which further confirmed that DLC1 does not inhibit nNOS activity. These findings suggest that the DLC1 binding may have other implications for the nNOS function in the cell. - Highlights: • The effect of interaction of nNOS with DLC1 has been debatable with contradicting reports in literature. • Purified DLC1 has no effect on electron transport between reductase and oxygenase domain of purified nNOS-CaM. • The NO release activity of nNOS was not altered by DLC1, supporting that DLC1 does not inhibit the enzyme. • These findings suggest that the DLC1 binding may have other implications for the nNOS function in the cell.

  13. The interrelationship between ligand binding and self-association of the folate binding protein

    DEFF Research Database (Denmark)

    Holm, Jan; Schou, Christian; Babol, Linnea N.

    2011-01-01

    The folate binding protein (FBP) regulates homeostasis and intracellular trafficking of folic acid, a vitamin of decisive importance in cell division and growth. We analyzed whether interrelationship between ligand binding and self-association of FBP plays a significant role in the physiology of ...

  14. Studies of the silencing of Baculovirus DNA binding protein

    NARCIS (Netherlands)

    Quadt, I.; Lent, van J.W.M.; Knebel-Morsdorf, D.

    2007-01-01

    Baculovirus DNA binding protein (DBP) binds preferentially single-stranded DNA in vitro and colocalizes with viral DNA replication sites. Here, its putative role as viral replication factor has been addressed by RNA interference. Silencing of DBP in Autographa californica multiple

  15. Synergistic inhibition of the intrinsic factor X activation by protein S and C4b-binding protein

    NARCIS (Netherlands)

    Koppelman, S. J.; van't Veer, C.; Sixma, J. J.; Bouma, B. N.

    1995-01-01

    The complement protein C4b-binding protein plays an important role in the regulation of the protein C anticoagulant pathway. C4b-binding protein can bind to protein S, thereby inhibiting the cofactor activity of protein S for activated protein C. In this report, we describe a new role for

  16. Identification of Putative Vero Cell Protein(s) that Bind Specifically to ...

    African Journals Online (AJOL)

    Results: The 45 KDa, 43 KDa and 30 KDa plasma membrane proteins were identified as viral envelope targets. Competitive binding assay showed these proteins competing with dengue virus binding. MTT assay indicate that viability of vero cells increases in cultures pretreated with 45 KDa, 43 KDa and 30 KDa proteins ...

  17. Protein kinase Calpha contains two activator binding sites that bind phorbol esters and diacylglycerols with opposite affinities.

    Science.gov (United States)

    Slater, S J; Ho, C; Kelly, M B; Larkin, J D; Taddeo, F J; Yeager, M D; Stubbs, C D

    1996-03-01

    Based on marked differences in the enzymatic properties of diacylglycerols compared with phorbol ester-activated protein kinase C (PKC), we recently proposed that activation induced by these compounds may not be equivalent (Slater, S. J., Kelly, M. B., Taddeo, F. J., Rubin, E., and Stubbs, C. D. (1994) J. Biol. Chem. 269, 17160-17165). In the present study, direct evidence is provided showing that phorbol esters and diacylglycerols bind simultaneously to PKC alpha. Using a novel binding assay employing the fluorescent phorbol ester, sapintoxin-D (SAPD), evidence for two sites of high and low affinity was obtained. Thus, both binding and activation dose-response curves for SAPD were double sigmoidal, which was also observed for dose-dependent activation by the commonly used phorbol ester, 4beta-12-O-tetradecanoylphorbol-13-acetate (TPA). TPA removed high affinity SAPD binding and also competed for the low affinity site. By contrast with TPA, low affinity binding of SAPD was inhibited by sn-1,2-dioleoylglycerol (DAG), while binding to the high affinity site was markedly enhanced. Again contrasting with both TPA and DAG, the potent PKC activator, bryostatin-I (B-I), inhibited SAPD binding to its high affinity site, while low affinity binding was unaffected. Based on these findings, a model for PKC activation is proposed in which binding of one activator to the low affinity site allosterically promotes binding of a second activator to the high affinity site, resulting in an enhanced level of activity. Overall, the results provide direct evidence that PKCalpha contains two distinct binding sites, with affinities that differ for each activator in the order: DAG > phorbol ester > B-I and B-I > phorbol ester > DAG, respectively.

  18. Cell Context Dependent p53 Genome-Wide Binding Patterns and Enrichment at Repeats

    Science.gov (United States)

    Botcheva, Krassimira; McCorkle, Sean R.

    2014-01-01

    The p53 ability to elicit stress specific and cell type specific responses is well recognized, but how that specificity is established remains to be defined. Whether upon activation p53 binds to its genomic targets in a cell type and stress type dependent manner is still an open question. Here we show that the p53 binding to the human genome is selective and cell context-dependent. We mapped the genomic binding sites for the endogenous wild type p53 protein in the human cancer cell line HCT116 and compared them to those we previously determined in the normal cell line IMR90. We report distinct p53 genome-wide binding landscapes in two different cell lines, analyzed under the same treatment and experimental conditions, using the same ChIP-seq approach. This is evidence for cell context dependent p53 genomic binding. The observed differences affect the p53 binding sites distribution with respect to major genomic and epigenomic elements (promoter regions, CpG islands and repeats). We correlated the high-confidence p53 ChIP-seq peaks positions with the annotated human repeats (UCSC Human Genome Browser) and observed both common and cell line specific trends. In HCT116, the p53 binding was specifically enriched at LINE repeats, compared to IMR90 cells. The p53 genome-wide binding patterns in HCT116 and IMR90 likely reflect the different epigenetic landscapes in these two cell lines, resulting from cancer-associated changes (accumulated in HCT116) superimposed on tissue specific differences (HCT116 has epithelial, while IMR90 has mesenchymal origin). Our data support the model for p53 binding to the human genome in a highly selective manner, mobilizing distinct sets of genes, contributing to distinct pathways. PMID:25415302

  19. The pancreatic zymogen granule membrane protein, GP2, binds Escherichia coli type 1 Fimbriae

    Directory of Open Access Journals (Sweden)

    Lowe Anson W

    2009-07-01

    Full Text Available Abstract Background GP2 is the major membrane protein present in the pancreatic zymogen granule, and is cleaved and released into the pancreatic duct along with exocrine secretions. The function of GP2 is unknown. GP2's amino acid sequence is most similar to that of uromodulin, which is secreted by the kidney. Recent studies have demonstrated uromodulin binding to bacterial Type 1 fimbria. The fimbriae serve as adhesins to host receptors. The present study examines whether GP2 also shares similar binding properties to bacteria with Type 1 fimbria. Commensal and pathogenic bacteria, including E. coli and Salmonella, express type 1 fimbria. Methods An in vitro binding assay was used to assay the binding of recombinant GP2 to defined strains of E. coli that differ in their expression of Type 1 fimbria or its subunit protein, FimH. Studies were also performed to determine whether GP2 binding is dependent on the presence of mannose residues, which is a known determinant for FimH binding. Results GP2 binds E. coli that express Type 1 fimbria. Binding is dependent on GP2 glycosylation, and specifically the presence of mannose residues. Conclusion GP2 binds to Type 1 fimbria, a bacterial adhesin that is commonly expressed by members of the Enterobacteriacae family.

  20. Human GIP(3-30)NH inhibits G protein-dependent as well as G protein-independent signaling and is selective for the GIP receptor with high-affinity binding to primate but not rodent GIP receptors

    DEFF Research Database (Denmark)

    Gabe, Maria Buur Nordskov; Sparre-Ulrich, Alexander Hovard; Pedersen, Mie Fabricius

    2018-01-01

    antagonism with a pA2and Hill slope of 16.8 nM and 1.11 ± 0.02 in cAMP, 10.6 nM and 1.15 ± 0.05 in β-arrestin 1 recruitment, and 10.2 nM and 1.06 ± 0.05 in β-arrestin 2 recruitment. Efficient internalization of the GIPR was dependent on the presence of either β-arrestin 1 or 2. Moreover, GIP(3-30)NH2...

  1. Water-binding of protein particles

    NARCIS (Netherlands)

    Peters, J.P.C.M.

    2016-01-01

    As overweight and obesity become more prevalent in society, the demand for food products that can help maintain body weight increases. One way to make such products is by decreasing the protein and fat content through increasing the water content. This thesis describes the potential of protein

  2. Estrogen receptor diminishes DNA-binding activities of chicken GATA-1 and CACCC-binding proteins.

    Science.gov (United States)

    Holth, L T; Sun, J M; Coutts, A S; Murphy, L C; Davie, J R

    1997-12-01

    The estrogen receptor (ER) repressed erythroid differentiation and erythroid-specific gene expression. In this study, we investigated the effect of ER alpha (referred to throughout as ER) on DNA-binding activities of transcription factors involved in regulating the expression of erythroid-specific genes, and, in particular, the histone H5 gene. Using electrophoretic mobility shift assays, we found that in the presence of rabbit reticulocyte lysate, human ER reduced the binding activities of chicken immature erythrocyte nuclear extracted proteins to GATA and CACCC sites in the H5 promoter and enhancer. In contrast, the binding activities of NF1 and Sp1 were not affected by ER. Binding of ER to an estrogen response element was enhanced by addition of rabbit reticulocyte lysate. This lysate was also necessary for ER to diminish the DNA-binding activity of GATA-1. These results suggest that additional factor(s) are necessary for full ER function. Both GATA-1 and CACCC-binding proteins are critical for the developmentally regulated expression of erythroid-specific genes. We hypothesize that interference in DNA-binding activities of GATA-1 and CACCC-binding proteins is the mechanism by which the ER inhibits regulation of these genes.

  3. Aspirin and salicylate bind to immunoglobulin heavy chain binding protein (BiP) and inhibit its ATPase activity in human fibroblasts.

    Science.gov (United States)

    Deng, W G; Ruan, K H; Du, M; Saunders, M A; Wu, K K

    2001-11-01

    Salicylic acid (SA), an endogenous signaling molecule of plants, possesses anti-inflammatory and anti-neoplastic actions in human. Its derivative, aspirin, is the most commonly used anti-inflammatory and analgesic drug. Aspirin and sodium salicylate (salicylates) have been reported to have multiple pharmacological actions. However, it is unclear whether they bind to a cellular protein. Here, we report for the first time the purification from human fibroblasts of a approximately 78 kDa salicylate binding protein with sequence identity to immunoglobulin heavy chain binding protein (BiP). The Kd values of SA binding to crude extract and to recombinant BiP were 45.2 and 54.6 microM, respectively. BiP is a chaperone protein containing a polypeptide binding site recognizing specific heptapeptide sequence and an ATP binding site. A heptapeptide with the specific sequence displaced SA binding in a concentration-dependent manner whereas a control heptapeptide did not. Salicylates inhibited ATPase activity stimulated by this specific heptapeptide but did not block ATP binding or induce BiP expression. These results indicate that salicylates bind specifically to the polypeptide binding site of BiP in human cells that may interfere with folding and transport of proteins important in inflammation.

  4. Anomalous DNA binding by E2 regulatory protein driven by spacer sequence TATA.

    Science.gov (United States)

    Xi, Zhiqun; Zhang, Yongli; Hegde, Rashmi S; Shakked, Zippora; Crothers, Donald M

    2010-06-01

    We have investigated the anomalously weak binding of human papillomavirus (HPV) regulatory protein E2 to a DNA target containing the spacer sequence TATA. Experiments in magnesium (Mg(2+)) and calcium (Ca(2+)) ion buffers revealed a marked reduction in cutting by DNase I at the CpG sequence in the protein-binding site 3' to the TATA spacer sequence, Studies of the cation dependence of DNA-E2 affinities showed that upon E2 binding the TATA sequence releases approximately twice as many Mg(2+) ions as the average of the other spacer sequences. Binding experiments for TATA spacer relative to ATAT showed that in potassium ion (K(+)) the E2 affinity of the two sequences is nearly equal, but the relative dissociation constant (K(d)) for TATA increases in the order K(+ )TATA relative to ATAT is independent of ion concentration, whereas for Mg(2+) the affinity for TATA drops sharply as ion concentration increases. Thus, ions of increasing positive charge density increasingly distort the E2 binding site, weakening the affinity for protein. In the case of Mg(2+), additional ions are bound to TATA that require displacement for protein binding. We suggest that the TATA sequence may bias the DNA structure towards a conformation that binds the protein relatively weakly.

  5. Mannan-binding lectin in cerebrospinal fluid: a leptomeningeal protein

    Directory of Open Access Journals (Sweden)

    Reiber Hansotto

    2012-08-01

    Full Text Available Abstract Background Mannan-binding lectin (MBL, a protein of the innate immune response is attracting increasing clinical interest, in particularly in relation to its deficiency. Due to its involvement in brain diseases, identifying the source of MBL in CSF is important. Analysis of cerebrospinal fluid (CSF can provide data that discriminates between blood-, brain-, and leptomeninges-derived proteins. To detect the source of MBL in CSF we need to consider three variables: the molecular size-dependent concentration gradient between CSF and blood, the variation in transfer between blood and CSF, and the CSF MBL concentration correlation with the albumin CSF/serum quotient (QAlb, i.e., with CSF flow rate. Methods MBL was assayed in samples of CSF and serum with an ELISA, coated with anti MBL antibodies. Routine parameters such as albumin-, immunoglobulin- CSF/serum quotients, oligoclonal IgG and cell count were used to characterize the patient groups. Groups comprised firstly, control patients without organic brain disease with normal CSF and normal barrier function and secondly, patients without inflammatory diseases but with increased QAlb, i.e. with a blood CSF barrier dysfunction. Results MBL concentration in CSF was at least five-fold higher than expected for a molecular-size-dependent passage from blood. Secondly, in a QIgM/QAlb quotient diagram (Reibergram 9/13 cases showed an intrathecal fraction in some cases over 80% of total CSF MBL concentration 3 The smaller inter-individual variation of MBL concentrations in CSF of the control group (CV = 66% compared to the MBL concentrations in serum (CV = 146% indicate an independent source of MBL in CSF. 4 The absolute MBL concentration in CSF increases with increasing QAlb. Among brain-derived proteins in CSF only the leptomeningeal proteins showed a (linear increase with decreasing CSF flow rate, neuronal and glial proteins are invariant to changes of QAlb. Conclusions MBL in CSF is

  6. N-Acetylgalactosaminyltransferase 14, a novel insulin-like growth factor binding protein-3 binding partner

    International Nuclear Information System (INIS)

    Wu, Chen; Yao, Guangyin; Zou, Minji; Chen, Guangyu; Wang, Min; Liu, Jingqian; Wang, Jiaxi; Xu, Donggang

    2007-01-01

    Insulin-like growth factor binding protein-3 (IGFBP-3) is known to inhibit cell proliferation and induce apoptosis in IGF-dependent and IGF-independent manners, but the mechanism underlying IGF-independent effects is not yet clear. In a yeast two-hybrid assay, IGFBP-3 was used as the bait to screen a human fetal liver cDNA library for it interactors that may potentially mediate IGFBP-3-regulated functions. N-Acetylgalactosaminyltransferase 14 (GalNAc-T14), a member of the GalNAc-Tases family, was identified as a novel IGFBP-3 binding partner. This interaction involved the ricin-type beta-trefoil domain of GalNAc-T14. The interaction between IGFBP-3 and GalNAc-T14 was reconfirmed in vitro and in vivo, using GST pull-down, co-immunoprecipitation and mammalian two-hybrid assays. Our findings may provide new clues for further study on the mechanism behind the IGF-independent effects of IGFBP-3 promoting apoptosis. The role of GalNAc-T14 as an intracellular mediator of the effects of IGFBP-3 need to be verified in future studies

  7. Ail Protein Binds Ninth Type III Fibronectin Repeat (9FNIII) within Central 120-kDa Region of Fibronectin to Facilitate Cell Binding by Yersinia pestis*

    Science.gov (United States)

    Tsang, Tiffany M.; Annis, Douglas S.; Kronshage, Malte; Fenno, Jesse T.; Usselman, Lisa D.; Mosher, Deane F.; Krukonis, Eric S.

    2012-01-01

    The Yersinia pestis adhesin molecule Ail interacts with the extracellular matrix protein fibronectin (Fn) on host cells to facilitate efficient delivery of cytotoxic Yop proteins, a process essential for plague virulence. A number of bacterial pathogens are known to bind to the N-terminal region of Fn, comprising type I Fn (FNI) repeats. Using proteolytically generated Fn fragments and purified recombinant Fn fragments, we demonstrated that Ail binds the centrally located 120-kDa fragment containing type III Fn (FNIII) repeats. A panel of monoclonal antibodies (mAbs) that recognize specific epitopes within the 120-kDa fragment demonstrated that mAb binding to 9FNIII blocks Ail-mediated bacterial binding to Fn. Epitopes of three mAbs that blocked Ail binding to Fn were mapped to a similar face of 9FNIII. Antibodies directed against 9FNIII also inhibited Ail-dependent cell binding activity, thus demonstrating the biological relevance of this Ail binding region on Fn. Bacteria expressing Ail on their surface could also bind a minimal fragment of Fn containing repeats 9–10FNIII, and this binding was blocked by a mAb specific for 9FNIII. These data demonstrate that Ail binds to 9FNIII of Fn and presents Fn to host cells to facilitate cell binding and delivery of Yops (cytotoxins of Y. pestis), a novel interaction, distinct from other bacterial Fn-binding proteins. PMID:22447929

  8. Ail protein binds ninth type III fibronectin repeat (9FNIII) within central 120-kDa region of fibronectin to facilitate cell binding by Yersinia pestis.

    Science.gov (United States)

    Tsang, Tiffany M; Annis, Douglas S; Kronshage, Malte; Fenno, Jesse T; Usselman, Lisa D; Mosher, Deane F; Krukonis, Eric S

    2012-05-11

    The Yersinia pestis adhesin molecule Ail interacts with the extracellular matrix protein fibronectin (Fn) on host cells to facilitate efficient delivery of cytotoxic Yop proteins, a process essential for plague virulence. A number of bacterial pathogens are known to bind to the N-terminal region of Fn, comprising type I Fn (FNI) repeats. Using proteolytically generated Fn fragments and purified recombinant Fn fragments, we demonstrated that Ail binds the centrally located 120-kDa fragment containing type III Fn (FNIII) repeats. A panel of monoclonal antibodies (mAbs) that recognize specific epitopes within the 120-kDa fragment demonstrated that mAb binding to (9)FNIII blocks Ail-mediated bacterial binding to Fn. Epitopes of three mAbs that blocked Ail binding to Fn were mapped to a similar face of (9)FNIII. Antibodies directed against (9)FNIII also inhibited Ail-dependent cell binding activity, thus demonstrating the biological relevance of this Ail binding region on Fn. Bacteria expressing Ail on their surface could also bind a minimal fragment of Fn containing repeats (9-10)FNIII, and this binding was blocked by a mAb specific for (9)FNIII. These data demonstrate that Ail binds to (9)FNIII of Fn and presents Fn to host cells to facilitate cell binding and delivery of Yops (cytotoxins of Y. pestis), a novel interaction, distinct from other bacterial Fn-binding proteins.

  9. Mutation of lysine residues in the nucleotide binding segments of the poliovirus RNA-dependent RNA polymerase.

    OpenAIRE

    Richards, O C; Baker, S; Ehrenfeld, E

    1996-01-01

    The poliovirus 3D RNA-dependent RNA polymerase contains two peptide segments previously shown to cross-link to nucleotide substrates via lysine residues. To determine which lysine residue(s) might be implicated in catalytic function, we engineered mutations to generate proteins with leucine residues substituted individually for each of the lysine residues in the NTP binding regions. These proteins were expressed in Escherichia coli and were examined for their abilities to bind nucleotides and...

  10. Relating the shape of protein binding sites to binding affinity profiles: is there an association?

    Directory of Open Access Journals (Sweden)

    Bitter István

    2010-10-01

    Full Text Available Abstract Background Various pattern-based methods exist that use in vitro or in silico affinity profiles for classification and functional examination of proteins. Nevertheless, the connection between the protein affinity profiles and the structural characteristics of the binding sites is still unclear. Our aim was to investigate the association between virtual drug screening results (calculated binding free energy values and the geometry of protein binding sites. Molecular Affinity Fingerprints (MAFs were determined for 154 proteins based on their molecular docking energy results for 1,255 FDA-approved drugs. Protein binding site geometries were characterized by 420 PocketPicker descriptors. The basic underlying component structure of MAFs and binding site geometries, respectively, were examined by principal component analysis; association between principal components extracted from these two sets of variables was then investigated by canonical correlation and redundancy analyses. Results PCA analysis of the MAF variables provided 30 factors which explained 71.4% of the total variance of the energy values while 13 factors were obtained from the PocketPicker descriptors which cumulatively explained 94.1% of the total variance. Canonical correlation analysis resulted in 3 statistically significant canonical factor pairs with correlation values of 0.87, 0.84 and 0.77, respectively. Redundancy analysis indicated that PocketPicker descriptor factors explain 6.9% of the variance of the MAF factor set while MAF factors explain 15.9% of the total variance of PocketPicker descriptor factors. Based on the salient structures of the factor pairs, we identified a clear-cut association between the shape and bulkiness of the drug molecules and the protein binding site descriptors. Conclusions This is the first study to investigate complex multivariate associations between affinity profiles and the geometric properties of protein binding sites. We found that

  11. Ubiquitin-binding proteins: similar, but different

    DEFF Research Database (Denmark)

    Andersen, Katrine M; Hofmann, Kay; Hartmann-Petersen, Rasmus

    2005-01-01

    of ubiquitin conjugation to endoplasmic reticulum degradation), UEV [ubiquitin E2 (ubiquitin-conjugating enzyme) variant] and NZF (nuclear protein localization gene 4 zinc finger) domain-containing proteins appear to have more specialized functions. Here we discuss functional and structural properties......Covalent modification of proteins with ubiquitin is a common regulatory mechanism in eukaryotic cells. Typically, ubiquitinated proteins are targeted for degradation by the 26 S proteasome. However, more recently the ubiquitin signal has also been connected with many other cell processes, including...... endocytosis, vesicle fusion, DNA repair and transcriptional silencing. Hence ubiquitination may be comparable with phosphorylation in its importance as an intracellular switch, controlling various signal-transduction pathways. Similar to the regulation of the extent of phosphorylation by kinases...

  12. Species specificity for HBsAg binding protein endonexin II

    NARCIS (Netherlands)

    deBruin, WCC; Leenders, WPJ; Moshage, H; vanHaelst, UJGM

    Background/Aims: Hepatitis B virus displays a distinct species and tissue tropism, Previously we have demonstrated that a human liver plasma membrane protein,vith a molecular weight of approximately 34 kiloDalton specifically binds to HBsAg. This protein was identified as endonexin II, a Ca2+

  13. Selectivity determinants of GPCR-G-protein binding

    DEFF Research Database (Denmark)

    Flock, Tilman; Hauser, Alexander S; Lund, Nadia

    2017-01-01

    The selective coupling of G-protein-coupled receptors (GPCRs) to specific G proteins is critical to trigger the appropriate physiological response. However, the determinants of selective binding have remained elusive. Here we reveal the existence of a selectivity barcode (that is, patterns of ami...

  14. Electrostatics, structure prediction, and the energy landscapes for protein folding and binding.

    Science.gov (United States)

    Tsai, Min-Yeh; Zheng, Weihua; Balamurugan, D; Schafer, Nicholas P; Kim, Bobby L; Cheung, Margaret S; Wolynes, Peter G

    2016-01-01

    While being long in range and therefore weakly specific, electrostatic interactions are able to modulate the stability and folding landscapes of some proteins. The relevance of electrostatic forces for steering the docking of proteins to each other is widely acknowledged, however, the role of electrostatics in establishing specifically funneled landscapes and their relevance for protein structure prediction are still not clear. By introducing Debye-Hückel potentials that mimic long-range electrostatic forces into the Associative memory, Water mediated, Structure, and Energy Model (AWSEM), a transferable protein model capable of predicting tertiary structures, we assess the effects of electrostatics on the landscapes of thirteen monomeric proteins and four dimers. For the monomers, we find that adding electrostatic interactions does not improve structure prediction. Simulations of ribosomal protein S6 show, however, that folding stability depends monotonically on electrostatic strength. The trend in predicted melting temperatures of the S6 variants agrees with experimental observations. Electrostatic effects can play a range of roles in binding. The binding of the protein complex KIX-pKID is largely assisted by electrostatic interactions, which provide direct charge-charge stabilization of the native state and contribute to the funneling of the binding landscape. In contrast, for several other proteins, including the DNA-binding protein FIS, electrostatics causes frustration in the DNA-binding region, which favors its binding with DNA but not with its protein partner. This study highlights the importance of long-range electrostatics in functional responses to problems where proteins interact with their charged partners, such as DNA, RNA, as well as membranes. © 2015 The Protein Society.

  15. Human vaccination against Plasmodium vivax Duffy-binding protein induces strain-transcending antibodies

    OpenAIRE

    Payne, Ruth O.; Silk, Sarah E.; Elias, Sean C.; Milne, Kathryn H.; Rawlinson, Thomas A.; Llewellyn, David; Shakri, A. Rushdi; Jin, Jing; Labb?, Genevi?ve M.; Edwards, Nick J.; Poulton, Ian D.; Roberts, Rachel; Farid, Ryan; J?rgensen, Thomas; Alanine, Daniel G.W.

    2017-01-01

    BACKGROUND: Plasmodium vivax is the most widespread human malaria geographically; however, no effective vaccine exists. Red blood cell invasion by the P. vivax merozoite depends on an interaction between the Duffy antigen receptor for chemokines (DARC) and region II of the parasite's Duffy-binding protein (PvDBP_RII). Naturally acquired binding-inhibitory antibodies against this interaction associate with clinical immunity, but it is unknown whether these responses can be induced by human vac...

  16. Acyl-CoA-binding protein/diazepam-binding inhibitor gene and pseudogenes

    DEFF Research Database (Denmark)

    Mandrup, S; Hummel, R; Ravn, S

    1992-01-01

    modulator of the GABAA receptor in brain membranes. ACBP/DBI, or proteolytically derived polypeptides of ACBP/DBI, have also been implicated in the control of steroidogenesis in mitochondria and glucose-stimulated insulin secretion. Thus, it appears that ACBP/DBI is a remarkable, versatile protein. Now we......Acyl-CoA-binding protein (ACBP) is a 10 kDa protein isolated from bovine liver by virtue of its ability to bind and induce the synthesis of medium-chain acyl-CoA esters. Surprisingly, it turned out to be identical to a protein named diazepam-binding Inhibitor (DBI) claimed to be an endogenous....... There is a remarkable correspondence between the structural modules of ACBP/DBI as determined by 1H nuclear magnetic resonance spectroscopy and the exon-intron architecture of the ACBP/DBI gene. Detailed analyses of transcription of the ACBP/DBI gene in brain and liver were performed to map transcription initiation...

  17. Detergent activation of the binding protein in the folate radioassay

    International Nuclear Information System (INIS)

    Hansen, S.I.; Holm, J.; Lyngbye, J.

    1982-01-01

    A minor cow's whey protein associated with β-lactoglobulin is used as binding protein in the competitive radioassay for serum and erythrocyte folate. Seeking to optimize the assay, we tested the performance of binder solutions of increasing purity. The folate binding protein was isolated from cow's whey by means of CM-Sepharose CL-6B cation-exchange chromatography, and further purified on a methotrexate-AH-Sepharose 4B affinity matrix. In contrast to β-lactoglobulin, the purified protein did not bind folate unless the detergents cetyltrimethylammonium (10 mmol/Ll) or Triton X-100 (1 g/L) were present. Such detergent activation was not needed in the presence of serum. There seems to be a striking analogy between these phenomena and the well-known reactivation of certain purified membrane-derived enzymes by surfactants

  18. RNA-Binding Domain Proteins in Kinetoplastids: a Comparative Analysis†

    Science.gov (United States)

    De Gaudenzi, Javier; Frasch, Alberto C.; Clayton, Christine

    2005-01-01

    RNA-binding proteins are important in many aspects of RNA processing, function, and destruction. One class of such proteins contains the RNA recognition motif (RRM), which consists of about 90 amino acid residues, including the canonical RNP1 octapeptide: (K/R)G(F/Y)(G/A)FVX(F/Y). We used a variety of homology searches to classify all of the RRM proteins of the three kinetoplastids Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major. All three organisms have similar sets of RRM-containing protein orthologues, suggesting common posttranscriptional processing and regulatory pathways. Of the 75 RRM proteins identified in T. brucei, only 13 had clear homologues in other eukaryotes, although 8 more could be given putative functional assignments. A comparison with the 18 RRM proteins of the obligate intracellular parasite Encephalitozoon cuniculi revealed just 3 RRM proteins which appear to be conserved at the primary sequence level throughout eukaryotic evolution: poly(A) binding protein, the rRNA-processing protein MRD1, and the nuclear cap binding protein. PMID:16339728

  19. Drug Promiscuity in PDB: Protein Binding Site Similarity Is Key.

    Science.gov (United States)

    Haupt, V Joachim; Daminelli, Simone; Schroeder, Michael

    2013-01-01

    Drug repositioning applies established drugs to new disease indications with increasing success. A pre-requisite for drug repurposing is drug promiscuity (polypharmacology) - a drug's ability to bind to several targets. There is a long standing debate on the reasons for drug promiscuity. Based on large compound screens, hydrophobicity and molecular weight have been suggested as key reasons. However, the results are sometimes contradictory and leave space for further analysis. Protein structures offer a structural dimension to explain promiscuity: Can a drug bind multiple targets because the drug is flexible or because the targets are structurally similar or even share similar binding sites? We present a systematic study of drug promiscuity based on structural data of PDB target proteins with a set of 164 promiscuous drugs. We show that there is no correlation between the degree of promiscuity and ligand properties such as hydrophobicity or molecular weight but a weak correlation to conformational flexibility. However, we do find a correlation between promiscuity and structural similarity as well as binding site similarity of protein targets. In particular, 71% of the drugs have at least two targets with similar binding sites. In order to overcome issues in detection of remotely similar binding sites, we employed a score for binding site similarity: LigandRMSD measures the similarity of the aligned ligands and uncovers remote local similarities in proteins. It can be applied to arbitrary structural binding site alignments. Three representative examples, namely the anti-cancer drug methotrexate, the natural product quercetin and the anti-diabetic drug acarbose are discussed in detail. Our findings suggest that global structural and binding site similarity play a more important role to explain the observed drug promiscuity in the PDB than physicochemical drug properties like hydrophobicity or molecular weight. Additionally, we find ligand flexibility to have a minor

  20. Mannose-Binding Lectin Binds to Amyloid Protein and Modulates Inflammation

    Directory of Open Access Journals (Sweden)

    Mykol Larvie

    2012-01-01

    Full Text Available Mannose-binding lectin (MBL, a soluble factor of the innate immune system, is a pattern recognition molecule with a number of known ligands, including viruses, bacteria, and molecules from abnormal self tissues. In addition to its role in immunity, MBL also functions in the maintenance of tissue homeostasis. We present evidence here that MBL binds to amyloid β peptides. MBL binding to other known carbohydrate ligands is calcium-dependent and has been attributed to the carbohydrate-recognition domain, a common feature of other C-type lectins. In contrast, we find that the features of MBL binding to Aβ are more similar to the reported binding characteristics of the cysteine-rich domain of the unrelated mannose receptor and therefore may involve the MBL cysteine-rich domain. Differences in MBL ligand binding may contribute to modulation of inflammatory response and may correlate with the function of MBL in processes such as coagulation and tissue homeostasis.

  1. MIT domain of Vps4 is a Ca2+-dependent phosphoinositide-binding domain.

    Science.gov (United States)

    Iwaya, Naoko; Takasu, Hirotoshi; Goda, Natsuko; Shirakawa, Masahiro; Tanaka, Toshiki; Hamada, Daizo; Hiroaki, Hidekazu

    2013-05-01

    The microtubule interacting and trafficking (MIT) domain is a small protein module that is conserved in proteins of diverged function, such as Vps4, spastin and sorting nexin 15 (SNX15). The molecular function of the MIT domain is protein-protein interaction, in which the domain recognizes peptides containing MIT-interacting motifs. Recently, we identified an evolutionarily related domain, 'variant' MIT domain at the N-terminal region of the microtubule severing enzyme katanin p60. We found that the domain was responsible for binding to microtubules and Ca(2+). Here, we have examined whether the authentic MIT domains also bind Ca(2+). We found that the loop between the first and second α-helices of the MIT domain binds a Ca(2+) ion. Furthermore, the MIT domains derived from Vps4b and SNX15a showed phosphoinositide-binding activities in a Ca(2+)-dependent manner. We propose that the MIT domain is a novel membrane-associating domain involved in endosomal trafficking.

  2. TATA-binding protein and the retinoblastoma gene product bind to overlapping epitopes on c-Myc and adenovirus E1A protein

    NARCIS (Netherlands)

    Hateboer, G.; Timmers, H.T.M.; Rustgi, A.K.; Billaud, Marc; Veer, L.J. Van 't; Bernards, R.A.

    1993-01-01

    Using a protein binding assay, we show that the amino-teminal 204 amino acids of the c-Myc protein interact di y with a key component of the basal p tdon factor TFID, the TATA box-binding protein (TBP). Essentialy the same region of the c-Myc protein alo binds the product of the retinoblatoma

  3. Effect of cobratoxin binding on the normal mode vibration within acetylcholine binding protein.

    Science.gov (United States)

    Bertaccini, Edward J; Lindahl, Erik; Sixma, Titia; Trudell, James R

    2008-04-01

    Recent crystal structures of the acetylcholine binding protein (AChBP) have revealed surprisingly small structural alterations upon ligand binding. Here we investigate the extent to which ligand binding may affect receptor dynamics. AChBP is a homologue of the extracellular component of ligand-gated ion channels (LGICs). We have previously used an elastic network normal-mode analysis to propose a gating mechanism for the LGICs and to suggest the effects of various ligands on such motions. However, the difficulties with elastic network methods lie in their inability to account for the modest effects of a small ligand or mutation on ion channel motion. Here, we report the successful application of an elastic network normal mode technique to measure the effects of large ligand binding on receptor dynamics. The present calculations demonstrate a clear alteration in the native symmetric motions of a protein due to the presence of large protein cobratoxin ligands. In particular, normal-mode analysis revealed that cobratoxin binding to this protein significantly dampened the axially symmetric motion of the AChBP that may be associated with channel gating in the full nAChR. The results suggest that alterations in receptor dynamics could be a general feature of ligand binding.

  4. Differential binding of heavy chain variable domain 3 antigen binding fragments to protein A chromatography resins.

    Science.gov (United States)

    Bach, Julia; Lewis, Nathaniel; Maggiora, Kathy; Gillespie, Alison J; Connell-Crowley, Lisa

    2015-08-28

    This work examines the binding of 15 different VH3 IgGs and their corresponding F(ab')2 fragments to two different protein A chromatography resins: MabSelect(®), which utilizes a recombinant protein A ligand, and MabSelect SuRe(®) (SuRe), which utilizes a tetrameric Z domain ligand. The results show that VH3 F(ab')2 fragments can exhibit a variety of binding behaviours for the two resins. Contrary to previously published data, a subset of these molecules show strong interaction with the Z domain of SuRe(®). Furthermore, the results show that sequence variability of residue 57 in the VH3 heavy chain CDR2 domain correlates with binding behaviour on MabSelect(®) and SuRe(®). Site-directed mutagenesis of this residue confers gain or loss of VH3 F(ab')2 binding to these resins in 3 mAbs, demonstrating that it plays a key role in both recombinant protein A and Z domain interaction. A fourth mAb with a longer CDR2 loop was not affected by mutation of residue 57, indicating that CDR2 domain length may alter the binding interface and lead to the involvement of other residues in protein A binding. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. CLIPZ: a database and analysis environment for experimentally determined binding sites of RNA-binding proteins.

    Science.gov (United States)

    Khorshid, Mohsen; Rodak, Christoph; Zavolan, Mihaela

    2011-01-01

    The stability, localization and translation rate of mRNAs are regulated by a multitude of RNA-binding proteins (RBPs) that find their targets directly or with the help of guide RNAs. Among the experimental methods for mapping RBP binding sites, cross-linking and immunoprecipitation (CLIP) coupled with deep sequencing provides transcriptome-wide coverage as well as high resolution. However, partly due to their vast volume, the data that were so far generated in CLIP experiments have not been put in a form that enables fast and interactive exploration of binding sites. To address this need, we have developed the CLIPZ database and analysis environment. Binding site data for RBPs such as Argonaute 1-4, Insulin-like growth factor II mRNA-binding protein 1-3, TNRC6 proteins A-C, Pumilio 2, Quaking and Polypyrimidine tract binding protein can be visualized at the level of the genome and of individual transcripts. Individual users can upload their own sequence data sets while being able to limit the access to these data to specific users, and analyses of the public and private data sets can be performed interactively. CLIPZ, available at http://www.clipz.unibas.ch, aims to provide an open access repository of information for post-transcriptional regulatory elements.

  6. Computational design of binding proteins to EGFR domain II.

    Directory of Open Access Journals (Sweden)

    Yoon Sup Choi

    Full Text Available We developed a process to produce novel interactions between two previously unrelated proteins. This process selects protein scaffolds and designs protein interfaces that bind to a surface patch of interest on a target protein. Scaffolds with shapes complementary to the target surface patch were screened using an exhaustive computational search of the human proteome and optimized by directed evolution using phage display. This method was applied to successfully design scaffolds that bind to epidermal growth factor receptor (EGFR domain II, the interface of EGFR dimerization, with high reactivity toward the target surface patch of EGFR domain II. One potential application of these tailor-made protein interactions is the development of therapeutic agents against specific protein targets.

  7. CNE, a collagen-binding protein of Streptococcus equi.

    Science.gov (United States)

    Lannergård, Jonas; Frykberg, Lars; Guss, Bengt

    2003-05-16

    Streptococcus equi subspecies equi is an important horse pathogenic bacterium causing a serious disease called strangles. Using bioinformatics we identified a gene denoted cne (gene encoding collagen-binding protein from S. equi) coding for a novel potential virulence factor of this species called protein CNE. The protein is composed of 657 amino acids and has the typical features found in cell surface-anchored proteins in Gram-positive bacteria. CNE displays amino acid sequence similarities to the previously well-studied collagen-binding protein CNA from Staphylococcus aureus, a proven virulence factor in septic arthritis. Based on similarity to CNA the structure of the mature CNE protein can be divided into an N-terminal A domain and a C-terminal B domain. The highest similarity between CNA and CNE is found in the A domains. The A domain in CNA is known to be the collagen-binding domain. Two parts of cne were amplified using polymerase chain reaction (PCR) and ligated into an expression vector, and recombinant CNE proteins were produced in Escherichia coli. The purified CNE proteins were shown to display collagen-binding activity in a Western ligand blot and to inhibit collagen binding to cells of subsp. equi and to CNE-coated microtitre wells. Furthermore, the A domain of CNE was sufficient for binding collagen, and was shown to compete for the same site on collagen as CNA in inhibition studies. Using PCR, the cne gene was detected in all studied strains of subsp. equi and S. equi subsp. zooepidemicus.

  8. SCM, the M Protein of Streptococcus canis Binds Immunoglobulin G.

    Science.gov (United States)

    Bergmann, Simone; Eichhorn, Inga; Kohler, Thomas P; Hammerschmidt, Sven; Goldmann, Oliver; Rohde, Manfred; Fulde, Marcus

    2017-01-01

    The M protein of Streptococcus canis (SCM) is a virulence factor and serves as a surface-associated receptor with a particular affinity for mini-plasminogen, a cleavage product of the broad-spectrum serine protease plasmin. Here, we report that SCM has an additional high-affinity immunoglobulin G (IgG) binding activity. The ability of a particular S. canis isolate to bind to IgG significantly correlates with a scm -positive phenotype, suggesting a dominant role of SCM as an IgG receptor. Subsequent heterologous expression of SCM in non-IgG binding S. gordonii and Western Blot analysis with purified recombinant SCM proteins confirmed its IgG receptor function. As expected for a zoonotic agent, the SCM-IgG interaction is species-unspecific, with a particular affinity of SCM for IgGs derived from human, cats, dogs, horses, mice, and rabbits, but not from cows and goats. Similar to other streptococcal IgG-binding proteins, the interaction between SCM and IgG occurs via the conserved Fc domain and is, therefore, non-opsonic. Interestingly, the interaction between SCM and IgG-Fc on the bacterial surface specifically prevents opsonization by C1q, which might constitute another anti-phagocytic mechanism of SCM. Extensive binding analyses with a variety of different truncated SCM fragments defined a region of 52 amino acids located in the central part of the mature SCM protein which is important for IgG binding. This binding region is highly conserved among SCM proteins derived from different S. canis isolates but differs significantly from IgG-Fc receptors of S. pyogenes and S. dysgalactiae sub. equisimilis , respectively. In summary, we present an additional role of SCM in the pathogen-host interaction of S. canis . The detailed analysis of the SCM-IgG interaction should contribute to a better understanding of the complex roles of M proteins in streptococcal pathogenesis.

  9. RNA-binding region of Macrobrachium rosenbergii nodavirus capsid protein.

    Science.gov (United States)

    Goh, Zee Hong; Mohd, Nur Azmina Syakirin; Tan, Soon Guan; Bhassu, Subha; Tan, Wen Siang

    2014-09-01

    White tail disease (WTD) kills prawn larvae and causes drastic losses to the freshwater prawn (Macrobrachium rosenbergii) industry. The main causative agent of WTD is Macrobrachium rosenbergii nodavirus (MrNV). The N-terminal end of the MrNV capsid protein is very rich in positively charged amino acids and is postulated to interact with RNA molecules. N-terminal and internal deletion mutagenesis revealed that the RNA-binding region is located at positions 20-29, where 80 % of amino acids are positively charged. Substitution of all these positively charged residues with alanine abolished the RNA binding. Mutants without the RNA-binding region still assembled into virus-like particles, suggesting that this region is not a part of the capsid assembly domain. This paper is, to the best of our knowledge, the first to report the specific RNA-binding region of MrNV capsid protein. © 2014 The Authors.

  10. Metal binding proteins, recombinant host cells and methods

    Science.gov (United States)

    Summers, Anne O.; Caguiat, Jonathan J.

    2004-06-15

    The present disclosure provides artificial heavy metal binding proteins termed chelons by the inventors. These chelons bind cadmium and/or mercuric ions with relatively high affinity. Also disclosed are coding sequences, recombinant DNA molecules and recombinant host cells comprising those recombinant DNA molecules for expression of the chelon proteins. In the recombinant host cells or transgenic plants, the chelons can be used to bind heavy metals taken up from contaminated soil, groundwater or irrigation water and to concentrate and sequester those ions. Recombinant enteric bacteria can be used within the gastrointestinal tracts of animals or humans exposed to toxic metal ions such as mercury and/or cadmium, where the chelon recombinantly expressed in chosen in accordance with the ion to be rededicated. Alternatively, the chelons can be immobilized to solid supports to bind and concentrate heavy metals from a contaminated aqueous medium including biological fluids.

  11. Twin-arginine-dependent translocation of folded proteins

    Science.gov (United States)

    Fröbel, Julia; Rose, Patrick; Müller, Matthias

    2012-01-01

    Twin-arginine translocation (Tat) denotes a protein transport pathway in bacteria, archaea and plant chloroplasts, which is specific for precursor proteins harbouring a characteristic twin-arginine pair in their signal sequences. Many Tat substrates receive cofactors and fold prior to translocation. For a subset of them, proofreading chaperones coordinate maturation and membrane-targeting. Tat translocases comprise two kinds of membrane proteins, a hexahelical TatC-type protein and one or two members of the single-spanning TatA protein family, called TatA and TatB. TatC- and TatA-type proteins form homo- and hetero-oligomeric complexes. The subunits of TatABC translocases are predominantly recovered from two separate complexes, a TatBC complex that might contain some TatA, and a homomeric TatA complex. TatB and TatC coordinately recognize twin-arginine signal peptides and accommodate them in membrane-embedded binding pockets. Advanced binding of the signal sequence to the Tat translocase requires the proton-motive force (PMF) across the membranes and might involve a first recruitment of TatA. When targeted in this manner, folded twin-arginine precursors induce homo-oligomerization of TatB and TatA. Ultimately, this leads to the formation of a transmembrane protein conduit that possibly consists of a pore-like TatA structure. The translocation step again is dependent on the PMF. PMID:22411976

  12. Retinoblastoma-binding protein 1 has an interdigitated double Tudor domain with DNA binding activity.

    Science.gov (United States)

    Gong, Weibin; Wang, Jinfeng; Perrett, Sarah; Feng, Yingang

    2014-02-21

    Retinoblastoma-binding protein 1 (RBBP1) is a tumor and leukemia suppressor that binds both methylated histone tails and DNA. Our previous studies indicated that RBBP1 possesses a Tudor domain, which cannot bind histone marks. In order to clarify the function of the Tudor domain, the solution structure of the RBBP1 Tudor domain was determined by NMR and is presented here. Although the proteins are unrelated, the RBBP1 Tudor domain forms an interdigitated double Tudor structure similar to the Tudor domain of JMJD2A, which is an epigenetic mark reader. This indicates the functional diversity of Tudor domains. The RBBP1 Tudor domain structure has a significant area of positively charged surface, which reveals a capability of the RBBP1 Tudor domain to bind nucleic acids. NMR titration and isothermal titration calorimetry experiments indicate that the RBBP1 Tudor domain binds both double- and single-stranded DNA with an affinity of 10-100 μM; no apparent DNA sequence specificity was detected. The DNA binding mode and key interaction residues were analyzed in detail based on a model structure of the Tudor domain-dsDNA complex, built by HADDOCK docking using the NMR data. Electrostatic interactions mediate the binding of the Tudor domain with DNA, which is consistent with NMR experiments performed at high salt concentration. The DNA-binding residues are conserved in Tudor domains of the RBBP1 protein family, resulting in conservation of the DNA-binding function in the RBBP1 Tudor domains. Our results provide further insights into the structure and function of RBBP1.

  13. T3: Targeted Proteomics of DNA-Binding Proteins

    OpenAIRE

    Nagore, Linda I.; Jarrett, Harry W.

    2015-01-01

    A technique that allows the inclusion of a specific DNA to enrich and direct proteomic identification of transcription factors (TF) while providing a route for high throughput screening on a single platform would be valuable in investigations of gene expression and regulation. Polyvinylpyrrolidone binds DNA avidly while binding negligible amounts of protein. This observation is used in a proof-of-concept method to enrich for TF by combining nuclear extract with a specific DNA sequence and imm...

  14. Transduction proteins of olfactory receptor cells: identification of guanine nucleotide binding proteins and protein kinase C

    International Nuclear Information System (INIS)

    Anholt, R.R.H.; Mumby, S.M.; Stoffers, D.A.; Girard, P.R.; Kuo, J.F.; Snyder, S.H.

    1987-01-01

    The authors have analyzed guanine nucleotide binding proteins (G-proteins) in the olfactory epithelium of Rana catesbeiana using subunit-specific antisera. The olfactory epithelium contained the α subunits of three G-proteins, migrating on polyacrylamide gels in SDS with apparent molecular weights of 45,000, 42,000, and 40,000, corresponding to G/sub s/, G/sub i/, and G/sub o/, respectively. A single β subunit with an apparent molecular weight of 36,000 was detected. An antiserum against the α subunit of retinal transducin failed to detect immunoreactive proteins in olfactory cilia detached from the epithelium. The olfactory cilia appeared to be enriched in immunoreactive G/sub sα/ relative to G/sub ichemical bond/ and G/sub ochemical bond/ when compared to membranes prepared from the olfactory epithelium after detachment of the cilia. Bound antibody was detected by autoradiography after incubation with [ 125 I]protein. Immunohistochemical studies using an antiserum against the β subunit of G-proteins revealed intense staining of the ciliary surface of the olfactory epithelium and of the axon bundles in the lamina propria. In contrast, an antiserum against a common sequence of the α subunits preferentially stained the cell membranes of the olfactory receptor cells and the acinar cells of Bowman's glands and the deep submucosal glands. In addition to G-proteins, they have identified protein kinase C in olfactory cilia via a protein kinase C specific antiserum and via phorbol ester binding. However, in contrast to the G-proteins, protein kinase C occurred also in cilia isolated from respiratory epithelium

  15. Chromate Binding and Removal by the Molybdate-Binding Protein ModA.

    Science.gov (United States)

    Karpus, Jason; Bosscher, Michael; Ajiboye, Ifedayo; Zhang, Liang; He, Chuan

    2017-04-04

    Effective and cheap methods and techniques for the safe removal of hexavalent chromate from the environment are in increasingly high demand. High concentrations of hexavalent chromate have been shown to have numerous harmful effects on human biology. We show that the E. coli molybdate-binding protein ModA is a genetically encoded tool capable of removing chromate from aqueous solutions. Although previously reported to not bind chromate, we show that ModA binds chromate tightly and is capable of removing chromate to levels well below current US federal standards. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Differential plasma protein binding to metal oxide nanoparticles

    International Nuclear Information System (INIS)

    Deng, Zhou J; Mortimer, Gysell; Minchin, Rodney F; Schiller, Tara; Musumeci, Anthony; Martin, Darren

    2009-01-01

    Nanoparticles rapidly interact with the proteins present in biological fluids, such as blood. The proteins that are adsorbed onto the surface potentially dictate the biokinetics of the nanomaterials and their fate in vivo. Using nanoparticles with different sizes and surface characteristics, studies have reported the effects of physicochemical properties on the composition of adsorbed plasma proteins. However, to date, few studies have been conducted focusing on the nanoparticles that are commonly exposed to the general public, such as the metal oxides. Using previously established ultracentrifugation approaches, two-dimensional gel electrophoresis and mass spectrometry, the current study investigated the binding of human plasma proteins to commercially available titanium dioxide, silicon dioxide and zinc oxide nanoparticles. We found that, despite these particles having similar surface charges in buffer, they bound different plasma proteins. For TiO 2 , the shape of the nanoparticles was also an important determinant of protein binding. Agglomeration in water was observed for all of the nanoparticles and both TiO 2 and ZnO further agglomerated in biological media. This led to an increase in the amount and number of different proteins bound to these nanoparticles. Proteins with important biological functions were identified, including immunoglobulins, lipoproteins, acute-phase proteins and proteins involved in complement pathways and coagulation. These results provide important insights into which human plasma proteins bind to particular metal oxide nanoparticles. Because protein absorption to nanoparticles may determine their interaction with cells and tissues in vivo, understanding how and why plasma proteins are adsorbed to these particles may be important for understanding their biological responses.

  17. Controlled formation of emissive silver nanoclusters using rationally designed metal-binding proteins.

    Science.gov (United States)

    Morozov, Vasily A; Ogawa, Michael Y

    2013-08-19

    The metal-binding properties of rationally designed, synthetic proteins were used to prepare a series of emissive silver nanoclusters having predictable sizes and emission energies. Metal-binding α-helical coiled coils were designed to exist as peptide trimers, tetramers, and hexamers and found to uniquely bind 6, 8, and 12 Ag(+) ions, respectively. Subsequent treatment with a chemical reducing agent produced a series of peptide-bound Ag(0) nanoclusters that display a strong visible fluorescence whose emission energies depend on the number of bound metal ions in excellent agreement with theory.

  18. Solvation structure of ice-binding antifreeze proteins

    Science.gov (United States)

    Hansen-Goos, Hendrik; Wettlaufer, John

    2009-03-01

    Antifreeze proteins (AFPs) can be found in organisms which survive at subzero temperatures. They were first discovered in polar fishes since the 1950's [1] and have been isolated meanwhile also from insects, plants, and bacteria. While AFPs shift the freezing point of water below the bulk melting point and hence can prevent recrystallization; the effect is non-colligative and there is a pronounced hysteresis between freezing and melting. For many AFPs it is generally accepted that they function through an irreversible binding to the ice-water interface which leads to a piecewise convex growth front with a lower nonequilibrium freezing point due to the Kelvin effect. Recent molecular dynamics simulations of the AFP from Choristoneura fumiferana reveal that the solvation structures of water at ice-binding and non-ice-binding faces of the protein are crucial for understanding how the AFP binds to the ice surface and how it is protected from being overgrown [2]. We use density functional theory of classical fluids in order to assess the microscopic solvent structure in the vicinity of protein faces with different surface properties. With our method, binding energies of different protein faces to the water-ice-interface can be computed efficiently in a simplified model. [1] Y. Yeh and R.E. Feeney, Chem. Rev. 96, 601 (1996). [2] D.R. Nutt and J.C. Smith, J. Am. Chem. Soc. 130, 13066 (2008).

  19. Heterologously expressed Staphylococcus aureus fibronectin-binding proteins are sufficient for invasion of host cells

    NARCIS (Netherlands)

    Sinha, B; Francois, P; Que, Y A; Hussain, M; Heilmann, C; Moreillon, P; Lew, D; Krause, K H; Peters, Georg; Herrmann, M

    2000-01-01

    Staphylococcus aureus invasion of mammalian cells, including epithelial, endothelial, and fibroblastic cells, critically depends on fibronectin bridging between S. aureus fibronectin-binding proteins (FnBPs) and the host fibronectin receptor integrin alpha(5)beta(1) (B. Sinha et al., Cell.

  20. Studies on two calcium-binding proteins from squid optic lobe

    International Nuclear Information System (INIS)

    Sheldon, A.

    1988-01-01

    Investigations focused on the physicochemical and functional properties of squid calcium-binding protein (SCaBP) and squid calmodulin (SCaM). The physiochemical studies included characterization of Ca 2+ - and Mg 2+ -binding properties, and the effects of metal ion-binding on protein conformation. These studies were performed using various ionic conditions, including those physiological for the squid. Ca 2+ -binding by SCaBP, SCaM, and bovine brain calmodulin (BCaM) (for comparison) was measured by equilibrium dialysis. All three proteins bound 4 Ca 2+ per mol protein under each set of ionic conditions. Under conditions physiological for the squid, both the squid proteins bound Ca 2+ over a similar range of free Ca 2+ concentrations. Ca 2+ - and Mg 2+ -induced changes in the conformation of the proteins were studied by polyacrylamide gel electrophoresis, fluphenazine-Sepharose affinity chromatography, and ultraviolet absorption difference spectroscopy. The functional studies focused on the identification of squid optic lobe proteins which interacted with SCaBP and SCaM in a Ca 2+ - dependent manner. These proteins were identified by SCaBP- and SCaM-Sepharose affinity chromatography, an immunoblotting technique using a polyclonal anti-SCaBP antibody, and a 125 I-calmodulin overlay procedure

  1. The Structure of the Iron Binding Protein, FutA1, from Synechocystis 6803*

    International Nuclear Information System (INIS)

    Koropatkin, Nicole; Randich, Amelia M.; Bhattacharyya-Pakrasi, Maitrayee; Pakrasi, Himadri B.; Smith, Thomas J.

    2007-01-01

    Cyanobacteria account for a significant percentage of aquatic primary productivity even in areas where the concentrations of essential micronutrients are extremely low. To better understand the mechanism of iron selectivity and transport, the structure of the solute-binding domain of an ABC iron transporter, FutA1, was determined in the presence and absence of iron. The iron ion is bound within the 'C-clamp' structure via four tyrosine and one histidine residues. There are extensive interactions between these ligating residues and the rest of the protein such that the conformations of the side chains remain relatively unchanged as the iron is released by the opening of the metal binding cleft. This is in stark contrast to the zinc binding protein, ZnuA, where the domains of the metal binding protein remain relatively fixed while the ligating residues rotate out of the binding pocket upon metal release. The rotation of the domains in FutA1 is facilitated by two flexible β-strands running along the back of the protein that act like a hinge during domain motion. This motion may require relatively little energy since total contact area between the domains is the same whether the protein is in the open or closed conformation. Consistent with the pH dependency of iron binding, the main trigger for iron release is likely the histidine in the iron-binding site. Finally, neither FutA1 nor FutA2 binds iron as a siderophore complex or in the presence of anions and both preferentially bind ferrous over ferric ions

  2. Evidence for a lactose-mediated association between two nuclear carbohydrate-binding proteins.

    Science.gov (United States)

    Sève, A P; Felin, M; Doyennette-Moyne, M A; Sahraoui, T; Aubery, M; Hubert, J

    1993-02-01

    Nuclear proteins were extracted in 2 M NaCl from membrane-depleted nuclei isolated from HL60 cells. Extracted proteins were submitted to affinity chromatography columns containing immobilized glucose, galactose or lactose. The polypeptides present in the different eluted fractions were resolved by SDS-PAGE and were either silver stained or analysed by immunoblotting with monoclonal or polyclonal antibodies, respectively, raised against the glucose-binding protein CBP67 and the galactose-binding proteins CBP35 and L14. The results presented here show that HL60 cell nuclei contain CBP35 and a glucose-binding lectin of 70 kDa (CBP70). These data account for the previously reported binding of neoglycoproteins containing glucosyl and galactosyl residues to HL60 cell nuclei. Furthermore, the present study provides the new information that CBP35 can associate with CBP70 by interactions dependent on the binding of CBP35 to lactose, and the results of some affinity chromatography experiments strongly suggest that CBP35 and CBP70 associate by protein-protein interactions. The potential function of this lactose-mediated interaction is discussed with respect to data recently reported by others showing that CBP35 is involved in in vitro mRNA splicing and that lactose inhibits the processing of the pre-RNA substrate.

  3. Phosphorus Binding Sites in Proteins: Structural Preorganization and Coordination

    DEFF Research Database (Denmark)

    Gruber, Mathias Felix; Greisen, Per Junior; Junker, Märta Caroline

    2014-01-01

    to individual structures that bind to phosphate groups; here, we investigate a total of 8307 structures obtained from the RCSB Protein Data Bank (PDB). An analysis of the binding site amino acid propensities reveals very characteristic first shell residue distributions, which are found to be influenced...... by the characteristics of the phosphorus compound and by the presence of cobound cations. The second shell, which supports the coordinating residues in the first shell, is found to consist mainly of protein backbone groups. Our results show how the second shell residue distribution is dictated mainly by the first shell...

  4. Computational scheme for pH-dependent binding free energy calculation with explicit solvent.

    Science.gov (United States)

    Lee, Juyong; Miller, Benjamin T; Brooks, Bernard R

    2016-01-01

    We present a computational scheme to compute the pH-dependence of binding free energy with explicit solvent. Despite the importance of pH, the effect of pH has been generally neglected in binding free energy calculations because of a lack of accurate methods to model it. To address this limitation, we use a constant-pH methodology to obtain a true ensemble of multiple protonation states of a titratable system at a given pH and analyze the ensemble using the Bennett acceptance ratio (BAR) method. The constant pH method is based on the combination of enveloping distribution sampling (EDS) with the Hamiltonian replica exchange method (HREM), which yields an accurate semi-grand canonical ensemble of a titratable system. By considering the free energy change of constraining multiple protonation states to a single state or releasing a single protonation state to multiple states, the pH dependent binding free energy profile can be obtained. We perform benchmark simulations of a host-guest system: cucurbit[7]uril (CB[7]) and benzimidazole (BZ). BZ experiences a large pKa shift upon complex formation. The pH-dependent binding free energy profiles of the benchmark system are obtained with three different long-range interaction calculation schemes: a cutoff, the particle mesh Ewald (PME), and the isotropic periodic sum (IPS) method. Our scheme captures the pH-dependent behavior of binding free energy successfully. Absolute binding free energy values obtained with the PME and IPS methods are consistent, while cutoff method results are off by 2 kcal mol(-1) . We also discuss the characteristics of three long-range interaction calculation methods for constant-pH simulations. © 2015 The Protein Society.

  5. Concentration Dependent Ion-Protein Interaction Patterns Underlying Protein Oligomerization Behaviours

    Science.gov (United States)

    Batoulis, Helena; Schmidt, Thomas H.; Weber, Pascal; Schloetel, Jan-Gero; Kandt, Christian; Lang, Thorsten

    2016-04-01

    Salts and proteins comprise two of the basic molecular components of biological materials. Kosmotropic/chaotropic co-solvation and matching ion water affinities explain basic ionic effects on protein aggregation observed in simple solutions. However, it is unclear how these theories apply to proteins in complex biological environments and what the underlying ionic binding patterns are. Using the positive ion Ca2+ and the negatively charged membrane protein SNAP25, we studied ion effects on protein oligomerization in solution, in native membranes and in molecular dynamics (MD) simulations. We find that concentration-dependent ion-induced protein oligomerization is a fundamental chemico-physical principle applying not only to soluble but also to membrane-anchored proteins in their native environment. Oligomerization is driven by the interaction of Ca2+ ions with the carboxylate groups of aspartate and glutamate. From low up to middle concentrations, salt bridges between Ca2+ ions and two or more protein residues lead to increasingly larger oligomers, while at high concentrations oligomers disperse due to overcharging effects. The insights provide a conceptual framework at the interface of physics, chemistry and biology to explain binding of ions to charged protein surfaces on an atomistic scale, as occurring during protein solubilisation, aggregation and oligomerization both in simple solutions and membrane systems.

  6. Photo-isomerization and oxidation of bilirubin in mammals is dependent on albumin binding.

    Science.gov (United States)

    Goncharova, Iryna; Jašprová, Jana; Vítek, Libor; Urbanová, Marie

    2015-12-01

    The bilirubin (BR) photo-conversion in the human body is a protein-dependent process; an effective photo-isomerization of the potentially neurotoxic Z,Z-BR as well as its oxidation to biliverdin in the antioxidant redox cycle is possible only when BR is bound on serum albumin. We present a novel analytical concept in the study of linear tetrapyrroles metabolic processes based on an in-depth mapping of binding sites in the structure of human serum albumin (HSA). A combination of fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular modeling methods was used for recognition of the binding site for BR, its derivatives (mesobilirubin and bilirubin ditaurate), and the products of the photo-isomerization and oxidation (lumirubin, biliverdin, and xanthobilirubic acid) on HSA. The CD spectra and fluorescent quenching of the Trp-HSA were used to calculate the binding constants. The results of the CD displacement experiments performed with hemin were interpreted together with the findings of molecular docking performed on the pigment-HSA complexes. We estimated that Z,Z-BR and its metabolic products bind on two independent binding sites. Our findings support the existence of a reversible antioxidant redox cycle for BR and explain an additional pathway of the photo-isomerization process (increase of HSA binding capacity; the excess free [unbound] BR can be converted and also bound to HSA). Copyright © 2015 Elsevier Inc. All rights reserved.

  7. The RNA-binding protein repertoire of Arabidopsis thaliana

    KAUST Repository

    Marondedze, Claudius

    2016-07-11

    RNA-binding proteins (RBPs) have essential roles in determining the fate of RNA from synthesis to decay and have been studied on a protein-by-protein basis, or computationally based on a number of well-characterised RNA-binding domains. Recently, high-throughput methods enabled the capture of mammalian RNA-binding proteomes. To gain insight into the role of Arabidopsis thaliana RBPs at the systems level, we have employed interactome capture techniques using cells from different ecotypes grown in cultures and leaves. In vivo UV-crosslinking of RNA to RBPs, oligo(dT) capture and mass spectrometry yielded 1,145 different proteins including 550 RBPs that either belong to the functional category ‘RNA-binding’, have known RNA-binding domains or have orthologs identified in mammals, C. elegans, or S. cerevisiae in addition to 595 novel candidate RBPs. We noted specific subsets of RBPs in cultured cells and leaves and a comparison of Arabidopsis, mammalian, C. elegans, and S. cerevisiae RBPs reveals a common set of proteins with a role in intermediate metabolism, as well as distinct differences suggesting that RBPs are also species and tissue specific. This study provides a foundation for studies that will advance our understanding of the biological significance of RBPs in plant developmental and stimulus specific responses.

  8. Low level chemiluminescence measurement of the binding of 8-methoxypsoralen to proteins and lymphocytic surfaces

    International Nuclear Information System (INIS)

    Lange, B.

    1980-01-01

    Photochemotherapy with 8-methoxypsoralen (8-MOP) and longwave ultraviolet light is beneficial in such different disorders like psoriasis, lichen planus, and mykosis fungoides. In contrast to a widely accepted hypothesis 8-MOP does not solely bind to nucleic acid, but also to certain proteins. The mechanism of this binding as well as the precise binding area are unknown. Therefore the UV-provoked reactions of 8-MOP with a lipid mixture, a glucosaminoglycan solution, a protein solution, and lymphocyte suspensions, respectively were investigated using low level chemiluminescence (LLCL). It was found an 8-MOP concentration-dependent decrease of LLCL intensity in the lymphocyte suspensions (10 3 to 10 4 cells/μl). This effect is result of the diminution of the photoactive 8-MOP content of the solution. 8-MOP binds quickly and in the course of a free radical reaction to lymphocytic surfaces and coincidentally loses its potency to start LLCL-detectable free radical chain responses. (author)

  9. Binding of plasma proteins to titanium dioxide nanotubes with different diameters

    Science.gov (United States)

    Kulkarni, Mukta; Flašker, Ajda; Lokar, Maruša; Mrak-Poljšak, Katjuša; Mazare, Anca; Artenjak, Andrej; Čučnik, Saša; Kralj, Slavko; Velikonja, Aljaž; Schmuki, Patrik; Kralj-Iglič, Veronika; Sodin-Semrl, Snezna; Iglič, Aleš

    2015-01-01

    Titanium and titanium alloys are considered to be one of the most applicable materials in medical devices because of their suitable properties, most importantly high corrosion resistance and the specific combination of strength with biocompatibility. In order to improve the biocompatibility of titanium surfaces, the current report initially focuses on specifying the topography of titanium dioxide (TiO2) nanotubes (NTs) by electrochemical anodization. The zeta potential (ζ-potential) of NTs showed a negative value and confirmed the agreement between the measured and theoretically predicted dependence of ζ-potential on salt concentration, whereby the absolute value of ζ-potential diminished with increasing salt concentrations. We investigated binding of various plasma proteins with different sizes and charges using the bicinchoninic acid assay and immunofluorescence microscopy. Results showed effective and comparatively higher protein binding to NTs with 100 nm diameters (compared to 50 or 15 nm). We also showed a dose-dependent effect of serum amyloid A protein binding to NTs. These results and theoretical calculations of total available surface area for binding of proteins indicate that the largest surface area (also considering the NT lengths) is available for 100 nm NTs, with decreasing surface area for 50 and 15 nm NTs. These current investigations will have an impact on increasing the binding ability of biomedical devices in the body leading to increased durability of biomedical devices. PMID:25733829

  10. Deoxyribonucleic-binding homeobox proteins are augmented in human cancer

    DEFF Research Database (Denmark)

    Wewer, U M; Mercurio, A M; Chung, S Y

    1990-01-01

    Homeobox genes encode sequence-specific DNA-binding proteins that are involved in the regulation of gene expression during embryonic development. In this study, we examined the expression of homeobox proteins in human cancer. Antiserum was obtained against a synthetic peptide derived from...... the same patients exhibited little immunoreactivity. Both the peptide antiserum and the polyclonal antiserum against the native protein immunoblotted a molecular weight 63,000 protein in nuclear extracts of tumor tissue, but not significantly in extracts of normal tissue. At the molecular level......, the presence of the homeobox transcript in human carcinoma was documented by in situ hybridization and RNase protection mapping. These results demonstrate that human cancer is associated with the expression of homeobox proteins. Such homeobox proteins, as well as other regulatory proteins, could be involved...

  11. Tritium NMR spectroscopy of ligand binding to maltose-binding protein

    Energy Technology Data Exchange (ETDEWEB)

    Gehring, K.; Williams, P.G.; Pelton, J.G.; Morimoto, H.; Wemmer, D.E. (Lawrence Berkeley Lab., CA (United States))

    1991-06-04

    Tritium-labeled {alpha}- and {beta}-maltodextrins have been used to study their complexes with maltose-binding protein (MBP), a 40-kDa bacterial protein. Five substrates, from maltose to maltohexaose, were labeled at their reducing ends and their binding studied. Tritium NMR specctroscopy of the labeled sugars showed large upfield chamical shift changes upon binding and strong anomeric specficity. At 10{degrees}C, MBP bound {alpha}-maltose with 2.7 {plus minus} 0.5-fold higher affinity than {beta}-maltose, and, for longer maltodextrins, the ratio of affinities was even larger. The maximum chemical shift change was 2.2 ppm, suggesting that the reducing end of bound {alpha}-maltodextrin makes close contact with an aromatic residue in the MBP-binding site. Experiments with maltotriose (and longer maltodextrins) also revealed the presence of two bound {beta}-maltotriose resonances in rapid exchange. The authors interpret these two resonances as arising from two distinct sugar-protein complexes. In one complex, the {beta}-maltodextrin is bound by its reducing end, and, in the other complex, the {beta}-maltodextrin is bound by the middle glucose residue(s). This interpretation also suggests how MBP is able to bind both linear and circular maltodextrins.

  12. Tritium NMR spectroscopy of ligand binding to maltose-binding protein

    International Nuclear Information System (INIS)

    Gehring, K.; Williams, P.G.; Pelton, J.G.; Morimoto, H.; Wemmer, D.E.

    1991-01-01

    Tritium-labeled α- and β-maltodextrins have been used to study their complexes with maltose-binding protein (MBP), a 40-kDa bacterial protein. Five substrates, from maltose to maltohexaose, were labeled at their reducing ends and their binding studied. Tritium NMR specctroscopy of the labeled sugars showed large upfield chamical shift changes upon binding and strong anomeric specficity. At 10 degrees C, MBP bound α-maltose with 2.7 ± 0.5-fold higher affinity than β-maltose, and, for longer maltodextrins, the ratio of affinities was even larger. The maximum chemical shift change was 2.2 ppm, suggesting that the reducing end of bound α-maltodextrin makes close contact with an aromatic residue in the MBP-binding site. Experiments with maltotriose (and longer maltodextrins) also revealed the presence of two bound β-maltotriose resonances in rapid exchange. The authors interpret these two resonances as arising from two distinct sugar-protein complexes. In one complex, the β-maltodextrin is bound by its reducing end, and, in the other complex, the β-maltodextrin is bound by the middle glucose residue(s). This interpretation also suggests how MBP is able to bind both linear and circular maltodextrins

  13. Fluorescence properties of porcine odorant binding protein Trp 16 residue

    Energy Technology Data Exchange (ETDEWEB)

    Albani, Jihad Rene, E-mail: Jihad-Rene.Albani@univ-lille1.f [Laboratoire de Biophysique Moleculaire, Universite des Sciences et Technologies de Lille, F-59655 Villeneuve d' Ascq Cedex (France)

    2010-11-15

    Summary: The present work deals with fluorescence studies of adult porcine odorant binding protein at pH=7.5. At this pH, the protein is a dimer, each monomer contains one tryptophan residue. Our results show that tryptophan residue displays significant motions and emits with three fluorescence lifetimes. Decay associated spectra showed that the three lifetime's components emanate from sub-structures surrounded by the same microenvironment.

  14. A dipeptide with enhanced anion binding affinity enables cell uptake and protein delivery.

    Science.gov (United States)

    Li, Mao; Mosel, Stefanie; Knauer, Shirley K; Schmuck, Carsten

    2018-03-14

    Herein, we report a rather simple strategy to enhance the anion binding ability of a dipeptide to achieve cell uptake and also protein delivery. Peptide 1, composed of only two synthetic amino acids with an artificial anion binding site in the side chains, has an overall molecular weight of only 630 Da and demonstrated strong binding affinity (10 7 M -1 ) and clustering ability with heparin as a model for cell surface sugars. Furthermore, peptide 1 is also efficiently taken up by cells most likely via endocytosis. The uptake efficiency is dependent on the amount of glycosaminoglycans on the cell surface. Cells with reduced amounts of surface bound glycosaminoglycans show significantly less uptake of peptide 1. Moreover, 1 induced the uptake of a model protein (avidin, around 67 kDa) into cells, which makes 1 a highly attractive candidate for drug and protein delivery, especially as 1 has negligible cytotoxicity.

  15. Cooperative Binding and Activation of Fibronectin by a Bacterial Surface Protein*

    Science.gov (United States)

    Marjenberg, Zoe R.; Ellis, Ian R.; Hagan, Robert M.; Prabhakaran, Sabitha; Höök, Magnus; Talay, Susanne R.; Potts, Jennifer R.; Staunton, David; Schwarz-Linek, Ulrich

    2011-01-01

    Integrin-dependent cell invasion of some pathogenic bacteria is mediated by surface proteins targeting the extracellular matrix protein fibronectin (FN). Although the structural basis for bacterial FN recognition is well understood, it has been unclear why proteins such as streptococcal SfbI contain several FN-binding sites. We used microcalorimetry to reveal cooperative binding of FN fragments to arrays of binding sites in SfbI. In combination with thermodynamic analyses, functional cell-based assays show that SfbI induces conformational changes in the N-terminal 100-kDa region of FN (FN100kDa), most likely by competition with intramolecular interactions defining an inactive state of FN100kDa. This study provides insights into how long range conformational changes resulting in FN activation may be triggered by bacterial pathogens. PMID:21059652

  16. Cooperative binding of copper(I) to the metal binding domains in Menkes disease protein

    DEFF Research Database (Denmark)

    Jensen, P Y; Bonander, N; Møller, L B

    1999-01-01

    spectroscopy, and their copper(I) binding properties have been determined. Structure prediction derived from far-UV CD indicates that the secondary structure is similar in the three proteins and dominated by beta-sheet. The tryptophan fluorescence maximum is blue-shifted in the constructs containing two...... and six MBDs relative to the monomer, suggesting more structurally buried tryptophan(s), compared to the single MBD construct. Copper(I) binding has been studied by equilibrium dialysis under anaerobic conditions. We show that the copper(I) binding to constructs containing two and six domains...... is cooperative, with Hill coefficients of 1.5 and 4, respectively. The apparent affinities are described by K(0.5), determined to be 65 microM and 19 microM for constructs containing two and six domains, respectively. Our data reveal a unique regulation of Menkes protein upon a change in copper(I) concentration...

  17. RBPmap: a web server for mapping binding sites of RNA-binding proteins.

    Science.gov (United States)

    Paz, Inbal; Kosti, Idit; Ares, Manuel; Cline, Melissa; Mandel-Gutfreund, Yael

    2014-07-01

    Regulation of gene expression is executed in many cases by RNA-binding proteins (RBPs) that bind to mRNAs as well as to non-coding RNAs. RBPs recognize their RNA target via specific binding sites on the RNA. Predicting the binding sites of RBPs is known to be a major challenge. We present a new webserver, RBPmap, freely accessible through the website http://rbpmap.technion.ac.il/ for accurate prediction and mapping of RBP binding sites. RBPmap has been developed specifically for mapping RBPs in human, mouse and Drosophila melanogaster genomes, though it supports other organisms too. RBPmap enables the users to select motifs from a large database of experimentally defined motifs. In addition, users can provide any motif of interest, given as either a consensus or a PSSM. The algorithm for mapping the motifs is based on a Weighted-Rank approach, which considers the clustering propensity of the binding sites and the overall tendency of regulatory regions to be conserved. In addition, RBPmap incorporates a position-specific background model, designed uniquely for different genomic regions, such as splice sites, 5' and 3' UTRs, non-coding RNA and intergenic regions. RBPmap was tested on high-throughput RNA-binding experiments and was proved to be highly accurate. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  18. Macromolecular crowding enhances the binding of superoxide dismutase to xanthine oxidase: implications for protein-protein interactions in intracellular environments.

    Science.gov (United States)

    Zhou, Yu-Ling; Liao, Jun-Ming; Chen, Jie; Liang, Yi

    2006-01-01

    Physiological medium constitutes a crowded environment that serves as the field of action for protein-protein interaction in vivo. Measuring protein-protein interaction in crowded solutions can mimic this environment. Here we report the application of fluorescence spectroscopy and resonant mirror biosensor to investigate the interactions of bovine milk xanthine oxidase and bovine erythrocyte copper, zinc-superoxide dismutase in crowded solutions. Four nonspecific high molecular mass crowding agents, poly(ethylene glycol) 2000 and 20,000, Ficoll 70, and dextran 70, and one low molecular mass compound, glycerol, are used. Superoxide dismutase shows a strong and macromolecular crowding agent concentration-dependent binding affinity to xanthine oxidase. Addition of high concentrations of such high molecular mass crowding agents increases the binding constant remarkably and thus stabilizes superoxide dismutase activity, compared to those in the absence of crowding agents. In contrast, glycerol has little effect on the binding constant and decreases superoxide dismutase activity over the same concentration range. Such a pattern suggests that the enhancing effects of polymers and polysaccharides on the binding are due to macromolecular crowding. Taken together, these results indicate that macromolecular crowding enhances the binding of superoxide dismutase to xanthine oxidase and is favorable to the function of superoxide dismutase.

  19. Cloning and expression analysis of a blue copper- binding protein ...

    African Journals Online (AJOL)

    Jane

    2011-07-20

    Jul 20, 2011 ... Full Length Research Paper. Cloning and expression analysis of a blue copper- binding protein gene from Dasypyrum Villosum. Huagang He1*, Shanying Zhu1, Wenbing Wang1, Tongde Bie2 and Peidu Chen3. 1Jiangsu University. Zhenjiang 212013, P. R. China. 2Yangzhou Academy of Agricultural ...

  20. Lipopolysaccharide (LPS)-binding protein mediates LPS detoxification by chylomicrons

    NARCIS (Netherlands)

    Vreugdenhil, Anita C. E.; Rousseau, Corine H.; Hartung, Thomas; Greve, Jan Willem M.; van 't Veer, Cornelis; Buurman, Wim A.

    2003-01-01

    Chylomicrons have been shown to protect against endotoxin-induced lethality. LPS-binding protein (LBP) is involved in the inactivation of bacterial toxin by lipoproteins. The current study examined the interaction among LBP, chylomicrons, and bacterial toxin. LBP was demonstrated to associate with

  1. MTBindingSim: simulate protein binding to microtubules.

    Science.gov (United States)

    Philip, Julia T; Pence, Charles H; Goodson, Holly V

    2012-02-01

    Many protein-protein interactions are more complex than can be accounted for by 1:1 binding models. However, biochemists have few tools available to help them recognize and predict the behaviors of these more complicated systems, making it difficult to design experiments that distinguish between possible binding models. MTBindingSim provides researchers with an environment in which they can rapidly compare different models of binding for a given scenario. It is written specifically with microtubule polymers in mind, but many of its models apply equally well to any polymer or any protein-protein interaction. MTBindingSim can thus both help in training intuition about binding models and with experimental design. MTBindingSim is implemented in MATLAB and runs either within MATLAB (on Windows, Mac or Linux) or as a binary without MATLAB (on Windows or Mac). The source code (licensed under the GNU General Public License) and binaries are freely available at http://mtbindingsim.googlecode.com. jphilip@nd.edu; cpence@nd.edu.

  2. Genome-wide regulation of TATA-binding protein activity

    NARCIS (Netherlands)

    van Werven, F.J.

    2009-01-01

    Transcription, the synthesis of RNA from a DNA template, is a well-controlled process. TATA binding protein (TBP) recruitment to promoters is essential for transcription by all three RNA polymerases, and often is the rate-limiting step of transcription initiation. TBP is incorporated into different

  3. Cyclic AMP response element binding protein and brain-derived ...

    Indian Academy of Sciences (India)

    Madhu

    the role of CREB and BDNF in depression and as targets/mediators of antidepressant action. [Nair A and Vaidya V A 2006 Cyclic AMP response element binding protein and brain-derived neurotrophic factor: Molecules that modulate our mood?; J. Biosci. 31 423–434]. Keywords. Antidepressant; depression; hippocampus ...

  4. Controlling transcription fidelity via TATA-binding protein dynamics

    NARCIS (Netherlands)

    Koster, M.J.E.

    2015-01-01

    Transcription underlies all cellular processes and responses to internal and external cues. Deregulation of transcription has implications for the fitness of the cell or organism. During my PhD I have investigated the importance of proper TATA-binding protein (TBP) regulation as a mechanism to

  5. Evidence for covalent binding of epicocconone with proteins from ...

    Indian Academy of Sciences (India)

    TECS

    Evidence for covalent binding of epicocconone with proteins from synchronous fluorescence spectra ... the interaction of epicocconone with human serum albumin is significantly different from its interaction with surfactant assemblies. .... at 620 nm is collected at right angles to the direction of the excitation beam, at magic ...

  6. Immunoglobulin classes, metal binding proteins, and trace metals in ...

    African Journals Online (AJOL)

    , IgA and IgM), metal binding proteins (Transferrin, Caeruloplasmin, Alpha-2- Macroglobulin and Haptoglobin) and nutritionally essential trace metals/heavy metals (Zn, Fe, Se, Cu, Mg, Cd and Pb) in Nigerian cassava processors using single ...

  7. molecular interactions of the TATA-binding protein

    Indian Academy of Sciences (India)

    Unknown

    variants and lacking a UASGAL, showed that TATA-binding protein (TBP)-TATA complex gets stabilized in the presence of the acidic activator GAL4-VP16. Activator also greatly suppressed the non-specific TBP-DNA complex formation. The effects were more pronounced over weaker TATA boxes. Activator also reduced the.

  8. PRODIGY: a web server for predicting the binding affinity of protein-protein complexes.

    Science.gov (United States)

    Xue, Li C; Rodrigues, João Pglm; Kastritis, Panagiotis L; Bonvin, Alexandre Mjj; Vangone, Anna

    2016-12-01

    Gaining insights into the structural determinants of protein-protein interactions holds the key for a deeper understanding of biological functions, diseases and development of therapeutics. An important aspect of this is the ability to accurately predict the binding strength for a given protein-protein complex. Here we present PROtein binDIng enerGY prediction (PRODIGY), a web server to predict the binding affinity of protein-protein complexes from their 3D structure. The PRODIGY server implements our simple but highly effective predictive model based on intermolecular contacts and properties derived from non-interface surface. PRODIGY is freely available at: http://milou.science.uu.nl/services/PRODIGY CONTACT: a.m.j.j.bonvin@uu.nl, a.vangone@uu.nl. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  9. Small world network strategies for studying protein structures and binding.

    Science.gov (United States)

    Taylor, Neil R

    2013-01-01

    Small world network concepts provide many new opportunities to investigate the complex three dimensional structures of protein molecules. This mini-review explores the published literature on using small-world network approaches to study protein structure, with emphasis on the different combinations of descriptors that have been tested, on studies involving ligand binding in protein-ligand complexes, and on protein-protein complexes. The benefits and success of small world network approaches, which change the focus from specific interactions to the local environment, even to non-local phenomenon, are described. The purpose is to show the different ways that small world network concepts have been used for building new computational models for studying protein structure and function, and for extending and improving existing modelling approaches.

  10. Sampling and energy evaluation challenges in ligand binding protein design.

    Science.gov (United States)

    Dou, Jiayi; Doyle, Lindsey; Jr Greisen, Per; Schena, Alberto; Park, Hahnbeom; Johnsson, Kai; Stoddard, Barry L; Baker, David

    2017-12-01

    The steroid hormone 17α-hydroxylprogesterone (17-OHP) is a biomarker for congenital adrenal hyperplasia and hence there is considerable interest in development of sensors for this compound. We used computational protein design to generate protein models with binding sites for 17-OHP containing an extended, nonpolar, shape-complementary binding pocket for the four-ring core of the compound, and hydrogen bonding residues at the base of the pocket to interact with carbonyl and hydroxyl groups at the more polar end of the ligand. Eight of 16 designed proteins experimentally tested bind 17-OHP with micromolar affinity. A co-crystal structure of one of the designs revealed that 17-OHP is rotated 180° around a pseudo-two-fold axis in the compound and displays multiple binding modes within the pocket, while still interacting with all of the designed residues in the engineered site. Subsequent rounds of mutagenesis and binding selection improved the ligand affinity to nanomolar range, while appearing to constrain the ligand to a single bound conformation that maintains the same "flipped" orientation relative to the original design. We trace the discrepancy in the design calculations to two sources: first, a failure to model subtle backbone changes which alter the distribution of sidechain rotameric states and second, an underestimation of the energetic cost of desolvating the carbonyl and hydroxyl groups of the ligand. The difference between design model and crystal structure thus arises from both sampling limitations and energy function inaccuracies that are exacerbated by the near two-fold symmetry of the molecule. © 2017 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

  11. HYPOTHESIS: PARALOG FORMATION FROM PROGENITOR PROTEINS AND PARALOG MUTAGENESIS SPUR THE RAPID EVOLUTION OF TELOMERE BINDING PROTEINS

    Directory of Open Access Journals (Sweden)

    Arthur J Lustig

    2016-02-01

    Full Text Available Through elegant studies in fungal cells and complex organisms, we propose a unifying paradigm for the rapid evolution of telomere binding proteins (TBPs that associate with either (or both telomeric DNA and telomeric proteins. TBPs protect and regulate telomere structure and function. Four critical factors are involved. First, TBPs that commonly bind to telomeric DNA include the c-Myb binding proteins, OB-fold single-stranded binding proteins, and G-G base paired Hoogsteen structure (G4 binding proteins. Each contributes independently or, in some cases, cooperatively, to provide a minimum level of telomere function. As a result of these minimal requirements and the great abundance of homologs of these motifs in the proteome, DNA telomere-binding activity may be generated more easily than expected. Second, telomere dysfunction gives rise to genome instability, through the elevation of recombination rates, genome ploidy, and the frequency of gene mutations. The formation of paralogs that diverge from their progenitor proteins ultimately can form a high frequency of altered TBPs with altered functions. Third, TBPs that assemble into complexes (e.g. mammalian shelterin derive benefits from the novel emergent functions. Fourth, a limiting factor in the evolution of TBP complexes is the formation of mutually compatible interaction surfaces amongst the TBPs. These factors may have different degrees of importance in the evolution of different phyla, illustrated by the apparently simpler telomeres in complex plants. Selective pressures that can utilize the mechanisms of paralog formation and mutagenesis to drive TBP evolution along routes dependent on the requisite physiologic changes.

  12. Engineering periplasmic ligand binding proteins as glucose nanosensors

    Directory of Open Access Journals (Sweden)

    Constance J. Jeffery

    2011-01-01

    Full Text Available Diabetes affects over 100 million people worldwide. Better methods for monitoring blood glucose levels are needed for improving disease management. Several labs have previously made glucose nanosensors by modifying members of the periplasmic ligand binding protein superfamily. This minireview summarizes recent developments in constructing new versions of these proteins that are responsive within the physiological range of blood glucose levels, employ new reporter groups, and/or are more robust. These experiments are important steps in the development of novel proteins that have the characteristics needed for an implantable glucose nanosensor for diabetes management: specificity for glucose, rapid response, sensitivity within the physiological range of glucose concentrations, reproducibility, and robustness.

  13. Treponema pallidum receptor binding proteins interact with fibronectin

    Energy Technology Data Exchange (ETDEWEB)

    Peterson, K.M.; Baseman, J.B.; Alderete, J.F.

    1983-06-01

    Analysis of plasma proteins avidly bound to T. pallidum surfaces revealed the ability of T. pallidum to acquire numerous host macromolecules. No acquisition was evident by the avirulent spirochete, T. phagedenis biotype Reiter. Western blotting technology using hyperimmune antifibronectin serum as a probe revealed the ability of virulent treponemes to avidly bind fibronectin from a complex medium such as plasma. The specificity of the tiplike adherence of motile T. pallidum to fibronectin-coated glass surfaces and to fibronectin on HEp-2 cells was reinforced by the observation that pretreatment of coverslips or cell monolayers with monospecific antiserum against fibronectin substantially reduced T. pallidum attachment. The stoichiometric binding of T. pallidum to fibronectin-coated coverslips and the inability of unlabeled or /sup 35/S-radiolabeled treponemes to interact with glass surfaces treated with other plasma proteins further established the specific nature of the interaction between virulent T. pallidum and fibronectin. The avid association between three outer envelope proteins of T. pallidum and fibronectin was also demonstrated. These treponemal surface proteins have been previously identified as putative receptor-binding proteins responsible for T. pallidum parasitism of host cells. The data suggest that surface fibronectin mediates tip-oriented attachment of T. pallidum to host cells via a receptor-ligand mechanism of recognition.

  14. The binding of in vitro synthesized adenovirus DNA binding protein to single-stranded DNA is stimulated by zinc ions

    NARCIS (Netherlands)

    Vos, H.L.; Lee, F.M. van der; Sussenbach, J.S.

    1988-01-01

    We have synthesized wild type DNA binding protein (DBP) of adenovirus type 5 (Ad5) and several truncated forms of this protein by a combination of in vitro transcription and translation. The proteins obtained were tested for binding to a single-stranded DNA-cellulose column. It could be shown that

  15. An angiogenin-binding protein from endothelial cells

    International Nuclear Information System (INIS)

    Hu, Guofu; Chang, Sooik; Riordan, J.F.; Vallee, B.L.

    1991-01-01

    A 42-kDa bovine protein that binds bovine angiogenin [angiogenin binding protein (AngBP)] has been identified as a dissociable cell-surface component of calf pulmonary artery endothelial cells and a transformed bovine endothelial cell line, GM7373. 125 I-Ang can be crosslinked efficiently to AngBP by a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbo-diimide. Bovine ribonuclease A competes with the binding of 125 I-Ang to AngBP, but lysozyme does not. Direct binding to AngBP of 125 I-labeled bovine ribonuclease A is, however, much weaker than that of 125 I-Ang. Two enzymatically active derivatives of angiogenin cleaved at residues 60-61 and 67-68, respectively, fail to induce angiogenesis and also bind to AngBP only weakly. AngBP has been isolated by treatment of cells with heparan sulfate, affinity chromatography on angiogenin-Sepharose of the material dissociated from the cell surface, and gel filtration HPLC. The results suggest that AngBP has the characteristics of a receptor that may likely function in angiogenesis

  16. Insulin-like growth factor binding proteins: a structural perspective

    Directory of Open Access Journals (Sweden)

    Briony eForbes

    2012-03-01

    Full Text Available Insulin-like growth factor binding proteins (IGFBP-1 to -6 bind insulin-like growth factors-I and -II (IGF-I and IGF-II with high affinity. These binding proteins maintain IGFs in the circulation and direct them to target tissues, where they promote cell growth, proliferation, differentiation and survival via the type 1 IGF receptor (IGF-1R. IGFBPs also interact with many other molecules, which not only influence their modulation of IGF action but also mediate IGF-independent activities that influence processes such as cell migration and apoptosis by influencing gene transcription.IGFBPs-1 to -6 are structurally similar proteins consisting of three distinct domains, N-terminal, Linker and C-terminal. There have been major advances in our understanding of IGFBP structure in the last decade and a half. While there is still no structure of an intact IGFBP to date, several structures of individual N- and C-domains have been solved. The structure of a complex of N-BP-4:IGF-I:C-BP-4 has also been solved, providing a detailed picture of the structural features of the IGF binding site and the mechanism of binding. Structural studies have also identified features important for interaction with extracellular matrix components and integrins. This review summarises structural studies reported so far and highlights features important for binding not only IGF but also other partners. It also highlights future directions in which structural studies will add to our knowledge of the role played by the IGFBP family in normal growth and development, as well as in disease.

  17. Sampling protein motion and solvent effect during ligand binding

    Science.gov (United States)

    Limongelli, Vittorio; Marinelli, Luciana; Cosconati, Sandro; La Motta, Concettina; Sartini, Stefania; Mugnaini, Laura; Da Settimo, Federico; Novellino, Ettore; Parrinello, Michele

    2012-01-01

    An exhaustive description of the molecular recognition mechanism between a ligand and its biological target is of great value because it provides the opportunity for an exogenous control of the related process. Very often this aim can be pursued using high resolution structures of the complex in combination with inexpensive computational protocols such as docking algorithms. Unfortunately, in many other cases a number of factors, like protein flexibility or solvent effects, increase the degree of complexity of ligand/protein interaction and these standard techniques are no longer sufficient to describe the binding event. We have experienced and tested these limits in the present study in which we have developed and revealed the mechanism of binding of a new series of potent inhibitors of Adenosine Deaminase. We have first performed a large number of docking calculations, which unfortunately failed to yield reliable results due to the dynamical character of the enzyme and the complex role of the solvent. Thus, we have stepped up the computational strategy using a protocol based on metadynamics. Our approach has allowed dealing with protein motion and solvation during ligand binding and finally identifying the lowest energy binding modes of the most potent compound of the series, 4-decyl-pyrazolo[1,5-a]pyrimidin-7-one. PMID:22238423

  18. Species dependence of [64Cu]Cu-Bis(thiosemicarbazone) radiopharmaceutical binding to serum albumins

    International Nuclear Information System (INIS)

    Basken, Nathan E.; Mathias, Carla J.; Lipka, Alexander E.; Green, Mark A.

    2008-01-01

    Introduction: Interactions of three copper(II) bis(thiosemicarbazone) positron emission tomography radiopharmaceuticals with human serum albumin, and the serum albumins of four additional mammalian species, were evaluated. Methods: 64 Cu-labeled diacetyl bis(N 4 -methylthiosemicarbazonato)copper(II) (Cu-ATSM), pyruvaldehyde bis(N 4 -methylthiosemicarbazonato)copper(II) (Cu-PTSM) and ethylglyoxal bis(thiosemicarbazonato)copper(II) (Cu-ETS) were synthesized and their binding to human, canine, rat, baboon and porcine serum albumins quantified by ultrafiltration. Protein binding was also measured for each tracer in human, porcine, rat and mouse serum. Results: The interaction of these neutral, lipophilic copper chelates with serum albumin is highly compound- and species-dependent. Cu-PTSM and Cu-ATSM exhibit particularly high affinity for human serum albumin (HSA), while the albumin binding of Cu-ETS is relatively insensitive to species. At HSA concentrations of 40 mg/ml, '% free' (non-albumin-bound) levels of radiopharmaceutical were 4.0±0.1%, 5.3±0.2% and 38.6±0.8% for Cu-PTSM, Cu-ATSM and Cu-ETS, respectively. Conclusions: Species-dependent variations in radiopharmaceutical binding to serum albumin may need to be considered when using animal models to predict the distribution and kinetics of these compounds in humans

  19. Binding free energy analysis of protein-protein docking model structures by evERdock.

    Science.gov (United States)

    Takemura, Kazuhiro; Matubayasi, Nobuyuki; Kitao, Akio

    2018-03-14

    To aid the evaluation of protein-protein complex model structures generated by protein docking prediction (decoys), we previously developed a method to calculate the binding free energies for complexes. The method combines a short (2 ns) all-atom molecular dynamics simulation with explicit solvent and solution theory in the energy representation (ER). We showed that this method successfully selected structures similar to the native complex structure (near-native decoys) as the lowest binding free energy structures. In our current work, we applied this method (evERdock) to 100 or 300 model structures of four protein-protein complexes. The crystal structures and the near-native decoys showed the lowest binding free energy of all the examined structures, indicating that evERdock can successfully evaluate decoys. Several decoys that show low interface root-mean-square distance but relatively high binding free energy were also identified. Analysis of the fraction of native contacts, hydrogen bonds, and salt bridges at the protein-protein interface indicated that these decoys were insufficiently optimized at the interface. After optimizing the interactions around the interface by including interfacial water molecules, the binding free energies of these decoys were improved. We also investigated the effect of solute entropy on binding free energy and found that consideration of the entropy term does not necessarily improve the evaluations of decoys using the normal model analysis for entropy calculation.

  20. Binding free energy analysis of protein-protein docking model structures by evERdock

    Science.gov (United States)

    Takemura, Kazuhiro; Matubayasi, Nobuyuki; Kitao, Akio

    2018-03-01

    To aid the evaluation of protein-protein complex model structures generated by protein docking prediction (decoys), we previously developed a method to calculate the binding free energies for complexes. The method combines a short (2 ns) all-atom molecular dynamics simulation with explicit solvent and solution theory in the energy representation (ER). We showed that this method successfully selected structures similar to the native complex structure (near-native decoys) as the lowest binding free energy structures. In our current work, we applied this method (evERdock) to 100 or 300 model structures of four protein-protein complexes. The crystal structures and the near-native decoys showed the lowest binding free energy of all the examined structures, indicating that evERdock can successfully evaluate decoys. Several decoys that show low interface root-mean-square distance but relatively high binding free energy were also identified. Analysis of the fraction of native contacts, hydrogen bonds, and salt bridges at the protein-protein interface indicated that these decoys were insufficiently optimized at the interface. After optimizing the interactions around the interface by including interfacial water molecules, the binding free energies of these decoys were improved. We also investigated the effect of solute entropy on binding free energy and found that consideration of the entropy term does not necessarily improve the evaluations of decoys using the normal model analysis for entropy calculation.

  1. Protein-folding location can regulate manganese-binding versus copper- or zinc-binding.

    Science.gov (United States)

    Tottey, Steve; Waldron, Kevin J; Firbank, Susan J; Reale, Brian; Bessant, Conrad; Sato, Katsuko; Cheek, Timothy R; Gray, Joe; Banfield, Mark J; Dennison, Christopher; Robinson, Nigel J

    2008-10-23

    Metals are needed by at least one-quarter of all proteins. Although metallochaperones insert the correct metal into some proteins, they have not been found for the vast majority, and the view is that most metalloproteins acquire their metals directly from cellular pools. However, some metals form more stable complexes with proteins than do others. For instance, as described in the Irving-Williams series, Cu(2+) and Zn(2+) typically form more stable complexes than Mn(2+). Thus it is unclear what cellular mechanisms manage metal acquisition by most nascent proteins. To investigate this question, we identified the most abundant Cu(2+)-protein, CucA (Cu(2+)-cupin A), and the most abundant Mn(2+)-protein, MncA (Mn(2+)-cupin A), in the periplasm of the cyanobacterium Synechocystis PCC 6803. Each of these newly identified proteins binds its respective metal via identical ligands within a cupin fold. Consistent with the Irving-Williams series, MncA only binds Mn(2+) after folding in solutions containing at least a 10(4) times molar excess of Mn(2+) over Cu(2+) or Zn(2+). However once MncA has bound Mn(2+), the metal does not exchange with Cu(2+). MncA and CucA have signal peptides for different export pathways into the periplasm, Tat and Sec respectively. Export by the Tat pathway allows MncA to fold in the cytoplasm, which contains only tightly bound copper or Zn(2+) (refs 10-12) but micromolar Mn(2+) (ref. 13). In contrast, CucA folds in the periplasm to acquire Cu(2+). These results reveal a mechanism whereby the compartment in which a protein folds overrides its binding preference to control its metal content. They explain why the cytoplasm must contain only tightly bound and buffered copper and Zn(2+).

  2. Functional reconstitution of prostaglandin E receptor from bovine adrenal medulla with guanine nucleotide binding proteins

    International Nuclear Information System (INIS)

    Negishi, M.; Ito, S.; Yokohama, H.; Hayashi, H.; Katada, T.; Ui, M.; Hayaishi, O.

    1988-01-01

    Prostaglandin E 2 (PEG 2 ) was found to bind specifically to a 100,000 x g pellet prepared from bovine adrenal medulla. The PGE receptor was associated with a GTP-binding protein (G-protein) and could be covalently cross-linked with this G-protein by dithiobis(succinimidyl propionate) in the 100,000 x g pellet. In order to characterize the G-protein associated with the PGE receptor and reconstitute these proteins in phospholipid vesicles, the authors purified the G-protein to apparent homogeneity from the 100,000 x g pellet. The G-protein served as a substrate of pertussis toxin but differed in its α subunit from two known pertussis toxin substrate G-proteins (G/sub i/ and G 0 ) purified from bovine brain. The molecular weight of the α subunit was 40,000, which is between those of G/sub i/ and G 0 . The purified protein was also distinguished immunologically from G/sub i/ and G 0 and was referred to as G/sub am/. Reconstitution of the PGE receptor with pure C/sub am/, G/sub i/, or G 0 in phospholipid vesicles resulted in a remarkable restoration of [ 3 H]PGE 2 binding activity in a GTP-dependent manner. The efficiency of these three G-proteins in this capacity was roughly equal. When pertussis toxin- or N-ethylmaleimide-treated G-proteins, instead of the native ones, were reconstituted into vesicles, the restoration of binding activity was no longer observed. These results indicate that the PGE receptor can couple functionally with G/sub am/, G/sub i/, or G 0 in phospholipid vesicles and suggest that G/sub am/ may be involved in signal transduction of the PGE receptor in bovine adrenal medulla

  3. A mosquito hemolymph odorant-binding protein family member specifically binds juvenile hormone

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Il Hwan; Pham, Van; Jablonka, Willy; Goodman, Walter G.; Ribeiro, José M. C.; Andersen, John F.

    2017-07-27

    Juvenile hormone (JH) is a key regulator of insect development and reproduction. In adult mosquitoes, it is essential for maturation of the ovary and normal male reproductive behavior, but how JH distribution and activity is regulated after secretion is unclear. Here, we report a new type of specific JH-binding protein, given the name mosquito juvenile hormone-binding protein (mJHBP), which circulates in the hemolymph of pupal and adult Aedes aegypti males and females. mJHBP is a member of the odorant-binding protein (OBP) family, and orthologs are present in the genomes of Aedes, Culex, and Anopheles mosquito species. Using isothermal titration calorimetry, we show that mJHBP specifically binds JH II and JH III but not eicosanoids or JH derivatives. mJHBP was crystallized in the presence of JH III and found to have a double OBP domain structure reminiscent of salivary “long” D7 proteins of mosquitoes. We observed that a single JH III molecule is contained in the N-terminal domain binding pocket that is closed in an apparent conformational change by a C-terminal domain-derived α-helix. The electron density for the ligand indicated a high occupancy of the natural 10R enantiomer of JH III. Of note, mJHBP is structurally unrelated to hemolymph JHBP from lepidopteran insects. A low level of expression of mJHBP in Ae. aegypti larvae suggests that it is primarily active during the adult stage where it could potentially influence the effects of JH on egg development, mating behavior, feeding, or other processes.

  4. Identification of Putative Vero Cell Protein(s) that Bind Specifically to ...

    African Journals Online (AJOL)

    with a cellular membrane [3,4]. The initial binding of dengue virus to target cells is mediated by binding of the envelope protein to a specific and unidentified cell surface receptor(s) [5]. Viral interaction with its targets observed by electron microscopy has been already reported. [6]. The structure of the ectodomain of DEN-2E.

  5. Guanylate kinase domains of the MAGUK family scaffold proteins as specific phospho-protein-binding modules

    OpenAIRE

    Zhu, Jinwei; Shang, Yuan; Xia, Caihao; Wang, Wenning; Wen, Wenyu; Zhang, Mingjie

    2011-01-01

    Membrane-associated guanylate kinases (MAGUK) family proteins contain an inactive guanylate kinase (GK) domain, whose function has been elusive. Here, this domain is revealed as a new type of phospho-peptide-binding module, in which the GMP-binding site has evolved to accommodate phospho-serines or -threonines.

  6. Characterization of the S100A1 protein binding site on TRPC6 C-terminus.

    Directory of Open Access Journals (Sweden)

    Jan Bily

    Full Text Available The transient receptor potential (TRP protein superfamily consists of seven major groups, among them the "canonical TRP" family. The TRPC proteins are calcium-permeable nonselective cation channels activated after the emptying of intracellular calcium stores and appear to be gated by various types of messengers. The TRPC6 channel has been shown to be expressed in various tissues and cells, where it modulates the calcium level in response to external signals. Calcium binding proteins such as Calmodulin or the family of S100A proteins are regulators of TRPC channels. Here we characterized the overlapping integrative binding site for S100A1 at the C-tail of TRPC6, which is also able to accomodate various ligands such as Calmodulin and phosphatidyl-inositol-(4,5-bisphosphate. Several positively charged amino acid residues (Arg852, Lys856, Lys859, Arg860 and Arg864 were determined by fluorescence anisotropy measurements for their participation in the calcium-dependent binding of S100A1 to the C terminus of TRPC6. The triple mutation Arg852/Lys859/Arg860 exhibited significant disruption of the binding of S100A1 to TRPC6. This indicates a unique involvement of these three basic residues in the integrative overlapping binding site for S100A1 on the C tail of TRPC6.

  7. Characterization of the S100A1 protein binding site on TRPC6 C-terminus.

    Science.gov (United States)

    Bily, Jan; Grycova, Lenka; Holendova, Blanka; Jirku, Michaela; Janouskova, Hana; Bousova, Kristyna; Teisinger, Jan

    2013-01-01

    The transient receptor potential (TRP) protein superfamily consists of seven major groups, among them the "canonical TRP" family. The TRPC proteins are calcium-permeable nonselective cation channels activated after the emptying of intracellular calcium stores and appear to be gated by various types of messengers. The TRPC6 channel has been shown to be expressed in various tissues and cells, where it modulates the calcium level in response to external signals. Calcium binding proteins such as Calmodulin or the family of S100A proteins are regulators of TRPC channels. Here we characterized the overlapping integrative binding site for S100A1 at the C-tail of TRPC6, which is also able to accomodate various ligands such as Calmodulin and phosphatidyl-inositol-(4,5)-bisphosphate. Several positively charged amino acid residues (Arg852, Lys856, Lys859, Arg860 and Arg864) were determined by fluorescence anisotropy measurements for their participation in the calcium-dependent binding of S100A1 to the C terminus of TRPC6. The triple mutation Arg852/Lys859/Arg860 exhibited significant disruption of the binding of S100A1 to TRPC6. This indicates a unique involvement of these three basic residues in the integrative overlapping binding site for S100A1 on the C tail of TRPC6.

  8. DNA Binding Proteins of the Filamentous Phages CTXφ and VGJφ of Vibrio cholerae▿

    Science.gov (United States)

    Falero, Alina; Caballero, Andy; Ferrán, Beatriz; Izquierdo, Yovanny; Fando, Rafael; Campos, Javier

    2009-01-01

    The native product of open reading frame 112 (orf112) and a recombinant variant of the RstB protein, encoded by Vibrio cholerae pathogen-specific bacteriophages VGJφ and CTXφ, respectively, were purified to more than 90% homogeneity. Orf112 protein was shown to specifically bind single-stranded genomic DNA of VGJφ; however, RstB protein unexpectedly bound double-stranded DNA in addition to the single-stranded genomic DNA. The DNA binding properties of these proteins may explain their requirement for the rolling circle replication of the respective phages and RstB's requirement for single-stranded-DNA chromosomal integration of CTXφ phage dependent on XerCD recombinases. PMID:19617366

  9. DNA binding proteins of the filamentous phages CTXphi and VGJphi of Vibrio cholerae.

    Science.gov (United States)

    Falero, Alina; Caballero, Andy; Ferrán, Beatriz; Izquierdo, Yovanny; Fando, Rafael; Campos, Javier

    2009-09-01

    The native product of open reading frame 112 (orf112) and a recombinant variant of the RstB protein, encoded by Vibrio cholerae pathogen-specific bacteriophages VGJphi and CTXphi, respectively, were purified to more than 90% homogeneity. Orf112 protein was shown to specifically bind single-stranded genomic DNA of VGJphi; however, RstB protein unexpectedly bound double-stranded DNA in addition to the single-stranded genomic DNA. The DNA binding properties of these proteins may explain their requirement for the rolling circle replication of the respective phages and RstB's requirement for single-stranded-DNA chromosomal integration of CTXphi phage dependent on XerCD recombinases.

  10. Predicting DNA-binding proteins and binding residues by complex structure prediction and application to human proteome.

    Directory of Open Access Journals (Sweden)

    Huiying Zhao

    Full Text Available As more and more protein sequences are uncovered from increasingly inexpensive sequencing techniques, an urgent task is to find their functions. This work presents a highly reliable computational technique for predicting DNA-binding function at the level of protein-DNA complex structures, rather than low-resolution two-state prediction of DNA-binding as most existing techniques do. The method first predicts protein-DNA complex structure by utilizing the template-based structure prediction technique HHblits, followed by binding affinity prediction based on a knowledge-based energy function (Distance-scaled finite ideal-gas reference state for protein-DNA interactions. A leave-one-out cross validation of the method based on 179 DNA-binding and 3797 non-binding protein domains achieves a Matthews correlation coefficient (MCC of 0.77 with high precision (94% and high sensitivity (65%. We further found 51% sensitivity for 82 newly determined structures of DNA-binding proteins and 56% sensitivity for the human proteome. In addition, the method provides a reasonably accurate prediction of DNA-binding residues in proteins based on predicted DNA-binding complex structures. Its application to human proteome leads to more than 300 novel DNA-binding proteins; some of these predicted structures were validated by known structures of homologous proteins in APO forms. The method [SPOT-Seq (DNA] is available as an on-line server at http://sparks-lab.org.

  11. Identification of two nuclear N-acetylglucosamine-binding proteins.

    Science.gov (United States)

    Felin, M; Doyennette-Moyne, M A; Hadj-Sahraoui, Y; Aubery, M; Hubert, J; Sève, A P

    1994-12-01

    Using neoglycoproteins, lectins that recognize different sugars, including N-acetylglucosamine residues, were previously detected in animal cell nuclei. We report herein the isolation of two N-acetylglucosamine-binding proteins from HL60 cell nuclei: i) a 22 kDa polypeptide (CBP22) with an isoelectric point of 4.5 was isolated for the first time and ii) a 70 kDa polypeptide with an isoelectric point of 7.8. This latter protein corresponds to the glucose-binding protein (CBP70) previously isolated, based on the following similarities: i) they have the same molecular mass, ii) they have the same isoelectric point, iii) they are recognized by antibodies raised against CBP70, and iv) both are lectins from the C group of Drickamer's classification. CBP70 appeared to recognize glucose and N-acetylglucosamine; however, its affinity for N-acetylglucosamine was found to be twice that for glucose. The presence in the nucleus of two nuclear N-acetylglucosamine-binding proteins and their potential ligands, such as O-N-acetylglucosamine glycoproteins, strongly argues for possible intranuclear glycoprotein-lectin interactions.

  12. Role of adipocyte lipid-binding protein (ALBP) and acyl-coA binding protein (ACBP) in PPAR-mediated transactivation

    DEFF Research Database (Denmark)

    Helledie, Torben; Jørgensen, Claus; Antonius, Marianne

    2002-01-01

    lipid binding protein (ALBP), the keratinocyte lipid binding protein (KLBP) and the acyl-CoA binding protein (ACBP) exhibit a prominent nuclear localization in differentiating 3T3-L1 adipocytes. Similarly, ectopic expression of these proteins in CV-1 cells resulted in a primarily nuclear localization...... appears to sequester or increase the turn-over of the agonist. Similarly, our results are in keeping with a model in which ACBP increase the metabolism of these ligands....

  13. Regulation of IGF binding protein proteolysis by pregnancy-associated plasma protein-ARegulation of IGF binding protein proteolysis by pregnancy-associated plasma protein-A

    DEFF Research Database (Denmark)

    Gaidamauskas, Ervinas

    recognised role for PAPP-A in ageing and in the development of age-related disease. PAPP-A is a secreted metalloproteinase that cleaves insulin-like growth factor binding proteins (IGFBPs). Ervinas Gaidamauskas studied the mechanism of IGF-modulated proteolysis of IGFBPs by PAPP-A and the structural......During his PhD studies, Ervinas Gaidamauskas researched the proteins pregnancy-associated plasma protein-A (PAPP-A) and its homologue PAPP-A2 in vitro. As suggested by its name, PAPP-A plays an important role in pregnancy and fetal development. Additionally, recent studies indicate a newly...... determinants for cleavage. Using small-angle X-ray scattering (SAXS), he also analysed the intermodular structural organisation of the C-terminal domain of PAPP-A involved in substrate binding. Detailed knowledge of interactions between PAPP-A and its substrates is required to understand the modulatory role...

  14. DNABP: Identification of DNA-Binding Proteins Based on Feature Selection Using a Random Forest and Predicting Binding Residues.

    Science.gov (United States)

    Ma, Xin; Guo, Jing; Sun, Xiao

    2016-01-01

    DNA-binding proteins are fundamentally important in cellular processes. Several computational-based methods have been developed to improve the prediction of DNA-binding proteins in previous years. However, insufficient work has been done on the prediction of DNA-binding proteins from protein sequence information. In this paper, a novel predictor, DNABP (DNA-binding proteins), was designed to predict DNA-binding proteins using the random forest (RF) classifier with a hybrid feature. The hybrid feature contains two types of novel sequence features, which reflect information about the conservation of physicochemical properties of the amino acids, and the binding propensity of DNA-binding residues and non-binding propensities of non-binding residues. The comparisons with each feature demonstrated that these two novel features contributed most to the improvement in predictive ability. Furthermore, to improve the prediction performance of the DNABP model, feature selection using the minimum redundancy maximum relevance (mRMR) method combined with incremental feature selection (IFS) was carried out during the model construction. The results showed that the DNABP model could achieve 86.90% accuracy, 83.76% sensitivity, 90.03% specificity and a Matthews correlation coefficient of 0.727. High prediction accuracy and performance comparisons with previous research suggested that DNABP could be a useful approach to identify DNA-binding proteins from sequence information. The DNABP web server system is freely available at http://www.cbi.seu.edu.cn/DNABP/.

  15. Concentration-dependent binding of CdSe quantum dots on the SH3 domain.

    Science.gov (United States)

    Bell, David R; Kang, Seung-Gu; Huynh, Tien; Zhou, Ruhong

    2017-12-21

    Quantum dots (QDs) are being used increasingly in applications for solar panels, consumer electronics, and biomedical imaging. For biomedical applications, QDs are typically coated with a biocompatible molecule for the system of interest. Experiments have indicated a QD dose-dependent and surface coating-dependent toxicity, with a portion of the toxicity being ascribed to interference with biomolecules. In this work, the interaction of trioctylphosphine oxide (TOPO) coated (CdSe) 13 QDs with the SRC homology 3 domain (SH3) protein domain are explored using molecular dynamics simulations. The results of this research agree well with experiments that show that at the lowest concentration, the QDs have little affinity for the native proline-rich motif (PRM) binding site of SH3. At higher concentrations, the QDs aggregate and increasingly prefer the PRM binding site, indicating that the normal SH3 function is impeded. This binding dependence is attributed to changes in the local density of the surface coated TOPO molecules upon aggregation. These results present possible interesting QD toxicity patterns and reveal the interdependence between dose and surface coating effects in QD toxicity.

  16. Yersinia enterocolitica serum resistance proteins YadA and ail bind the complement regulator C4b-binding protein.

    Science.gov (United States)

    Kirjavainen, Vesa; Jarva, Hanna; Biedzka-Sarek, Marta; Blom, Anna M; Skurnik, Mikael; Meri, Seppo

    2008-08-29

    Many pathogens are equipped with factors providing resistance against the bactericidal action of complement. Yersinia enterocolitica, a Gram-negative enteric pathogen with invasive properties, efficiently resists the deleterious action of human complement. The major Y. enterocolitica serum resistance determinants include outer membrane proteins YadA and Ail. Lipopolysaccharide (LPS) O-antigen (O-ag) and outer core (OC) do not contribute directly to complement resistance. The aim of this study was to analyze a possible mechanism whereby Y. enterocolitica could inhibit the antibody-mediated classical pathway of complement activation. We show that Y. enterocolitica serotypes O:3, O:8, and O:9 bind C4b-binding protein (C4bp), an inhibitor of both the classical and lectin pathways of complement. To identify the C4bp receptors on Y. enterocolitica serotype O:3 surface, a set of mutants expressing YadA, Ail, O-ag, and OC in different combinations was tested for the ability to bind C4bp. The studies showed that both YadA and Ail acted as C4bp receptors. Ail-mediated C4bp binding, however, was blocked by the O-ag and OC, and could be observed only with mutants lacking these LPS structures. C4bp bound to Y. enterocolitica was functionally active and participated in the factor I-mediated degradation of C4b. These findings show that Y. enterocolitica uses two proteins, YadA and Ail, to bind C4bp. Binding of C4bp could help Y. enterocolitica to evade complement-mediated clearance in the human host.

  17. Yersinia enterocolitica serum resistance proteins YadA and ail bind the complement regulator C4b-binding protein.

    Directory of Open Access Journals (Sweden)

    Vesa Kirjavainen

    Full Text Available Many pathogens are equipped with factors providing resistance against the bactericidal action of complement. Yersinia enterocolitica, a Gram-negative enteric pathogen with invasive properties, efficiently resists the deleterious action of human complement. The major Y. enterocolitica serum resistance determinants include outer membrane proteins YadA and Ail. Lipopolysaccharide (LPS O-antigen (O-ag and outer core (OC do not contribute directly to complement resistance. The aim of this study was to analyze a possible mechanism whereby Y. enterocolitica could inhibit the antibody-mediated classical pathway of complement activation. We show that Y. enterocolitica serotypes O:3, O:8, and O:9 bind C4b-binding protein (C4bp, an inhibitor of both the classical and lectin pathways of complement. To identify the C4bp receptors on Y. enterocolitica serotype O:3 surface, a set of mutants expressing YadA, Ail, O-ag, and OC in different combinations was tested for the ability to bind C4bp. The studies showed that both YadA and Ail acted as C4bp receptors. Ail-mediated C4bp binding, however, was blocked by the O-ag and OC, and could be observed only with mutants lacking these LPS structures. C4bp bound to Y. enterocolitica was functionally active and participated in the factor I-mediated degradation of C4b. These findings show that Y. enterocolitica uses two proteins, YadA and Ail, to bind C4bp. Binding of C4bp could help Y. enterocolitica to evade complement-mediated clearance in the human host.

  18. Isolation and characterization of a novel calmodulin-binding protein from potato

    Science.gov (United States)

    Reddy, Anireddy S N.; Day, Irene S.; Narasimhulu, S. B.; Safadi, Farida; Reddy, Vaka S.; Golovkin, Maxim; Harnly, Melissa J.

    2002-01-01

    Tuberization in potato is controlled by hormonal and environmental signals. Ca(2+), an important intracellular messenger, and calmodulin (CaM), one of the primary Ca(2+) sensors, have been implicated in controlling diverse cellular processes in plants including tuberization. The regulation of cellular processes by CaM involves its interaction with other proteins. To understand the role of Ca(2+)/CaM in tuberization, we have screened an expression library prepared from developing tubers with biotinylated CaM. This screening resulted in isolation of a cDNA encoding a novel CaM-binding protein (potato calmodulin-binding protein (PCBP)). Ca(2+)-dependent binding of the cDNA-encoded protein to CaM is confirmed by (35)S-labeled CaM. The full-length cDNA is 5 kb long and encodes a protein of 1309 amino acids. The deduced amino acid sequence showed significant similarity with a hypothetical protein from another plant, Arabidopsis. However, no homologs of PCBP are found in nonplant systems, suggesting that it is likely to be specific to plants. Using truncated versions of the protein and a synthetic peptide in CaM binding assays we mapped the CaM-binding region to a 20-amino acid stretch (residues 1216-1237). The bacterially expressed protein containing the CaM-binding domain interacted with three CaM isoforms (CaM2, CaM4, and CaM6). PCBP is encoded by a single gene and is expressed differentially in the tissues tested. The expression of CaM, PCBP, and another CaM-binding protein is similar in different tissues and organs. The predicted protein contained seven putative nuclear localization signals and several strong PEST motifs. Fusion of the N-terminal region of the protein containing six of the seven nuclear localization signals to the reporter gene beta-glucuronidase targeted the reporter gene to the nucleus, suggesting a nuclear role for PCBP.

  19. Circulating growth hormone (GH)-binding protein complex: a major constituent of plasma GH in man

    International Nuclear Information System (INIS)

    Baumann, G.; Amburn, K.; Shaw, M.A.

    1988-01-01

    The recent discovery of a specific binding protein for human GH (hGH) in human plasma suggests that hGH circulates in part as a complex in association with the binding protein(s). However, the magnitude of the complexed fraction prevailing under physiological conditions is unknown because of 1) dissociation of the complex during analysis and 2) potential differences in the binding characteristics of radiolabeled and native hGH. We conducted experiments designed to minimize dissociation during analysis (gel filtration in prelabeled columns, frontal analysis, and batch molecular sieving) with both native and radioiodinated hGH. All three methods yielded similar estimates for the complexed fraction. In normal plasma the bound fraction for 22 K hGH averaged 50.1% (range, 39-59%), that for 20 K hGH averaged 28.5% (range, 26-31%). Above a hGH level of about 20 ng/ml the bound fraction declines in concentration-dependent manner due to saturation of the binding protein. We conclude that a substantial part of circulating hGH is complexed with carrier proteins. This concept has important implications for the metabolism, distribution, and biological activity of hGH

  20. Alpha-amylase inhibitor, CS-1036 binds to serum amylase in a concentration-dependent and saturable manner.

    Science.gov (United States)

    Honda, Tomohiro; Kaneno-Urasaki, Yoko; Ito, Takashi; Kimura, Takako; Matsushima, Nobuko; Okabe, Hiromi; Yamasaki, Atsushi; Izumi, Takashi

    2014-03-01

    (2R,3R,4R)-4-hydroxy-2-(hydroxymethyl)pyrrolidin-3-yl 4-O-(6-deoxy-β-D-glucopyranosyl)-α-D-glucopyranoside (CS-1036), which is an α-amylase inhibitor, exhibited biphasic and sustained elimination with a long t1/2 (18.4-30.0 hours) in rats and monkeys, but exhibited a short t1/2 (3.7-7.9 hours) in humans. To clarify the species differences in the t1/2, the plasma protein binding of CS-1036 was evaluated by ultrafiltration. A concentration-dependent and saturable plasma protein binding of CS-1036 was observed in rats and monkeys with the dissociation rate constant (KD) of 8.95 and 27.2 nM, and maximal binding capacity (Bmax) of 52.8 and 22.1 nM, respectively. By the assessments of the recombinant amylase and immunoprecipitation, the major binding protein of CS-1036 in rats was identified as salivary amylase (KD 5.64 nM). CS-1036 also showed concentration-dependent and saturable binding to human salivary and pancreatic amylase, with similar binding affinity in rats. However, the protein binding of CS-1036 was constant in human plasma (≤10.2%) due to the lower serum amylase level compared with rats and monkeys. From the calculation of the unbound fraction (fu) in plasma based on in vitro KD and Bmax, the dose-dependent increase in fu after oral administration is speculated to lead to a dose-dependent increase in total body clearance and a high area under the curve/dose at lower doses, such as 0.3 mg/kg in rats.

  1. Protein kinase A-dependent transactivation by the E2A-Pbx1 fusion protein.

    Science.gov (United States)

    Ogo, A; Waterman, M R; Kamps, M P; Kagawa, N

    1995-10-27

    The chimeric gene E2A-PBX1 is formed by the t(1;19) chromosomal translocation exclusively associated with pediatric pre-B cell acute lymphoblastic leukemia (pre-B ALL). The resultant fusion protein from this chimeric gene contains the DNA-binding homeodomain of Pbx1. The first and only functional Pbx1 binding site has been localized in bovine CYP17 to a sequence (CRS1) that participates in cAMP-dependent transcription of this gene encoding the steroid hydroxylase, 17 alpha-hydroxylase cytochrome P450. Because Pbx1 is not expressed in pre-B cells, it may be possible that the E2a-Pbx1 fusion protein expressed in pre-B cells having this translocation will activate, in response to cAMP, transcription of genes not normally expressed in these cells leading to arrest of differentiation at the pre-B cell stage. We have now shown that reporter genes comprising CRS1 are activated transcriptionally by protein kinase A (PKA) in the pre-B cell line 697, which endogenously expresses the fusion protein, and that overexpression of E2A-Pbx1 in additional cell lines enhances transcription of reporter genes in a PKA-dependent fashion. Thus, it seems plausible that arrest in the pre-B stage leading to pre-B ALL includes cAMP-dependent activation of E2A-Pbx1.

  2. Identification and characterization of intracellular proteins that bind oligonucleotides with phosphorothioate linkages.

    Science.gov (United States)

    Liang, Xue-hai; Sun, Hong; Shen, Wen; Crooke, Stanley T

    2015-03-11

    Although the RNase H-dependent mechanism of inhibition of gene expression by chemically modified antisense oligonucleotides (ASOs) has been well characterized, little is known about the interactions between ASOs and intracellular proteins that may alter cellular localization and/or potency of ASOs. Here, we report the identification of 56 intracellular ASO-binding proteins using multi-step affinity selection approaches. Many of the tested proteins had no significant effect on ASO activity; however, some proteins, including La/SSB, NPM1, ANXA2, VARS and PC4, appeared to enhance ASO activities, likely through mechanisms related to subcellular distribution. VARS and ANXA2 co-localized with ASOs in endocytic organelles, and reduction in the level of VARS altered lysosome/ASO localization patterns, implying that these proteins may facilitate ASO release from the endocytic pathway. Depletion of La and NPM1 reduced nuclear ASO levels, suggesting potential roles in ASO nuclear accumulation. On the other hand, Ku70 and Ku80 proteins inhibited ASO activity, most likely by competition with RNase H1 for ASO/RNA duplex binding. Our results demonstrate that phosphorothioate-modified ASOs bind a set of cellular proteins that affect ASO activity via different mechanisms. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. Fragile X mental retardation protein: A paradigm for translational control by RNA-binding proteins.

    Science.gov (United States)

    Chen, Eileen; Joseph, Simpson

    2015-07-01

    Translational control is a common mechanism used to regulate gene expression and occur in bacteria to mammals. Typically in translational control, an RNA-binding protein binds to a unique sequence in the mRNA to regulate protein synthesis by the ribosomes. Alternatively, a protein may bind to or modify a translation factor to globally regulate protein synthesis by the cell. Here, we review translational control by the fragile X mental retardation protein (FMRP), the absence of which causes the neurological disease, fragile X syndrome (FXS). Copyright © 2015 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.

  4. Anchored Clathrate Waters Bind Antifreeze Proteins to Ice

    Energy Technology Data Exchange (ETDEWEB)

    C Garnham; R Campbell; P Davies

    2011-12-31

    The mechanism by which antifreeze proteins (AFPs) irreversibly bind to ice has not yet been resolved. The ice-binding site of an AFP is relatively hydrophobic, but also contains many potential hydrogen bond donors/acceptors. The extent to which hydrogen bonding and the hydrophobic effect contribute to ice binding has been debated for over 30 years. Here we have elucidated the ice-binding mechanism through solving the first crystal structure of an Antarctic bacterial AFP. This 34-kDa domain, the largest AFP structure determined to date, folds as a Ca{sup 2+}-bound parallel beta-helix with an extensive array of ice-like surface waters that are anchored via hydrogen bonds directly to the polypeptide backbone and adjacent side chains. These bound waters make an excellent three-dimensional match to both the primary prism and basal planes of ice and in effect provide an extensive X-ray crystallographic picture of the AFP{vert_ellipsis}ice interaction. This unobstructed view, free from crystal-packing artefacts, shows the contributions of both the hydrophobic effect and hydrogen bonding during AFP adsorption to ice. We term this mode of binding the 'anchored clathrate' mechanism of AFP action.

  5. Boar seminal plasma proteins and their binding prperties. A review

    Czech Academy of Sciences Publication Activity Database

    Jonáková, Věra; Tichá, M.

    2004-01-01

    Roč. 69, - (2004), s. 461-475 ISSN 0010-0765 R&D Projects: GA ČR GA303/02/0433; GA ČR GP303/02/P069; GA MZd NJ7463 Institutional research plan: CEZ:AV0Z5052915; CEZ:MSM 113100001 Keywords : seminal plasma proteins * binding properties * spermadhesins Subject RIV: CE - Biochemistry Impact factor: 1.062, year: 2004

  6. Studies of Fibronectin-Binding Proteins of Streptococcus equi

    OpenAIRE

    Lannergård, Jonas; Flock, Margareta; Johansson, Staffan; Flock, Jan-Ingmar; Guss, Bengt

    2005-01-01

    Streptococcus equi subsp. equi is the causative agent of strangles, a disease of the upper respiratory tract in horses. The initiation of S. equi subsp. equi infection is likely to involve cell surface-anchored molecules mediating bacterial adhesion to the epithelium of the host. The present study describes the cloning and characterization of FNEB, a fibronectin-binding protein with cell wall-anchoring motifs. FNEB can thus be predicted as cell surface located, contrary to the two previously ...

  7. RNA Binding Proteins Posttranscriptionally Regulate Genes Involved In Oncogenesis

    Science.gov (United States)

    2010-06-01

    lysed in triple- detergent RIPA buffer with protease inhibitor cocktail (Roche, Pleasanton, CA). For nuclear and cytoplasmic fractionation, the NE-PER kit...Posttranscriptional regulation of IL-13 in T cells: role of the RNA-binding protein HuR. The Journal of allergy and clinical immunology 2008, 121(4):853-859...and western blot analysis. Western analysis was performed as described previously.12 For detection of VEGFα and TSP1 from tumors, triple- detergent

  8. Interaction of bacteriophage T4 and T7 single-stranded DNA-binding proteins with DNA

    International Nuclear Information System (INIS)

    Shokri, Leila; Williams, Mark C; Rouzina, Ioulia

    2009-01-01

    Bacteriophages T4 and T7 are well-studied model replication systems, which have allowed researchers to determine the roles of many proteins central to DNA replication, recombination and repair. Here we summarize and discuss the results from two recently developed single-molecule methods to determine the salt-dependent DNA-binding kinetics and thermodynamics of the single-stranded DNA (ssDNA)-binding proteins (SSBs) from these systems. We use these methods to characterize both the equilibrium double-stranded DNA (dsDNA) and ssDNA binding of the SSBs T4 gene 32 protein (gp32) and T7 gene 2.5 protein (gp2.5). Despite the overall two-orders-of-magnitude weaker binding of gp2.5 to both forms of DNA, we find that both proteins exhibit four-orders-of-magnitude preferential binding to ssDNA relative to dsDNA. This strong preferential ssDNA binding as well as the weak dsDNA binding is essential for the ability of both proteins to search dsDNA in one dimension to find available ssDNA-binding sites at the replication fork

  9. Mannan-binding proteins from boar seminal plasma

    Czech Academy of Sciences Publication Activity Database

    Jelínková-Slavíčková, Petra; Liberda, J.; Maňásková, Pavla; Ryšlavá, H.; Jonáková, Věra; Tichá, M.

    2004-01-01

    Roč. 62, 1-2 (2004), s. 167-182 ISSN 0165-0378. [Congress of the European Society for Reproductive & Developmental Immunology /4./. Rhodes, 04.06.2003-06.06.2003] R&D Projects: GA ČR GA303/02/0433; GA ČR GP303/02/P069; GA MŠk VS96141; GA MZd NJ7463 Institutional research plan: CEZ:AV0Z5052915 Keywords : boar seminal plasma proteins * mannan-binding proteins * oviductal epithelium Subject RIV: CE - Biochemistry Impact factor: 2.726, year: 2004

  10. Characterization of auxin-binding proteins from zucchini plasma membrane

    Science.gov (United States)

    Hicks, G. R.; Rice, M. S.; Lomax, T. L.

    1993-01-01

    We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948-4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or multimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may

  11. Binding properties and immunolocalization of a fatty acid-binding protein in Giardia lamblia.

    Science.gov (United States)

    Hassan, S M T; Maache, M; de la Guardia, R Díaz; Córdova, O M; García, J R Gil; Galiana, M; Acuña Castroviejo, D; Martins, M; Osuna, Antonio

    2005-04-01

    We describe here a fatty acid-binding protein (FABP) isolated and purified from the parasitic protozoon Giardia lamblia. The protein has a molecular mass of 8 kDa and an isoelectric point of 4.96. A Scatchard analysis of the data at equilibrium revealed a dissociation constant of 3.12 x 10(-8) M when the labeled oleic acid was displaced by a 10-fold greater concentration of unlabeled oleic acid. Testosterone, sodium desoxycholate, taurocholate, metronidazol, and alpha-tocopherol, together with butyric, arachidonic, palmitic, retinoic, and glycocholic acids, were also bound to the protein. Assays with polyclonal antibodies revealed that the protein is located in the ventral disk and also appears in the dorsal membrane, the cytoplasm, and in the vicinity of the lipid vacuoles.

  12. Fluctuations in Mass-Action Equilibrium of Protein Binding Networks

    Science.gov (United States)

    Yan, Koon-Kiu; Walker, Dylan; Maslov, Sergei

    2008-12-01

    We consider two types of fluctuations in the mass-action equilibrium in protein binding networks. The first type is driven by slow changes in total concentrations of interacting proteins. The second type (spontaneous) is caused by quickly decaying thermodynamic deviations away from equilibrium. We investigate the effects of network connectivity on fluctuations by comparing them to scenarios in which the interacting pair is isolated from the network and analytically derives bounds on fluctuations. Collective effects are shown to sometimes lead to large amplification of spontaneous fluctuations. The strength of both types of fluctuations is positively correlated with the complex connectivity and negatively correlated with complex concentration. Our general findings are illustrated using a curated network of protein interactions and multiprotein complexes in baker’s yeast, with empirical protein concentrations.

  13. Decreased protein binding of moxifloxacin in patients with sepsis?

    Directory of Open Access Journals (Sweden)

    Dorn, Christoph

    2017-02-01

    Full Text Available The mean (SD unbound fraction of moxifloxacin in plasma from patients with severe sepsis or septic shock was determined by ultrafiltration to 85.5±3.0% (range 81.9 and 91.6% indicating a decreased protein binding of moxifloxacin in this population compared with the value of 58–60% provided in the Summary of Product Characteristics. However, previous investigations neglected the influence of pH and temperature on the protein binding of moxifloxacin. Maintaining physiological conditions (pH 7.4, 37°C – as in the present study – the unbound fraction of moxifloxacin in plasma from healthy volunteers was 84%. In contrast, the unbound fraction of moxifloxacin was 77% at 4°C and 66–68% in unbuffered plasma or at pH 8.5 in fair agreement with previously published data. PK/PD parameters e.g. AUC/MIC or ratios between interstitial fluid and free plasma concentrations, which were obtained assuming a protein binding rate of moxifloxacin of 40% or more, should be revised.

  14. Improving the binding capacities of protein A chromatographic materials by means of ligand polymerization.

    Science.gov (United States)

    Freiherr von Roman, Matthias; Berensmeier, Sonja

    2014-06-20

    Protein A chromatography is one of the most important techniques used in the purification of monoclonal antibodies. Due to the low dynamic binding capacity of protein A chromatographic materials compared to other stationary phases, protein A chromatography is often discussed to be the bottleneck among current purification processes. Several approaches were tested within this study in order to maximize IgG binding capacities of current acrylamido-based based resins. Genetic engineering techniques were used in order to polymerize one of the IgG binding domains (B-domain) of protein A from Staphylococcus aureus (SpA) to achieve ligands with an increased length. The solution-binding ratio and the total size of ligand-antibody complexes were used to characterize the interaction potential of novel ligands, revealing a relatively linear dependency between the number of binding domains upon the amount of bound antibody molecules. This relationship was also valid up to a ligand which was comprised of 8 B-domains after attaching them onto acrylamido-based based stationary phases using epoxy coupling techniques. Equilibrium binding capacities of more than 80mghIgGmL(-1) were achieved using the B8 ligand. Furthermore, static binding capacities, especially for smaller ligands comprised of fewer B-domains, were improved up to 87mghIgGmL(-1) using site-specific coupling chemistry, which is an improvement of more than 20% compared to commercially available materials. In order to evaluate pore exclusion effects due to the use of prolonged affinity ligands, prepared materials were characterized regarding their effective intraparticle porosity and breakthrough capacity. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Hydrophobic erythrocyte folate binding proteins are converted to hydrophilic forms by trypsin in vitro.

    Science.gov (United States)

    Antony, A C; Verma, R S

    1989-02-13

    Human erythrocyte membranes contain high-affinity folate-binding proteins (FBPs) which on solubilization with detergents resolve into apparent 160,000 Mr moieties on Sephacryl S-200 gel filtration in Triton X-100. These FBPs share antigenic and ligand binding characteristics with particulate FBPs from other human tissues. During studies to define the vectorial orientation of these FBPs on the erythrocytes, we trypsinized intact cells with 250 micrograms trypsin per ml packed cells and quantitatively analysed the remaining cell-associated FBPs as well as the products of proteolysed FBPs in the supernatant. Incubation of intact cells with trypsin resulted in a dose-dependent decrease in their capacity to bind 125I-labelled pteroylglutamate (histamine derivative); at 250 micrograms/ml trypsin, folate binding was decreased by 77% compared to nontrypsin-treated control cells. While trypsinized cells contained proportionately lower quantities of apparent 160,000 Mr FBPs than untreated control cells, the supernatant of trypsinized cells (soluble phase) contained a single species of Mr = 40,000 which retained folate binding capacity. The sum of FBPs in trypsin supernatant and trypsin-treated cells was 87% of that found in untreated cells. Analysis of solubilized particulate erythrocyte FBPs and soluble (trypsin product) FBPs by sucrose density gradient ultracentrifugation in H2O and 2H2O above the critical micellar concentration of Triton X-100 revealed that apparent 160,000 Mr FBPs were detergent-binding (hydrophobic) species (which sedimented at Mr = 40,000 in H2O) while soluble FBPs (also sedimenting at Mr = 40,000) were hydrophilic and did not bind Triton X-100. These are the first data which show that hydrophobic FBPs can be directly converted to hydrophilic FBPs by a trypsin-mediated proteolytic event. The trypsin-sensitive site is likely to be at the junction between the detergent-binding site and the major body of the protein (Mr = 40,000) containing the folate

  16. An antifungal protein from Ginkgo biloba binds actin and can trigger cell death.

    Science.gov (United States)

    Gao, Ningning; Wadhwani, Parvesh; Mühlhäuser, Philipp; Liu, Qiong; Riemann, Michael; Ulrich, Anne S; Nick, Peter

    2016-07-01

    Ginkbilobin is a short antifungal protein that had been purified and cloned from the seeds of the living fossil Ginkgo biloba. Homologues of this protein can be detected in all seed plants and the heterosporic fern Selaginella and are conserved with respect to domain structures, peptide motifs, and specific cysteine signatures. To get insight into the cellular functions of these conserved motifs, we expressed green fluorescent protein fusions of full-length and truncated ginkbilobin in tobacco BY-2 cells. We show that the signal peptide confers efficient secretion of ginkbilobin. When this signal peptide is either cleaved or masked, ginkbilobin binds and visualizes the actin cytoskeleton. This actin-binding activity of ginkbilobin is mediated by a specific subdomain just downstream of the signal peptide, and this subdomain can also coassemble with actin in vitro. Upon stable overexpression of this domain, we observe a specific delay in premitotic nuclear positioning indicative of a reduced dynamicity of actin. To elucidate the cellular response to the binding of this subdomain to actin, we use chemical engineering based on synthetic peptides comprising different parts of the actin-binding subdomain conjugated with the cell-penetrating peptide BP100 and with rhodamine B as a fluorescent reporter. Binding of this synthetic construct to actin efficiently induces programmed cell death. We discuss these findings in terms of a working model, where ginkbilobin can activate actin-dependent cell death.

  17. Heart-type fatty-acid-binding protein (FABP3 is a lysophosphatidic acid-binding protein in human coronary artery endothelial cells

    Directory of Open Access Journals (Sweden)

    Ryoko Tsukahara

    2014-01-01

    Full Text Available Fatty-acid-binding protein 3, muscle and heart (FABP3, also known as heart-type FABP, is a member of the family of intracellular lipid-binding proteins. It is a small cytoplasmic protein with a molecular mass of about 15 kDa. FABPs are known to be carrier proteins for transporting fatty acids and other lipophilic substances from the cytoplasm to the nucleus, where these lipids are released to a group of nuclear receptors such as peroxisome proliferator-activated receptors (PPARs. In this study, using lysophosphatidic acid (LPA-coated agarose beads, we have identified FABP3 as an LPA carrier protein in human coronary artery endothelial cells (HCAECs. Administration of LPA to HCAECs resulted in a dose-dependent increase in PPARγ activation. Furthermore, the LPA-induced PPARγ activation was abolished when the FABP3 expression was reduced using small interfering RNA (siRNA. We further show that the nuclear fraction of control HCAECs contained a significant amount of exogenously added LPA, whereas FABP3 siRNA-transfected HCAECs had a decreased level of LPA in the nucleus. Taken together, these results suggest that FABP3 governs the transcriptional activities of LPA by targeting them to cognate PPARγ in the nucleus.

  18. PRODIGY : a web server for predicting the binding affinity of protein-protein complexes

    NARCIS (Netherlands)

    Xue, Li; Garcia Lopes Maia Rodrigues, João; Kastritis, Panagiotis L; Bonvin, Alexandre Mjj; Vangone, Anna

    2016-01-01

    Gaining insights into the structural determinants of protein-protein interactions holds the key for a deeper understanding of biological functions, diseases and development of therapeutics. An important aspect of this is the ability to accurately predict the binding strength for a given

  19. Haptoglobin-related protein is a high-affinity hemoglobin-binding plasma protein

    DEFF Research Database (Denmark)

    Nielsen, Marianne Jensby; Petersen, Steen Vang; Jacobsen, Christian

    2006-01-01

    Haptoglobin-related protein (Hpr) is a primate-specific plasma protein associated with apolipoprotein L-I (apoL-I)-containing high-density lipoprotein (HDL) particles shown to be a part of the innate immune defense. Despite the assumption hitherto that Hpr does not bind to hemoglobin, the present...

  20. A Venom Gland Extracellular Chitin-Binding-Like Protein from Pupal Endoparasitoid Wasps, Pteromalus Puparum, Selectively Binds Chitin

    Science.gov (United States)

    Chitin-binding proteins (CBPs) existed in various species and involved in different biology processes. In the present study, we cloned a full length cDNA of chitin-binding protein-like (PpCBP-like) from Pteromalus puparum, a pupal endoparasitoid of Pieris rapae. PpCBP-like encoded a 96 putative amin...

  1. ATP-Binding Cassette Proteins: Towards a Computational View of Mechanism

    Science.gov (United States)

    Liao, Jielou

    2004-03-01

    Many large machine proteins can generate mechanical force and undergo large-scale conformational changes (LSCC) to perform varying biological tasks in living cells by utilizing ATP. Important examples include ATP-binding cassette (ABC) transporters. They are membrane proteins that couple ATP binding and hydrolysis to the translocation of substrates across membranes [1]. To interpret how the mechanical force generated by ATP binding and hydrolysis is propagated, a coarse-grained ATP-dependent harmonic network model (HNM) [2,3] is applied to the ABC protein, BtuCD. This protein machine transports vitamin B12 across membranes. The analysis shows that subunits of the protein move against each other in a concerted manner. The lowest-frequency modes of the BtuCD protein are found to link the functionally critical domains, and are suggested to be responsible for large-scale ATP-coupled conformational changes. [1] K. P. Locher, A. T. Lee and D. C. Rees. Science 296, 1091-1098 (2002). [2] Atilgan, A. R., S. R. Durell, R. L. Jernigan, M. C. Demirel, O. Keskin, and I. Bahar. Biophys. J. 80, 505-515(2002); M. M Tirion, Phys. Rev. Lett. 77, 1905-1908 (1996). [3] J. -L. Liao and D. N. Beratan, 2003, to be published.

  2. Composite Structural Motifs of Binding Sites for Delineating Biological Functions of Proteins

    Science.gov (United States)

    Kinjo, Akira R.; Nakamura, Haruki

    2012-01-01

    Most biological processes are described as a series of interactions between proteins and other molecules, and interactions are in turn described in terms of atomic structures. To annotate protein functions as sets of interaction states at atomic resolution, and thereby to better understand the relation between protein interactions and biological functions, we conducted exhaustive all-against-all atomic structure comparisons of all known binding sites for ligands including small molecules, proteins and nucleic acids, and identified recurring elementary motifs. By integrating the elementary motifs associated with each subunit, we defined composite motifs that represent context-dependent combinations of elementary motifs. It is demonstrated that function similarity can be better inferred from composite motif similarity compared to the similarity of protein sequences or of individual binding sites. By integrating the composite motifs associated with each protein function, we define meta-composite motifs each of which is regarded as a time-independent diagrammatic representation of a biological process. It is shown that meta-composite motifs provide richer annotations of biological processes than sequence clusters. The present results serve as a basis for bridging atomic structures to higher-order biological phenomena by classification and integration of binding site structures. PMID:22347478

  3. Human pentraxin 3 binds to the complement regulator c4b-binding protein.

    Directory of Open Access Journals (Sweden)

    Anne Braunschweig

    Full Text Available The long pentraxin 3 (PTX3 is a soluble recognition molecule with multiple functions including innate immune defense against certain microbes and the clearance of apoptotic cells. PTX3 interacts with recognition molecules of the classical and lectin complement pathways and thus initiates complement activation. In addition, binding of PTX3 to the alternative complement pathway regulator factor H was shown. Here, we show that PTX3 binds to the classical and lectin pathway regulator C4b-binding protein (C4BP. A PTX3-binding site was identified within short consensus repeats 1-3 of the C4BP α-chain. PTX3 did not interfere with the cofactor activity of C4BP in the fluid phase and C4BP maintained its complement regulatory activity when bound to PTX3 on surfaces. While C4BP and factor H did not compete for PTX3 binding, the interaction of C4BP with PTX3 was inhibited by C1q and by L-ficolin. PTX3 bound to human fibroblast- and endothelial cell-derived extracellular matrices and recruited functionally active C4BP to these surfaces. Whereas PTX3 enhanced the activation of the classical/lectin pathway and caused enhanced C3 deposition on extracellular matrix, deposition of terminal pathway components and the generation of the inflammatory mediator C5a were not increased. Furthermore, PTX3 enhanced the binding of C4BP to late apoptotic cells, which resulted in an increased rate of inactivation of cell surface bound C4b and a reduction in the deposition of C5b-9. Thus, in addition to complement activators, PTX3 interacts with complement inhibitors including C4BP. This balanced interaction on extracellular matrix and on apoptotic cells may prevent excessive local complement activation that would otherwise lead to inflammation and host tissue damage.

  4. Nonspecific DNA Binding and Bending by HUαβ: Interfaces of the Three Binding Modes Characterized by Salt Dependent Thermodynamics

    Science.gov (United States)

    Koh, Junseock; Shkel, Irina; Saecker, Ruth M.; Record, M. Thomas

    2011-01-01

    Previous ITC and FRET studies demonstrated that Escherichia coli HUαβ binds nonspecifically to duplex DNA in three different binding modes: a tighter-binding 34 bp mode which interacts with DNA in large (>34 bp) gaps between bound proteins, reversibly bending it 140° and thereby increasing its flexibility, and two weaker, modestly cooperative small-site-size modes (10 bp, 6 bp) useful for filling gaps between bound proteins shorter than 34 bp. Here we use ITC to determine the thermodynamics of these binding modes as a function of salt concentration, and deduce that DNA in the 34 bp mode is bent around but not wrapped on the body of HU, in contrast to specific binding of IHF. Analyses of binding isotherms (8, 15, 34 bp DNA) and initial binding heats (34, 38, 160 bp DNA) reveal that all three modes have similar log-log salt concentration derivatives of the binding constants (Ski) even though their binding site sizes differ greatly; most probable values of Ski on 34 bp or larger DNA are − 7.5 ± 0.5. From the similarity of Ski values, we conclude that binding interfaces of all three modes involve the same region of the arms and saddle of HU. All modes are entropy-driven, as expected for nonspecific binding driven by the polyelectrolyte effect. The bent-DNA 34 bp mode is most endothermic, presumably because of the cost of HU-induced DNA bending, while the 6 bp mode is modestly exothermic at all salt concentrations examined. Structural models consistent with the observed Ski values are proposed. PMID:21513716

  5. RecO protein initiates DNA recombination and strand annealing through two alternative DNA binding mechanisms.

    Science.gov (United States)

    Ryzhikov, Mikhail; Gupta, Richa; Glickman, Michael; Korolev, Sergey

    2014-10-17

    Recombination mediator proteins (RMPs) are important for genome stability in all organisms. Several RMPs support two alternative reactions: initiation of homologous recombination and DNA annealing. We examined mechanisms of RMPs in both reactions with Mycobacterium smegmatis RecO (MsRecO) and demonstrated that MsRecO interacts with ssDNA by two distinct mechanisms. Zinc stimulates MsRecO binding to ssDNA during annealing, whereas the recombination function is zinc-independent and is regulated by interaction with MsRecR. Thus, different structural motifs or conformations of MsRecO are responsible for interaction with ssDNA during annealing and recombination. Neither annealing nor recombinase loading depends on MsRecO interaction with the conserved C-terminal tail of single-stranded (ss) DNA-binding protein (SSB), which is known to bind Escherichia coli RecO. However, similarly to E. coli proteins, MsRecO and MsRecOR do not dismiss SSB from ssDNA, suggesting that RMPs form a complex with SSB-ssDNA even in the absence of binding to the major protein interaction motif. We propose that alternative conformations of such complexes define the mechanism by which RMPs initiate the repair of stalled replication and support two different functions during recombinational repair of DNA breaks. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. A conserved NAD+binding pocket that regulates protein-protein interactions during aging.

    Science.gov (United States)

    Li, Jun; Bonkowski, Michael S; Moniot, Sébastien; Zhang, Dapeng; Hubbard, Basil P; Ling, Alvin J Y; Rajman, Luis A; Qin, Bo; Lou, Zhenkun; Gorbunova, Vera; Aravind, L; Steegborn, Clemens; Sinclair, David A

    2017-03-24

    DNA repair is essential for life, yet its efficiency declines with age for reasons that are unclear. Numerous proteins possess Nudix homology domains (NHDs) that have no known function. We show that NHDs are NAD + (oxidized form of nicotinamide adenine dinucleotide) binding domains that regulate protein-protein interactions. The binding of NAD + to the NHD domain of DBC1 (deleted in breast cancer 1) prevents it from inhibiting PARP1 [poly(adenosine diphosphate-ribose) polymerase], a critical DNA repair protein. As mice age and NAD + concentrations decline, DBC1 is increasingly bound to PARP1, causing DNA damage to accumulate, a process rapidly reversed by restoring the abundance of NAD + Thus, NAD + directly regulates protein-protein interactions, the modulation of which may protect against cancer, radiation, and aging. Copyright © 2017, American Association for the Advancement of Science.

  7. Visualizing double-stranded RNA distribution and dynamics in living cells by dsRNA binding-dependent fluorescence complementation

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Xiaofei [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario N5V 4T3 (Canada); College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, Zhejiang 310036 (China); Deng, Ping; Cui, Hongguang [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario N5V 4T3 (Canada); Wang, Aiming, E-mail: aiming.wang@agr.gc.ca [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario N5V 4T3 (Canada)

    2015-11-15

    Double-stranded RNA (dsRNA) is an important type of RNA that plays essential roles in diverse cellular processes in eukaryotic organisms and a hallmark in infections by positive-sense RNA viruses. Currently, no in vivo technology has been developed for visualizing dsRNA in living cells. Here, we report a dsRNA binding-dependent fluorescence complementation (dRBFC) assay that can be used to efficiently monitor dsRNA distribution and dynamics in vivo. The system consists of two dsRNA-binding proteins, which are fused to the N- and C-terminal halves of the yellow fluorescent protein (YFP). Binding of the two fusion proteins to a common dsRNA brings the split YFP halves in close proximity, leading to the reconstitution of the fluorescence-competent structure and restoration of fluorescence. Using this technique, we were able to visualize the distribution and trafficking of the replicative RNA intermediates of positive-sense RNA viruses in living cells. - Highlights: • A live-cell imaging system was developed for visualizing dsRNA in vivo. • It uses dsRNA binding proteins fused with two halves of a fluorescent protein. • Binding to a common dsRNA enables the reporter to become fluorescent. • The system can efficiently monitor viral RNA replication in living cells.

  8. Visualizing double-stranded RNA distribution and dynamics in living cells by dsRNA binding-dependent fluorescence complementation

    International Nuclear Information System (INIS)

    Cheng, Xiaofei; Deng, Ping; Cui, Hongguang; Wang, Aiming

    2015-01-01

    Double-stranded RNA (dsRNA) is an important type of RNA that plays essential roles in diverse cellular processes in eukaryotic organisms and a hallmark in infections by positive-sense RNA viruses. Currently, no in vivo technology has been developed for visualizing dsRNA in living cells. Here, we report a dsRNA binding-dependent fluorescence complementation (dRBFC) assay that can be used to efficiently monitor dsRNA distribution and dynamics in vivo. The system consists of two dsRNA-binding proteins, which are fused to the N- and C-terminal halves of the yellow fluorescent protein (YFP). Binding of the two fusion proteins to a common dsRNA brings the split YFP halves in close proximity, leading to the reconstitution of the fluorescence-competent structure and restoration of fluorescence. Using this technique, we were able to visualize the distribution and trafficking of the replicative RNA intermediates of positive-sense RNA viruses in living cells. - Highlights: • A live-cell imaging system was developed for visualizing dsRNA in vivo. • It uses dsRNA binding proteins fused with two halves of a fluorescent protein. • Binding to a common dsRNA enables the reporter to become fluorescent. • The system can efficiently monitor viral RNA replication in living cells.

  9. Shrimp arginine kinase being a binding protein of WSSV envelope protein VP31

    Science.gov (United States)

    Ma, Cuiyan; Gao, Qiang; Liang, Yan; Li, Chen; Liu, Chao; Huang, Jie

    2016-11-01

    Viral entry into the host is the earliest stage of infection in the viral life cycle in which attachment proteins play a key role. VP31 (WSV340/WSSV396), an envelope protein of white spot syndrome virus (WSSV), contains an Arg-Gly-Asp (RGD) peptide domain known as a cellular attachment site. At present, the process of VP31 interacting with shrimp host cells has not been explored. Therefore, the VP31 gene was cloned into pET30a (+), expressed in Escherichia coli strain BL21 and purified with immobilized metal ion affinity chromatography. Four gill cellular proteins of shrimp ( Fenneropenaeus chinensis) were pulled down by an affinity column coupled with recombinant VP31 (rVP31), and the amino acid sequences were identified with MALDI-TOF/TOF mass spectrometry. Hemocyanin, beta-actin, arginine kinase (AK), and an unknown protein were suggested as the putative VP31 receptor proteins. SDS-PAGE showed that AK is the predominant binding protein of VP31. An i n vitro binding activity experiment indicated that recombinant AK's (rAK) binding activity with rVP31 is comparable to that with the same amount of WSSV. These results suggested that AK, as a member of the phosphagen kinase family, plays a role in WSSV infection. This is the first evidence showing that AK is a binding protein of VP31. Further studies on this topic will elucidate WSSV infection mechanism in the future.

  10. Binding proteins enhance specific uptake rate by increasing the substrate-transporter encounter rate.

    NARCIS (Netherlands)

    Bosdriesz, E.; Magnúsdóttir, S.; Bruggeman, F.J.; Teusink, B.; Molenaar, D.

    2015-01-01

    Microorganisms rely on binding-protein assisted, active transport systems to scavenge for scarce nutrients. Several advantages of using binding proteins in such uptake systems have been proposed. However, a systematic, rigorous and quantitative analysis of the function of binding proteins is

  11. Activity of cefixime against Helicobacter pylori and affinities for the penicillin-binding proteins.

    Science.gov (United States)

    Ikeda, F; Yokota, Y; Mine, Y; Tatsuta, M

    1990-12-01

    Cefixime induced the formation of rounded cells from the spiral bacillary form of Helicobacter pylori at the MIC or less. Three main penicillin-binding proteins, called A, B and C, were separated from H. pylori. Cefixime had the strongest affinity to penicillin-binding protein B. The binding of cefixime to this protein may induce the formation of rounded H. pylori cells.

  12. DMPD: LPS-binding proteins and receptors. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 9665271 LPS-binding proteins and receptors. Fenton MJ, Golenbock DT. J Leukoc Biol.... 1998 Jul;64(1):25-32. (.png) (.svg) (.html) (.csml) Show LPS-binding proteins and receptors. PubmedID 9665271 Title LPS-binding prot...eins and receptors. Authors Fenton MJ, Golenbock DT. Publication J Leukoc Biol. 199

  13. Galectin-3 directs antimicrobial guanylate binding proteins to vacuoles furnished with bacterial secretion systems.

    Science.gov (United States)

    Feeley, Eric M; Pilla-Moffett, Danielle M; Zwack, Erin E; Piro, Anthony S; Finethy, Ryan; Kolb, Joseph P; Martinez, Jennifer; Brodsky, Igor E; Coers, Jörn

    2017-02-28

    Many invasive bacteria establish pathogen-containing vacuoles (PVs) as intracellular niches for microbial growth. Immunity to these infections is dependent on the ability of host cells to recognize PVs as targets for host defense. The delivery of several host defense proteins to PVs is controlled by IFN-inducible guanylate binding proteins (GBPs), which themselves dock to PVs through poorly characterized mechanisms. Here, we demonstrate that GBPs detect the presence of bacterial protein secretion systems as "patterns of pathogenesis" associated with PVs. We report that the delivery of GBP2 to Legionella -containing vacuoles is dependent on the bacterial Dot/Icm secretion system, whereas the delivery of GBP2 to Yersinia- containing vacuoles (YCVs) requires hypersecretion of Yersinia translocon proteins. We show that the presence of bacterial secretion systems directs cytosolic carbohydrate-binding protein Galectin-3 to PVs and that the delivery of GBP1 and GBP2 to Legionella- containing vacuoles or YCVs is substantially diminished in Galectin-3-deficient cells. Our results illustrate that insertion of bacterial secretion systems into PV membranes stimulates Galectin-3-dependent recruitment of antimicrobial GBPs to PVs as part of a coordinated host defense program.

  14. Cloud computing for protein-ligand binding site comparison.

    Science.gov (United States)

    Hung, Che-Lun; Hua, Guan-Jie

    2013-01-01

    The proteome-wide analysis of protein-ligand binding sites and their interactions with ligands is important in structure-based drug design and in understanding ligand cross reactivity and toxicity. The well-known and commonly used software, SMAP, has been designed for 3D ligand binding site comparison and similarity searching of a structural proteome. SMAP can also predict drug side effects and reassign existing drugs to new indications. However, the computing scale of SMAP is limited. We have developed a high availability, high performance system that expands the comparison scale of SMAP. This cloud computing service, called Cloud-PLBS, combines the SMAP and Hadoop frameworks and is deployed on a virtual cloud computing platform. To handle the vast amount of experimental data on protein-ligand binding site pairs, Cloud-PLBS exploits the MapReduce paradigm as a management and parallelizing tool. Cloud-PLBS provides a web portal and scalability through which biologists can address a wide range of computer-intensive questions in biology and drug discovery.

  15. Folding energetics of ligand binding proteins. I. Theoretical model.

    Science.gov (United States)

    Rösgen, J; Hinz, H J

    2001-03-02

    Heat capacity curves as obtained from differential scanning calorimetry are an outstanding source for molecular information on protein folding and ligand-binding energetics. However, deconvolution of C(p) data of proteins in the presence of ligands can be compromised by indeterminacies concerning the correct choice of the statistical thermodynamic ensemble. By convent, the assumption of constant free ligand concentration has been used to derive formulae for the enthalpy. Unless the ligand occurs at large excess, this assumption is incorrect. Still the relevant ensemble is the grand canonical ensemble. We derive formulae for both constraints, constancy of total or free ligand concentration and illustrate the equations by application to the typical equilibrium Nx N + x D + x. It is demonstrated that as long as the thermodynamic properties of the ligand can be completely corrected for by performing a reference measurement, the grand canonical approach provides the proper and mathematically significantly simpler choice. We demonstrate on the two cases of sequential or independent ligand-binding the fact, that similar binding mechanisms result in different and distinguishable heat capacity equations. Finally, we propose adequate strategies for DSC experiments as well as for obtaining first estimates of the characteristic thermodynamic parameters, which can be used as starting values in a global fit of DSC data. Copyright 2001 Academic Press.

  16. Retinoic acid binding protein in normal and neopolastic rat prostate.

    Science.gov (United States)

    Gesell, M S; Brandes, M J; Arnold, E A; Isaacs, J T; Ueda, H; Millan, J C; Brandes, D

    1982-01-01

    Sucrose density gradient analysis of cytosol from normal and neoplastic rat prostatic tissues exhibited a peak of (3H) retinoic acid binding in the 2S region, corresponding to the cytoplasmic retinoic acid binding protein (cRABP). In the Fisher-Copenhagen F1 rat, cRABP was present in the lateral lobe, but could not be detected in the ventral nor in the dorsal prostatic lobes. Four sublines of the R-3327 rat prostatic tumor contained similar levels of this binding protein. The absence of cRABP in the normal tissue of origin of the R-3327 tumor, the rat dorsal prostate, and reappearance in the neoplastic tissues follows a pattern described in other human and animal tumors. The occurrence of cRABP in the well-differentiated as well as in the anaplastic R-3327 tumors in which markers which reflect a state of differentiation and hormonal regulation, such as androgen receptor, 5 alpha reductase, and secretory acid phosphatase are either markedly reduced or absent, points to cRABP as a marker of malignant transformation.

  17. The clinical significance of fatty acid binding proteins

    Directory of Open Access Journals (Sweden)

    Barbara Choromańska

    2011-11-01

    Full Text Available Excessive levels of free fatty acids are toxic to cells. The human body has evolved a defense mechanism in the form of small cytoplasmic proteins called fatty acid binding proteins (FABPs that bind long-chain fatty acids (LCFA, and then refer them to appropriate intracellular disposal sites (oxidation in mitochondria and peroxisomes or storage in the endoplasmic reticulum. So far, nine types of these proteins have been described, and their name refers to the place in which they were first identified or where they can be found in the greatest concentration. The most important FABPs were isolated from the liver (L-FABP, heart (H-FABP, intestine (I-FABP, brain (B-FABP, epidermis (E-FABP and adipocytes (A-FABP. Determination of H-FABP is used in the diagnosis of myocardial infarction, and L-FABP in kidney lesions of different etiologies. It is postulated that FABPs play an important role in the pathogenesis of metabolic diseases. Elevated levels of A-FABP have been found in the pericardial fat tissue and were associated with cardiac dysfunction in obese people. A rise in A-FABP has been observed in patients with type II diabetes. I-FABP is known as a marker of cell damage in the small intestine. Increased concentration of B-FABP has been associated with human brain tumors such as glioblastoma and astrocytoma, as well as with neurodegenerative diseases (Alzheimer’s, Parkinson’s and other disorders of cognitive function. The aim of this work was to present current data on the clinical significance of fatty acid binding proteins.

  18. A Rational Engineering Strategy for Designing Protein A-Binding Camelid Single-Domain Antibodies.

    Directory of Open Access Journals (Sweden)

    Kevin A Henry

    Full Text Available Staphylococcal protein A (SpA and streptococcal protein G (SpG affinity chromatography are the gold standards for purifying monoclonal antibodies (mAbs in therapeutic applications. However, camelid VHH single-domain Abs (sdAbs or VHHs are not bound by SpG and only sporadically bound by SpA. Currently, VHHs require affinity tag-based purification, which limits their therapeutic potential and adds considerable complexity and cost to their production. Here we describe a simple and rapid mutagenesis-based approach designed to confer SpA binding upon a priori non-SpA-binding VHHs. We show that SpA binding of VHHs is determined primarily by the same set of residues as in human mAbs, albeit with an unexpected degree of tolerance to substitutions at certain core and non-core positions and some limited dependence on at least one residue outside the SpA interface, and that SpA binding could be successfully introduced into five VHHs against three different targets with no adverse effects on expression yield or antigen binding. Next-generation sequencing of llama, alpaca and dromedary VHH repertoires suggested that species differences in SpA binding may result from frequency variation in specific deleterious polymorphisms, especially Ile57. Thus, the SpA binding phenotype of camelid VHHs can be easily modulated to take advantage of tag-less purification techniques, although the frequency with which this is required may depend on the source species.

  19. Interleukin-11 binds specific EF-hand proteins via their conserved structural motifs.

    Science.gov (United States)

    Kazakov, Alexei S; Sokolov, Andrei S; Vologzhannikova, Alisa A; Permyakova, Maria E; Khorn, Polina A; Ismailov, Ramis G; Denessiouk, Konstantin A; Denesyuk, Alexander I; Rastrygina, Victoria A; Baksheeva, Viktoriia E; Zernii, Evgeni Yu; Zinchenko, Dmitry V; Glazatov, Vladimir V; Uversky, Vladimir N; Mirzabekov, Tajib A; Permyakov, Eugene A; Permyakov, Sergei E

    2017-01-01

    Interleukin-11 (IL-11) is a hematopoietic cytokine engaged in numerous biological processes and validated as a target for treatment of various cancers. IL-11 contains intrinsically disordered regions that might recognize multiple targets. Recently we found that aside from IL-11RA and gp130 receptors, IL-11 interacts with calcium sensor protein S100P. Strict calcium dependence of this interaction suggests a possibility of IL-11 interaction with other calcium sensor proteins. Here we probed specificity of IL-11 to calcium-binding proteins of various types: calcium sensors of the EF-hand family (calmodulin, S100B and neuronal calcium sensors: recoverin, NCS-1, GCAP-1, GCAP-2), calcium buffers of the EF-hand family (S100G, oncomodulin), and a non-EF-hand calcium buffer (α-lactalbumin). A specific subset of the calcium sensor proteins (calmodulin, S100B, NCS-1, GCAP-1/2) exhibits metal-dependent binding of IL-11 with dissociation constants of 1-19 μM. These proteins share several amino acid residues belonging to conservative structural motifs of the EF-hand proteins, 'black' and 'gray' clusters. Replacements of the respective S100P residues by alanine drastically decrease its affinity to IL-11, suggesting their involvement into the association process. Secondary structure and accessibility of the hinge region of the EF-hand proteins studied are predicted to control specificity and selectivity of their binding to IL-11. The IL-11 interaction with the EF-hand proteins is expected to occur under numerous pathological conditions, accompanied by disintegration of plasma membrane and efflux of cellular components into the extracellular milieu.

  20. A Single Rainbow Trout Cobalamin-binding Protein Stands in for Three Human Binders

    DEFF Research Database (Denmark)

    Greibe, Eva Holm; Fedosov, Sergey; Sorensen, Boe S

    2012-01-01

    Cobalamin uptake and transport in mammals are mediated by three cobalamin-binding proteins: haptocorrin, intrinsic factor, and transcobalamin. The nature of cobalamin-binding proteins in lower vertebrates remains to be elucidated. The aim of this study was to characterize the cobalamin......-binding proteins of the rainbow trout (Oncorhynchus mykiss) and to compare their properties with those of the three human cobalamin-binding proteins. High cobalamin-binding capacity was found in trout stomach (210 pmol/g), roe (400 pmol/g), roe fluid (390 nmol/liter), and plasma (2500 nmol/liter). In all cases......, it appeared to be the same protein based on analysis of partial sequences and immunological responses. The trout cobalamin-binding protein was purified from roe fluid, sequenced, and further characterized. Like haptocorrin, the trout cobalamin-binding protein was stable at low pH and had a high binding...

  1. Integrating protein structures and precomputed genealogies in the Magnum database: Examples with cellular retinoid binding proteins

    Directory of Open Access Journals (Sweden)

    Bradley Michael E

    2006-02-01

    Full Text Available Abstract Background When accurate models for the divergent evolution of protein sequences are integrated with complementary biological information, such as folded protein structures, analyses of the combined data often lead to new hypotheses about molecular physiology. This represents an excellent example of how bioinformatics can be used to guide experimental research. However, progress in this direction has been slowed by the lack of a publicly available resource suitable for general use. Results The precomputed Magnum database offers a solution to this problem for ca. 1,800 full-length protein families with at least one crystal structure. The Magnum deliverables include 1 multiple sequence alignments, 2 mapping of alignment sites to crystal structure sites, 3 phylogenetic trees, 4 inferred ancestral sequences at internal tree nodes, and 5 amino acid replacements along tree branches. Comprehensive evaluations revealed that the automated procedures used to construct Magnum produced accurate models of how proteins divergently evolve, or genealogies, and correctly integrated these with the structural data. To demonstrate Magnum's capabilities, we asked for amino acid replacements requiring three nucleotide substitutions, located at internal protein structure sites, and occurring on short phylogenetic tree branches. In the cellular retinoid binding protein family a site that potentially modulates ligand binding affinity was discovered. Recruitment of cellular retinol binding protein to function as a lens crystallin in the diurnal gecko afforded another opportunity to showcase the predictive value of a browsable database containing branch replacement patterns integrated with protein structures. Conclusion We integrated two areas of protein science, evolution and structure, on a large scale and created a precomputed database, known as Magnum, which is the first freely available resource of its kind. Magnum provides evolutionary and structural

  2. Exploring the binding sites and binding mechanism for hydrotrope encapsulated griseofulvin drug on γ-tubulin protein.

    Science.gov (United States)

    Das, Shubhadip; Paul, Sandip

    2018-01-01

    The protein γ-tubulin plays an important role in centrosomal clustering and this makes it an attractive therapeutic target for treating cancers. Griseofulvin, an antifungal drug, has recently been used to inhibit proliferation of various types of cancer cells. It can also affect the microtubule dynamics by targeting the γ-tubulin protein. So far, the binding pockets of γ-tubulin protein are not properly identified and the exact mechanism by which the drug binds to it is an area of intense speculation and research. The aim of the present study is to investigate the binding mechanism and binding affinity of griseofulvin on γ-tubulin protein using classical molecular dynamics simulations. Since the drug griseofulvin is sparingly soluble in water, here we also present a promising approach for formulating and achieving delivery of hydrophobic griseofulvin drug via hydrotrope sodium cumene sulfonate (SCS) cluster. We observe that the binding pockets of γ-tubulin protein are mainly formed by the H8, H9 helices and S7, S8, S14 strands and the hydrophobic interactions between the drug and γ-tubulin protein drive the binding process. The release of the drug griseofulvin from the SCS cluster is confirmed by the coordination number analysis. We also find hydrotrope-induced alteration of the binding sites of γ-tubulin protein and the weakening of the drug-protein interactions.

  3. Exploring the binding sites and binding mechanism for hydrotrope encapsulated griseofulvin drug on γ-tubulin protein.

    Directory of Open Access Journals (Sweden)

    Shubhadip Das

    Full Text Available The protein γ-tubulin plays an important role in centrosomal clustering and this makes it an attractive therapeutic target for treating cancers. Griseofulvin, an antifungal drug, has recently been used to inhibit proliferation of various types of cancer cells. It can also affect the microtubule dynamics by targeting the γ-tubulin protein. So far, the binding pockets of γ-tubulin protein are not properly identified and the exact mechanism by which the drug binds to it is an area of intense speculation and research. The aim of the present study is to investigate the binding mechanism and binding affinity of griseofulvin on γ-tubulin protein using classical molecular dynamics simulations. Since the drug griseofulvin is sparingly soluble in water, here we also present a promising approach for formulating and achieving delivery of hydrophobic griseofulvin drug via hydrotrope sodium cumene sulfonate (SCS cluster. We observe that the binding pockets of γ-tubulin protein are mainly formed by the H8, H9 helices and S7, S8, S14 strands and the hydrophobic interactions between the drug and γ-tubulin protein drive the binding process. The release of the drug griseofulvin from the SCS cluster is confirmed by the coordination number analysis. We also find hydrotrope-induced alteration of the binding sites of γ-tubulin protein and the weakening of the drug-protein interactions.

  4. Mechanism of the G-protein mimetic nanobody binding to a muscarinic G-protein-coupled receptor.

    Science.gov (United States)

    Miao, Yinglong; McCammon, J Andrew

    2018-03-20

    Protein-protein binding is key in cellular signaling processes. Molecular dynamics (MD) simulations of protein-protein binding, however, are challenging due to limited timescales. In particular, binding of the medically important G-protein-coupled receptors (GPCRs) with intracellular signaling proteins has not been simulated with MD to date. Here, we report a successful simulation of the binding of a G-protein mimetic nanobody to the M 2 muscarinic GPCR using the robust Gaussian accelerated MD (GaMD) method. Through long-timescale GaMD simulations over 4,500 ns, the nanobody was observed to bind the receptor intracellular G-protein-coupling site, with a minimum rmsd of 2.48 Å in the nanobody core domain compared with the X-ray structure. Binding of the nanobody allosterically closed the orthosteric ligand-binding pocket, being consistent with the recent experimental finding. In the absence of nanobody binding, the receptor orthosteric pocket sampled open and fully open conformations. The GaMD simulations revealed two low-energy intermediate states during nanobody binding to the M 2 receptor. The flexible receptor intracellular loops contribute remarkable electrostatic, polar, and hydrophobic residue interactions in recognition and binding of the nanobody. These simulations provided important insights into the mechanism of GPCR-nanobody binding and demonstrated the applicability of GaMD in modeling dynamic protein-protein interactions.

  5. Properties of the periplasmic ModA molybdate-binding protein of Escherichia coli.

    Science.gov (United States)

    Rech, S; Wolin, C; Gunsalus, R P

    1996-02-02

    The modABCD operon, located at 17 min on the Escherichia coli chromosome, encodes the protein components of a high affinity molybdate uptake system. Sequence analysis of the modA gene (GenBank L34009) predicts that it encodes a periplasmic binding protein based on the presence of a leader-like sequence at its N terminus. To examine the properties of the ModA protein, the modA structural gene was overexpressed, and its product was purified. The ModA protein was localized to the periplasmic space of the cell, and it was released following a gentle osmotic shock. The N-terminal sequence of ModA confirmed that a leader region of 24 amino acids was removed upon export from the cell. The apparent size of ModA is 31.6 kDa as determined by gel sieve chromatography, whereas it is 22.5 kDa when examined by SDS-polyacrylamide gel electrophoresis. A ligand-dependent protein mobility shift assay was devised using a native polyacrylamide gel electrophoresis protocol to examine binding of molybdate and other anions to the ModA periplasmic protein. Whereas molybdate and tungstate were bound with high affinity (approximately 5 microM), sulfate, chromate, selenate, phosphate, and chlorate did not bind even when tested at 2 mM. A UV spectral assay revealed apparent Kd values of binding for molybdate and tungstate of 3 and 7 microM, respectively. Strains defective in the modA gene were unable to transport molybdate unless high levels of the anion were supplied in the medium. Therefore the modA gene product is essential for high affinity molybdate uptake by the cell. Tungstate interference of molybdate acquisition by the cell is apparently due in part to the high affinity of the ModA protein for this anion.

  6. A fluorescent glycolipid-binding peptide probe traces cholesterol dependent microdomain-derived trafficking pathways.

    Directory of Open Access Journals (Sweden)

    Steffen Steinert

    Full Text Available BACKGROUND: The uptake and intracellular trafficking of sphingolipids, which self-associate into plasma membrane microdomains, is associated with many pathological conditions, including viral and toxin infection, lipid storage disease, and neurodegenerative disease. However, the means available to label the trafficking pathways of sphingolipids in live cells are extremely limited. In order to address this problem, we have developed an exogenous, non-toxic probe consisting of a 25-amino acid sphingolipid binding domain, the SBD, derived from the amyloid peptide Abeta, and conjugated by a neutral linker with an organic fluorophore. The current work presents the characterization of the sphingolipid binding and live cell trafficking of this novel probe, the SBD peptide. SBD was the name given to a motif originally recognized by Fantini et al in a number of glycolipid-associated proteins, and was proposed to interact with sphingolipids in membrane microdomains. METHODOLOGY/PRINCIPAL FINDINGS: In accordance with Fantini's model, optimal SBD binding to membranes depends on the presence of sphingolipids and cholesterol. In synthetic membrane binding assays, SBD interacts preferentially with raft-like lipid mixtures containing sphingomyelin, cholesterol, and complex gangliosides in a pH-dependent manner, but is less glycolipid-specific than Cholera toxin B (CtxB. Using quantitative time-course colocalization in live cells, we show that the uptake and intracellular trafficking route of SBD is unlike that of either the non-raft marker Transferrin or the raft markers CtxB and Flotillin2-GFP. However, SBD traverses an endolysosomal route that partially intersects with raft-associated pathways, with a major portion being diverted at a late time point to rab11-positive recycling endosomes. Trafficking of SBD to acidified compartments is strongly disrupted by cholesterol perturbations, consistent with the regulation of sphingolipid trafficking by cholesterol

  7. Rbfox2 controls autoregulation in RNA-binding protein networks.

    Science.gov (United States)

    Jangi, Mohini; Boutz, Paul L; Paul, Prakriti; Sharp, Phillip A

    2014-03-15

    The tight regulation of splicing networks is critical for organismal development. To maintain robust splicing patterns, many splicing factors autoregulate their expression through alternative splicing-coupled nonsense-mediated decay (AS-NMD). However, as negative autoregulation results in a self-limiting window of splicing factor expression, it is unknown how variations in steady-state protein levels can arise in different physiological contexts. Here, we demonstrate that Rbfox2 cross-regulates AS-NMD events within RNA-binding proteins to alter their expression. Using individual nucleotide-resolution cross-linking immunoprecipitation coupled to high-throughput sequencing (iCLIP) and mRNA sequencing, we identified >200 AS-NMD splicing events that are bound by Rbfox2 in mouse embryonic stem cells. These "silent" events are characterized by minimal apparent splicing changes but appreciable changes in gene expression upon Rbfox2 knockdown due to degradation of the NMD-inducing isoform. Nearly 70 of these AS-NMD events fall within genes encoding RNA-binding proteins, many of which are autoregulated. As with the coding splicing events that we found to be regulated by Rbfox2, silent splicing events are evolutionarily conserved and frequently contain the Rbfox2 consensus UGCAUG. Our findings uncover an unexpectedly broad and multilayer regulatory network controlled by Rbfox2 and offer an explanation for how autoregulatory splicing networks are tuned.

  8. Global analysis of ion dependence unveils hidden steps in DNA binding and bending by integration host factor

    Science.gov (United States)

    Vivas, Paula; Velmurugu, Yogambigai; Kuznetsov, Serguei V.; Rice, Phoebe A.; Ansari, Anjum

    2013-09-01

    Proteins that recognize and bind to specific sites on DNA often distort the DNA at these sites. The rates at which these DNA distortions occur are considered to be important in the ability of these proteins to discriminate between specific and nonspecific sites. These rates have proven difficult to measure for most protein-DNA complexes in part because of the difficulty in separating the kinetics of unimolecular conformational rearrangements (DNA bending and kinking) from the kinetics of bimolecular complex association and dissociation. A notable exception is the Integration Host Factor (IHF), a eubacterial architectural protein involved in chromosomal compaction and DNA recombination, which binds with subnanomolar affinity to specific DNA sites and bends them into sharp U-turns. The unimolecular DNA bending kinetics has been resolved using both stopped-flow and laser temperature-jump perturbation. Here we expand our investigation by presenting a global analysis of the ionic strength dependence of specific binding affinity and relaxation kinetics of an IHF-DNA complex. This analysis enables us to obtain each of the underlying elementary rates (DNA bending/unbending and protein-DNA association/dissociation), and their ionic strength dependence, even under conditions where the two processes are coupled. Our analysis indicates interesting differences in the ionic strength dependence of the bi- versus unimolecular steps. At moderate [KCl] (100-500 mM), nearly all the ionic strength dependence to the overall equilibrium binding affinity appears in the bimolecular association/dissociation of an initial, presumably weakly bent, encounter complex, with a slope SKbi ≈ 8 describing the loglog-dependence of the equilibrium constant to form this complex on [KCl]. In contrast, the unimolecular equilibrium constant to form the fully wrapped specific complex from the initial complex is nearly independent of [KCl], with SKuni protein-DNA contacts in the fully wrapped complex

  9. Classification and purification of proteins of heterogeneous nuclear ribonucleoprotein particles by RNA-binding specificities.

    OpenAIRE

    Swanson, M S; Dreyfuss, G

    1988-01-01

    Several proteins of heterogeneous nuclear ribonucleoprotein (hnRNP) particles display very high binding affinities for different ribonucleotide homopolymers. The specificity of some of these proteins at high salt concentrations and in the presence of heparin allows for their rapid one-step purification from HeLa nucleoplasm. We show that the hnRNP C proteins are poly(U)-binding proteins and compare their specificity to that of the previously described cytoplasmic poly(A)-binding protein. Thes...

  10. Protein phosphatase PPM1G regulates protein translation and cell growth by dephosphorylating 4E binding protein 1 (4E-BP1).

    Science.gov (United States)

    Liu, Jianyu; Stevens, Payton D; Eshleman, Nichole E; Gao, Tianyan

    2013-08-09

    Protein translation initiation is a tightly controlled process responding to nutrient availability and mitogen stimulation. Serving as one of the most important negative regulators of protein translation, 4E binding protein 1 (4E-BP1) binds to translation initiation factor 4E and inhibits cap-dependent translation in a phosphorylation-dependent manner. Although it has been demonstrated previously that the phosphorylation of 4E-BP1 is controlled by mammalian target of rapamycin in the mammalian target of rapamycin complex 1, the mechanism underlying the dephosphorylation of 4E-BP1 remains elusive. Here, we report the identification of PPM1G as the phosphatase of 4E-BP1. A coimmunoprecipitation experiment reveals that PPM1G binds to 4E-BP1 in cells and that purified PPM1G dephosphorylates 4E-BP1 in vitro. Knockdown of PPM1G in 293E and colon cancer HCT116 cells results in an increase in the phosphorylation of 4E-BP1 at both the Thr-37/46 and Ser-65 sites. Furthermore, the time course of 4E-BP1 dephosphorylation induced by amino acid starvation or mammalian target of rapamycin inhibition is slowed down significantly in PPM1G knockdown cells. Functionally, the amount of 4E-BP1 bound to the cap-dependent translation initiation complex is decreased when the expression of PPM1G is depleted. As a result, the rate of cap-dependent translation, cell size, and protein content are increased in PPM1G knockdown cells. Taken together, our study has identified protein phosphatase PPM1G as a novel regulator of cap-dependent protein translation by negatively controlling the phosphorylation of 4E-BP1.

  11. Nucleic acid-binding properties of the RRM-containing protein RDM1

    International Nuclear Information System (INIS)

    Hamimes, Samia; Bourgeon, Dominique; Stasiak, Alicja Z.; Stasiak, Andrzej; Van Dyck, Eric

    2006-01-01

    RDM1 (RAD52 Motif 1) is a vertebrate protein involved in the cellular response to the anti-cancer drug cisplatin. In addition to an RNA recognition motif, RDM1 contains a small amino acid motif, named RD motif, which it shares with the recombination and repair protein, RAD52. RDM1 binds to single- and double-stranded DNA, and recognizes DNA distortions induced by cisplatin adducts in vitro. Here, we have performed an in-depth analysis of the nucleic acid-binding properties of RDM1 using gel-shift assays and electron microscopy. We show that RDM1 possesses acidic pH-dependent DNA-binding activity and that it binds RNA as well as DNA, and we present evidence from competition gel-shift experiments that RDM1 may be capable of discrimination between the two nucleic acids. Based on reported studies of RAD52, we have generated an RDM1 variant mutated in its RD motif. We find that the L 119 GF → AAA mutation affects the mode of RDM1 binding to single-stranded DNA

  12. High-affinity naloxone binding to filamin a prevents mu opioid receptor-Gs coupling underlying opioid tolerance and dependence.

    Directory of Open Access Journals (Sweden)

    Hoau-Yan Wang

    Full Text Available Ultra-low-dose opioid antagonists enhance opioid analgesia and reduce analgesic tolerance and dependence by preventing a G protein coupling switch (Gi/o to Gs by the mu opioid receptor (MOR, although the binding site of such ultra-low-dose opioid antagonists was previously unknown. Here we show that with approximately 200-fold higher affinity than for the mu opioid receptor, naloxone binds a pentapeptide segment of the scaffolding protein filamin A, known to interact with the mu opioid receptor, to disrupt its chronic opioid-induced Gs coupling. Naloxone binding to filamin A is demonstrated by the absence of [(3H]-and FITC-naloxone binding in the melanoma M2 cell line that does not contain filamin or MOR, contrasting with strong [(3H]naloxone binding to its filamin A-transfected subclone A7 or to immunopurified filamin A. Naloxone binding to A7 cells was displaced by naltrexone but not by morphine, indicating a target distinct from opioid receptors and perhaps unique to naloxone and its analogs. The intracellular location of this binding site was confirmed by FITC-NLX binding in intact A7 cells. Overlapping peptide fragments from c-terminal filamin A revealed filamin A(2561-2565 as the binding site, and an alanine scan of this pentapeptide revealed an essential mid-point lysine. Finally, in organotypic striatal slice cultures, peptide fragments containing filamin A(2561-2565 abolished the prevention by 10 pM naloxone of both the chronic morphine-induced mu opioid receptor-Gs coupling and the downstream cAMP excitatory signal. These results establish filamin A as the target for ultra-low-dose opioid antagonists previously shown to enhance opioid analgesia and to prevent opioid tolerance and dependence.

  13. High-Affinity Naloxone Binding to Filamin A Prevents Mu Opioid Receptor–Gs Coupling Underlying Opioid Tolerance and Dependence

    Science.gov (United States)

    Wang, Hoau-Yan; Frankfurt, Maya; Burns, Lindsay H.

    2008-01-01

    Ultra-low-dose opioid antagonists enhance opioid analgesia and reduce analgesic tolerance and dependence by preventing a G protein coupling switch (Gi/o to Gs) by the mu opioid receptor (MOR), although the binding site of such ultra-low-dose opioid antagonists was previously unknown. Here we show that with approximately 200-fold higher affinity than for the mu opioid receptor, naloxone binds a pentapeptide segment of the scaffolding protein filamin A, known to interact with the mu opioid receptor, to disrupt its chronic opioid-induced Gs coupling. Naloxone binding to filamin A is demonstrated by the absence of [3H]-and FITC-naloxone binding in the melanoma M2 cell line that does not contain filamin or MOR, contrasting with strong [3H]naloxone binding to its filamin A-transfected subclone A7 or to immunopurified filamin A. Naloxone binding to A7 cells was displaced by naltrexone but not by morphine, indicating a target distinct from opioid receptors and perhaps unique to naloxone and its analogs. The intracellular location of this binding site was confirmed by FITC-NLX binding in intact A7 cells. Overlapping peptide fragments from c-terminal filamin A revealed filamin A2561-2565 as the binding site, and an alanine scan of this pentapeptide revealed an essential mid-point lysine. Finally, in organotypic striatal slice cultures, peptide fragments containing filamin A2561-2565 abolished the prevention by 10 pM naloxone of both the chronic morphine-induced mu opioid receptor–Gs coupling and the downstream cAMP excitatory signal. These results establish filamin A as the target for ultra-low-dose opioid antagonists previously shown to enhance opioid analgesia and to prevent opioid tolerance and dependence. PMID:18253501

  14. High-affinity naloxone binding to filamin a prevents mu opioid receptor-Gs coupling underlying opioid tolerance and dependence.

    Science.gov (United States)

    Wang, Hoau-Yan; Frankfurt, Maya; Burns, Lindsay H

    2008-02-06

    Ultra-low-dose opioid antagonists enhance opioid analgesia and reduce analgesic tolerance and dependence by preventing a G protein coupling switch (Gi/o to Gs) by the mu opioid receptor (MOR), although the binding site of such ultra-low-dose opioid antagonists was previously unknown. Here we show that with approximately 200-fold higher affinity than for the mu opioid receptor, naloxone binds a pentapeptide segment of the scaffolding protein filamin A, known to interact with the mu opioid receptor, to disrupt its chronic opioid-induced Gs coupling. Naloxone binding to filamin A is demonstrated by the absence of [(3)H]-and FITC-naloxone binding in the melanoma M2 cell line that does not contain filamin or MOR, contrasting with strong [(3)H]naloxone binding to its filamin A-transfected subclone A7 or to immunopurified filamin A. Naloxone binding to A7 cells was displaced by naltrexone but not by morphine, indicating a target distinct from opioid receptors and perhaps unique to naloxone and its analogs. The intracellular location of this binding site was confirmed by FITC-NLX binding in intact A7 cells. Overlapping peptide fragments from c-terminal filamin A revealed filamin A(2561-2565) as the binding site, and an alanine scan of this pentapeptide revealed an essential mid-point lysine. Finally, in organotypic striatal slice cultures, peptide fragments containing filamin A(2561-2565) abolished the prevention by 10 pM naloxone of both the chronic morphine-induced mu opioid receptor-Gs coupling and the downstream cAMP excitatory signal. These results establish filamin A as the target for ultra-low-dose opioid antagonists previously shown to enhance opioid analgesia and to prevent opioid tolerance and dependence.

  15. Intracellular protein delivery activity of peptides derived from insulin-like growth factor binding proteins 3 and 5

    International Nuclear Information System (INIS)

    Goda, Natsuko; Tenno, Takeshi; Inomata, Kosuke; Shirakawa, Masahiro; Tanaka, Toshiki; Hiroaki, Hidekazu

    2008-01-01

    Insulin-like growth factor binding proteins (IGFBPs) have various IGF-independent cellular activities, including receptor-independent cellular uptake followed by transcriptional regulation, although mechanisms of cellular entry remain unclear. Herein, we focused on their receptor-independent cellular entry mechanism in terms of protein transduction domain (PTD) activity, which is an emerging technique useful for clinical applications. The peptides of 18 amino acid residues derived from IGFBP-3 and IGFBP-5, which involve heparin-binding regions, mediated cellular delivery of an exogenous protein into NIH3T3 and HeLa cells. Relative protein delivery activities of IGFBP-3/5-derived peptides were approximately 20-150% compared to that of the HIV-Tat peptide, a potent PTD. Heparin inhibited the uptake of the fusion proteins with IGFBP-3 and IGFBP-5, indicating that the delivery pathway is heparin-dependent endocytosis, similar to that of HIV-Tat. The delivery of GST fused to HIV-Tat was competed by either IGFBP-3 or IGFBP-5-derived synthetic peptides. Therefore, the entry pathways of the three PTDs are shared. Our data has shown a new approach for designing protein delivery systems using IGFBP-3/5 derived peptides based on the molecular mechanisms of IGF-independent activities of IGFBPs

  16. Comparison of two methods forecasting binding rate of plasma protein.

    Science.gov (United States)

    Hongjiu, Liu; Yanrong, Hu

    2014-01-01

    By introducing the descriptors calculated from the molecular structure, the binding rates of plasma protein (BRPP) with seventy diverse drugs are modeled by a quantitative structure-activity relationship (QSAR) technique. Two algorithms, heuristic algorithm (HA) and support vector machine (SVM), are used to establish linear and nonlinear models to forecast BRPP. Empirical analysis shows that there are good performances for HA and SVM with cross-validation correlation coefficients Rcv(2) of 0.80 and 0.83. Comparing HA with SVM, it was found that SVM has more stability and more robustness to forecast BRPP.

  17. Modification of DNA radiolysis by DNA-binding proteins: Structural aspects

    International Nuclear Information System (INIS)

    Davidkova, M.; Stisova, V.; Goffinont, S.; Gillard, N.; Castaing, B.; Spotheim-Maurizot, M.

    2006-01-01

    Formation of specific complexes between proteins and their cognate DNA modulates the yields and the location of radiation damage on both partners of the complex. The radiolysis of DNA-protein complexes is studied for: (1) the Escherichia coli lactose operator-repressor complex, (2) the complex between DNA bearing an analogue of an abasic site and the repair protein Fpg of Lactococcus lactis. Experimental patterns of DNA damages are presented and compared to predicted damage distribution obtained using an improved version of the stochastic model RADACK. The same method is used for predicting the location of damages on the proteins. At doses lower than a threshold that depends on the system, proteins protect their specific binding site on DNA while at high doses, the studied complexes are disrupted mainly through protein damage. The loss of binding ability is the functional consequence of the amino-acids modification by OH . radicals. Many of the most probably damaged amino acids are essential for the DNA-protein interaction and within a complex are protected by DNA. (authors)

  18. Poly(C)-binding proteins as transcriptional regulators of gene expression

    International Nuclear Information System (INIS)

    Choi, Hack Sun; Hwang, Cheol Kyu; Song, Kyu Young; Law, P.-Y.; Wei, L.-N.; Loh, Horace H.

    2009-01-01

    Poly(C)-binding proteins (PCBPs) are generally known as RNA-binding proteins that interact in a sequence-specific fashion with single-stranded poly(C). They can be divided into two groups: hnRNP K and PCBP1-4. These proteins are involved mainly in various posttranscriptional regulations (e.g., mRNA stabilization or translational activation/silencing). In this review, we summarize and discuss how PCBPs act as transcriptional regulators by binding to specific elements in gene promoters that interact with the RNA polymerase II transcription machinery. Transcriptional regulation of PCBPs might itself be regulated by their localization within the cell. For example, activation by p21-activated kinase 1 induces increased nuclear retention of PCBP1, as well as increased promoter activity. PCBPs can function as a signal-dependent and coordinated regulator of transcription in eukaryotic cells. We address the molecular mechanisms by which PCBPs binding to single- and double-stranded DNA mediates gene expression.

  19. Adenovirus DNA binding protein inhibits SrCap-activated CBP and CREB-mediated transcription

    International Nuclear Information System (INIS)

    Xu Xiequn; Tarakanova, Vera; Chrivia, John; Yaciuk, Peter

    2003-01-01

    The SNF2-related CBP activator protein (SrCap) is a potent activator of transcription mediated by CBP and CREB. We have previously demonstrated that the Adenovirus 2 DNA Binding Protein (DBP) binds to SrCap and inhibits the transcription mediated by the carboxyl-terminal region of SrCap (amino acids 1275-2971). We report here that DBP inhibits the ability of full-length SrCap (1-2971) to activate transcription mediated by Gal-CREB and Gal-CBP. In addition, DBP also inhibits the ability of SrCap to enhance Protein Kinase A (PKA) activated transcription of the enkaphalin promoter. DBP was found to dramatically inhibit transcription of a mammalian two-hybrid system that was dependent on the interaction of SrCap and CBP binding domains. We also found that DBP has no effect on transcription mediated by a transcriptional activator that is not related to SrCap, indicating that our reported transcriptional inhibition is specific for SrCap and not due to nonspecific effects of DBP's DNA binding activity on the CAT reporter plasmid. Taken together, these results suggest a model in which DBP inhibits cellular transcription mediated by the interaction between SrCap and CBP

  20. Thermostability and reversibility of silver nanoparticle-protein binding.

    Science.gov (United States)

    Wang, Bo; Seabrook, Shane A; Nedumpully-Govindan, Praveen; Chen, Pengyu; Yin, Hong; Waddington, Lynne; Epa, V Chandana; Winkler, David A; Kirby, Jason K; Ding, Feng; Ke, Pu Chun

    2015-01-21

    The interactions between nanoparticles (NPs) and proteins in living systems are a precursor to the formation of a NP-protein "corona" that underlies cellular and organism responses to nanomaterials. However, the thermodynamic properties and reversibility of NP-protein interactions have rarely been examined. Using an automated, high-throughput and temperature-controlled dynamic light scattering (DLS) technique we observed a distinct hysteresis in the hydrodynamic radius of branched polyethyleneimine (BPEI) coated-silver nanoparticles (bAgNPs) exposed to like-charged lysozyme during the processes of heating and cooling, in contrast to the irreversible interactions between bAgNPs and oppositely charged alpha lactalbumin (ALact). Our discrete molecular dynamics (DMD) simulations offered a new molecular insight into the differential structure, dynamics and thermodynamics of bAgNPs binding with the two protein homologs and further revealed the different roles of the capping agents of citrate and BPEI in NP-protein interactions. This study facilitates our understanding of the transformation of nanomaterials in living systems, whose implications range from the field study of nanotoxicology to nanomaterials synthesis, nanobiotechnology and nanomedicine.

  1. Bee venom phospholipase A2 as a membrane-binding vector for cell surface display or internalization of soluble proteins.

    Science.gov (United States)

    Babon, Aurélie; Wurceldorf, Thibault; Almunia, Christine; Pichard, Sylvain; Chenal, Alexandre; Buhot, Cécile; Beaumelle, Bruno; Gillet, Daniel

    2016-06-15

    We showed that bee venom phospholipase A2 can be used as a membrane-binding vector to anchor to the surface of cells a soluble protein fused to its C-terminus. ZZ, a two-domain derivative of staphylococcal protein A capable of binding constant regions of antibodies was fused to the C-terminus of the phospholipase or to a mutant devoid of enzymatic activity. The fusion proteins bound to the surface of cells and could themselves bind IgGs. Their fate depended on the cell type to which they bound. On the A431 carcinoma cell line the proteins remained exposed on the cell surface. In contrast, on human dendritic cells the proteins were internalized into early endosomes. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Protein-protein binding before and after photo-modification of albumin

    Science.gov (United States)

    Rozinek, Sarah C.; Glickman, Randolph D.; Thomas, Robert J.; Brancaleon, Lorenzo

    2016-03-01

    Bioeffects of directed-optical-energy encompass a wide range of applications. One aspect of photochemical interactions involves irradiating a photosensitizer with visible light in order to induce protein unfolding and consequent changes in function. In the past, irradiation of several dye-protein combinations has revealed effects on protein structure. Beta lactoglobulin, human serum albumin (HSA) and tubulin have all been photo-modified with meso-tetrakis(4- sulfonatophenyl)porphyrin (TSPP) bound, but only in the case of tubulin has binding caused a verified loss of biological function (loss of ability to form microtubules) as a result of this light-induced structural change. The current work questions if the photo-induced structural changes that occur to HSA, are sufficient to disable its biological function of binding to osteonectin. The albumin-binding protein, osteonectin, is about half the molecular weight of HSA, so the two proteins and their bound product can be separated and quantified by size exclusion high performance liquid chromatography. TSPP was first bound to HSA and irradiated, photo-modifying the structure of HSA. Then native HSA or photo-modified HSA (both with TSPP bound) were compared, to assess loss in HSA's innate binding ability as a result of light-induced structure modification.

  3. Efficient purification of recombinant proteins fused to maltose-binding protein by mixed-mode chromatography.

    Science.gov (United States)

    Cabanne, Charlotte; Pezzini, Jérôme; Joucla, Gilles; Hocquellet, Agnès; Barbot, Caroline; Garbay, Bertrand; Santarelli, Xavier

    2009-05-15

    Two mixed-mode resins were evaluated as an alternative to conventional affinity resins for the purification of recombinant proteins fused to maltose-binding protein (MPB). We purified recombinant MBP, MBP-LacZ and MBP-Leap2 from crude Escherichia coli extracts. Mixed-mode resins allowed the efficient purification of MBP-fused proteins. Indeed, the quantity of purified proteins was significantly higher with mixed-mode resins, and their purity was equivalent to that obtained with affinity resins. By using purified MBP, MBP-LacZ and MBP-Leap2, the dynamic binding capacity of mixed-mode resins was 5-fold higher than that of affinity resins. Moreover, the recovery for the three proteins studied was in the 50-60% range for affinity resins, and in the 80-85% range for mixed-mode resins. Mixed-mode resins thus represent a powerful alternative to the classical amylose or dextrin resins for the purification of recombinant proteins fused to maltose-binding protein.

  4. Light-activated DNA binding in a designed allosteric protein

    Energy Technology Data Exchange (ETDEWEB)

    Strickland, Devin; Moffat, Keith; Sosnick, Tobin R. (UC)

    2008-09-03

    An understanding of how allostery, the conformational coupling of distant functional sites, arises in highly evolvable systems is of considerable interest in areas ranging from cell biology to protein design and signaling networks. We reasoned that the rigidity and defined geometry of an {alpha}-helical domain linker would make it effective as a conduit for allosteric signals. To test this idea, we rationally designed 12 fusions between the naturally photoactive LOV2 domain from Avena sativa phototropin 1 and the Escherichia coli trp repressor. When illuminated, one of the fusions selectively binds operator DNA and protects it from nuclease digestion. The ready success of our rational design strategy suggests that the helical 'allosteric lever arm' is a general scheme for coupling the function of two proteins.

  5. The Role of Microtubule End Binding (EB) Proteins in Ciliogenesis

    DEFF Research Database (Denmark)

    Schrøder, Jacob Morville

    centrosomal MT array and abnormally long centriole-associated rootlet filaments. Cells lacking EB1 also had stumpy cilia and a disorganized centrosomal MT array, but rootlet filaments appeared normal. Further, live imaging revealed increased release frequency of MTs from the centrosome upon EB1 or EB3......EB1 is a small microtubule (MT)-binding protein that associates preferentially with MT plus ends. EB1 plays a role in regulating MT dynamics, localizing other MT-associated proteins to the plus end, and in regulating interactions of MTs with the cell cortex, mitotic kinetochores and different......, are required for assembly of primary cilia in cultured human cells. The EB3 - siRNA ciliary phenotype could be rescued by GFP-EB1 expression, and GFP-EB3 over expression resulted in elongated cilia. Transmission electron microscopy (TEM) revealed that EB3-depleted cells possess stumpy cilia, a disorganized...

  6. Ice cream structure modification by ice-binding proteins.

    Science.gov (United States)

    Kaleda, Aleksei; Tsanev, Robert; Klesment, Tiina; Vilu, Raivo; Laos, Katrin

    2018-04-25

    Ice-binding proteins (IBPs), also known as antifreeze proteins, were added to ice cream to investigate their effect on structure and texture. Ice recrystallization inhibition was assessed in the ice cream mixes using a novel accelerated microscope assay and the ice cream microstructure was studied using an ice crystal dispersion method. It was found that adding recombinantly produced fish type III IBPs at a concentration 3 mg·L -1 made ice cream hard and crystalline with improved shape preservation during melting. Ice creams made with IBPs (both from winter rye, and type III IBP) had aggregates of ice crystals that entrapped pockets of the ice cream mixture in a rigid network. Larger individual ice crystals and no entrapment in control ice creams was observed. Based on these results a model of ice crystals aggregates formation in the presence of IBPs was proposed. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. The nucleotide-binding site of the Escherichia coli DnaC protein: molecular topography of DnaC protein-nucleotide cofactor complexes.

    Science.gov (United States)

    Galletto, Roberto; Jezewska, Maria J; Maillard, Rodrigo; Bujalowski, Wlodzimierz

    2005-01-01

    The structure of the nucleotide-binding site of the Escherichia coli replication factor DnaC protein and the effect of the nucleotide cofactor on the protein structure have been examined using ultraviolet, steady-state, and time-dependent fluorescence spectroscopy. Emission spectra and quenching studies of the fluorescent nucleotide analogs, 3'-O-(N-methylantraniloyl)-5'-triphosphate (MANT-ATP) and 3'-O-(N-methylantraniloyl)-5'-diphosphate (MANT-ADP), bound to the DnaC protein indicate that the nucleotide-binding site forms a hydrophobic cleft on the surface of the protein. Fluorescence decays of free and bound MANT-ATP and MANT-ADP indicate that cofactors exist in two different conformations both, free and bound to the protein. However, the two conformations of the bound nucleotides differ in their solvent accessibilities. Moreover, there are significant differences in the solvent accessibility between ATP and ADP complexes. Specific binding of magnesium to the protein controls the structure of the binding site, particularly, in the case of the ATP complex, leading to additional opening of the binding site cleft. Both tyrosine and tryptophan residues are located on the surface of the protein. The tryptophans are clustered at a large distance from the nucleotide-binding site. However, in spite of a large spatial separation, binding of both cofactors induces significant and different changes in the structure of the environment of tryptophans, indicating long-range structural effects through the DnaC molecule. Moreover, only ATP induces changes in the distribution of the tyrosine residues on the surface of the protein. The data reveal that the nucleotide-DnaC protein complex is a sophisticated allosteric system, responding differently to the ATP and ADP binding.

  8. Immobilized sialyloligo-macroligand and its protein binding specificity.

    Science.gov (United States)

    Narla, Satya Nandana; Sun, Xue-Long

    2012-05-14

    We report a chemoenzymatic synthesis of chain-end functionalized sialyllactose-containing glycopolymers with different linkages and their oriented immobilization for glycoarray and SPR-based glyco-biosensor applications. Specifically, O-cyanate chain-end functionalized sialyllactose-containing glycopolymers were synthesized by enzymatic α2,3- and α2,6-sialylation of a lactose-containing glycopolymer that was synthesized by cyanoxyl-mediated free radical polymerization. (1)H NMR showed almost quantitative α2,3- and α2,6-sialylation. The O-cyanate chain-end functionalized sialyllactose-containing glycopolymers were printed onto amine-functionalized glass slides via isourea bond formation for glycoarray formation. Specific protein binding activity of the arrays was confirmed with α2,3- and α2,6-sialyl specific binding lectins together with inhibition assays. Further, immobilizing O-cyanate chain-end functionalized sialyllactose-containing glycopolymers onto amine-modified SPR chip via isourea bond formation afforded SPR-based glyco-biosensor, which showed specific binding activity for lectins and influenza viral hemagglutinins (HA). These sialyloligo-macroligand derived glycoarray and SPR-based glyco-biosensor are closely to mimic 3D nature presentation of sialyloligosaccharides and will provide important high-throughput tools for virus diagnosis and potential antiviral drug candidates screening applications.

  9. Comprehensive review and empirical analysis of hallmarks of DNA-, RNA- and protein-binding residues in protein chains.

    Science.gov (United States)

    Zhang, Jian; Ma, Zhiqiang; Kurgan, Lukasz

    2017-12-15

    Proteins interact with a variety of molecules including proteins and nucleic acids. We review a comprehensive collection of over 50 studies that analyze and/or predict these interactions. While majority of these studies address either solely protein-DNA or protein-RNA binding, only a few have a wider scope that covers both protein-protein and protein-nucleic acid binding. Our analysis reveals that binding residues are typically characterized with three hallmarks: relative solvent accessibility (RSA), evolutionary conservation and propensity of amino acids (AAs) for binding. Motivated by drawbacks of the prior studies, we perform a large-scale analysis to quantify and contrast the three hallmarks for residues that bind DNA-, RNA-, protein- and (for the first time) multi-ligand-binding residues that interact with DNA and proteins, and with RNA and proteins. Results generated on a well-annotated data set of over 23 000 proteins show that conservation of binding residues is higher for nucleic acid- than protein-binding residues. Multi-ligand-binding residues are more conserved and have higher RSA than single-ligand-binding residues. We empirically show that each hallmark discriminates between binding and nonbinding residues, even predicted RSA, and that combining them improves discriminatory power for each of the five types of interactions. Linear scoring functions that combine these hallmarks offer good predictive performance of residue-level propensity for binding and provide intuitive interpretation of predictions. Better understanding of these residue-level interactions will facilitate development of methods that accurately predict binding in the exponentially growing databases of protein sequences. © The Author(s) 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  10. A SILAC-based screen for Methyl-CpG binding proteins identifies RBP-J as a DNA methylation and sequence-specific binding protein.

    Directory of Open Access Journals (Sweden)

    Stefanie J J Bartels

    Full Text Available BACKGROUND: DNA methylation is an epigenetic modification that plays a crucial role in a variety of biological processes. Methylated DNA is specifically bound by Methyl-CpG Binding Proteins (MBPs. Three different types of MBPs have been identified so far: the Methyl-CpG Binding Domain (MBD family proteins, three BTB/POZ-Zn-finger proteins, and UHRF1. Most of the known MBPs have been identified via homology with the MBD and Zn-finger domains as present in MeCP2 and Kaiso, respectively. It is conceivable that other proteins are capable of recognizing methylated DNA. METHODOLOGY/PRINCIPAL FINDINGS: For the purpose of identifying novel 'readers' we set up a methyl-CpG pull-down assay combined with stable-isotope labeling by amino acids in cell culture (SILAC. In a methyl-CpG pull-down with U937 nuclear extracts, we recovered several known MBPs and almost all subunits of the MBD2/NuRD complex as methylation specific binders, providing proof-of-principle. Interestingly, RBP-J, the transcription factor downstream of Notch receptors, also bound the DNA in a methylation dependent manner. Follow-up pull-downs and electrophoretic mobility shift assays (EMSAs showed that RBP-J binds methylated DNA in the context of a mutated RBP-J consensus motif. CONCLUSIONS/SIGNIFICANCE: The here described SILAC/methyl-CpG pull-down constitutes a new approach to identify potential novel DNAme readers and will advance unraveling of the complete methyl-DNA interactome.

  11. On the prediction of DNA-binding proteins only from primary sequences: A deep learning approach.

    Directory of Open Access Journals (Sweden)

    Yu-Hui Qu

    Full Text Available DNA-binding proteins play pivotal roles in alternative splicing, RNA editing, methylating and many other biological functions for both eukaryotic and prokaryotic proteomes. Predicting the functions of these proteins from primary amino acids sequences is becoming one of the major challenges in functional annotations of genomes. Traditional prediction methods often devote themselves to extracting physiochemical features from sequences but ignoring motif information and location information between motifs. Meanwhile, the small scale of data volumes and large noises in training data result in lower accuracy and reliability of predictions. In this paper, we propose a deep learning based method to identify DNA-binding proteins from primary sequences alone. It utilizes two stages of convolutional neutral network to detect the function domains of protein sequences, and the long short-term memory neural network to identify their long term dependencies, an binary cross entropy to evaluate the quality of the neural networks. When the proposed method is tested with a realistic DNA binding protein dataset, it achieves a prediction accuracy of 94.2% at the Matthew's correlation coefficient of 0.961. Compared with the LibSVM on the arabidopsis and yeast datasets via independent tests, the accuracy raises by 9% and 4% respectively. Comparative experiments using different feature extraction methods show that our model performs similar accuracy with the best of others, but its values of sensitivity, specificity and AUC increase by 27.83%, 1.31% and 16.21% respectively. Those results suggest that our method is a promising tool for identifying DNA-binding proteins.

  12. Identification of Actin-Binding Proteins from Maize Pollen

    Energy Technology Data Exchange (ETDEWEB)

    Staiger, C.J.

    2004-01-13

    Specific Aims--The goal of this project was to gain an understanding of how actin filament organization and dynamics are controlled in flowering plants. Specifically, we proposed to identify unique proteins with novel functions by investigating biochemical strategies for the isolation and characterization of actin-binding proteins (ABPs). In particular, our hunt was designed to identify capping proteins and nucleation factors. The specific aims included: (1) to use F-actin affinity chromatography (FAAC) as a general strategy to isolate pollen ABPs (2) to produce polyclonal antisera and perform subcellular localization in pollen tubes (3) to isolate cDNA clones for the most promising ABPs (4) to further purify and characterize ABP interactions with actin in vitro. Summary of Progress By employing affinity chromatography on F-actin or DNase I columns, we have identified at least two novel ABPs from pollen, PrABP80 (gelsolin-like) and ZmABP30, We have also cloned and expressed recombinant protein, as well as generated polyclonal antisera, for 6 interesting ABPs from Arabidopsis (fimbrin AtFIM1, capping protein a/b (AtCP), adenylyl cyclase-associated protein (AtCAP), AtCapG & AtVLN1). We performed quantitative analyses of the biochemical properties for two of these previously uncharacterized ABPs (fimbrin and capping protein). Our studies provide the first evidence for fimbrin activity in plants, demonstrate the existence of barbed-end capping factors and a gelsolin-like severing activity, and provide the quantitative data necessary to establish and test models of F-actin organization and dynamics in plant cells.

  13. Immunochemical characterization of the brain glutamate binding protein

    International Nuclear Information System (INIS)

    Roy, S.

    1986-01-01

    A glutamate binding protein (GBP) was purified from bovine and rat brain to near homogeneity. Polyclonal antibodies were raised against this protein. An enzyme-linked-immunosorbent-assay was used to quantify and determine the specificity of the antibody response. The antibodies were shown to strongly react with bovine brain GBP and the analogous protein from rat brain. The antibodies did not show any crossreactivity with the glutamate metabolizing enzymes, glutamate dehydrogenase, glutamine synthetase and glutamyl transpeptidase, however it crossreacted moderately with glutamate decarboxylase. The antibodies were also used to define the possible physiologic activity of GBP in synaptic membranes. The antibodies were shown: (i) to inhibit the excitatory amino-acid stimulation of thiocyanate (SCN)flux, (ii) had no effect on transport of L-Glutamic acid across the synaptic membrane, and (iii) had no effect on the depolarization-induced release of L-glutamate. When the anti-GBP antibodies were used to localize and quantify the GBP distribution in various subcellular fractions and in brain tissue samples, it was found that the hippocampus had the highest immunoreactivity followed by the cerebral cortex, cerebellar cortex and caudate-putamen. The distribution of immunoreactivity in the subcellular fraction were as follows: synaptic membranes > crude mitochondrial fraction > homogenate > myelin. In conclusion these studies suggest that: (a) the rat brain GBP and the bovine brain GBP are immunologically homologous protein, (b) there are no structural similarities between the GBP and the glutamate metabolizing enzymes with the exception of glutamate decarboxylase and (c) the subcellular and regional distribution of the GBP immunoreactivity followed a similar pattern as observed for L-[ 3 H]-binding

  14. Thermodynamics parameters for binding of halogenated benzotriazole inhibitors of human protein kinase CK2α.

    Science.gov (United States)

    Winiewska, Maria; Kucińska, Katarzyna; Makowska, Małgorzata; Poznański, Jarosław; Shugar, David

    2015-10-01

    The interaction of human CK2α (hCK2α) with nine halogenated benzotriazoles, TBBt and its analogues representing all possible patterns of halogenation on the benzene ring of benzotriazole, was studied by biophysical methods. Thermal stability of protein-ligand complexes, monitored by calorimetric (DSC) and optical (DSF) methods, showed that the increase in the mid-point temperature for unfolding of protein-ligand complexes (i.e. potency of ligand binding to hCK2α) follow the inhibitory activities determined by biochemical assays. The dissociation constant for the ATP-hCK2α complex was estimated with the aid of microscale thermophoresis (MST) as 4.3±1.8 μM, and MST-derived dissociation constants determined for halogenated benzotriazoles, when converted according to known ATP concentrations, perfectly reconstruct IC50 values determined by the biochemical assays. Ligand-dependent quenching of tyrosine fluorescence, together with molecular modeling and DSC-derived heats of unfolding, support the hypothesis that halogenated benzotriazoles bind in at least two alternative orientations, and those that are efficient hCK2α inhibitors bind in the orientation which TBBt adopts in its complex with maize CK2α. DSC-derived apparent heat for ligand binding (ΔΔHbind) is driven by intermolecular electrostatic interactions between Lys68 and the triazole ring of the ligand, as indicated by a good correlation between ΔΔHbind and ligand pKa. Overall results, additionally supported by molecular modeling, confirm that a balance of hydrophobic and electrostatic interactions contribute predominantly (~40 kJ/mol), relative to possible intermolecular halogen/hydrogen bonding (less than 10 kJ/mol), in binding of halogenated benzotriazoles to the ATP-binding site of hCK2α. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Prediction of protein-protein binding site by using core interface residue and support vector machine

    Directory of Open Access Journals (Sweden)

    Sun Zhonghua

    2008-12-01

    Full Text Available Abstract Background The prediction of protein-protein binding site can provide structural annotation to the protein interaction data from proteomics studies. This is very important for the biological application of the protein interaction data that is increasing rapidly. Moreover, methods for predicting protein interaction sites can also provide crucial information for improving the speed and accuracy of protein docking methods. Results In this work, we describe a binding site prediction method by designing a new residue neighbour profile and by selecting only the core-interface residues for SVM training. The residue neighbour profile includes both the sequential and the spatial neighbour residues of an interface residue, which is a more complete description of the physical and chemical characteristics surrounding the interface residue. The concept of core interface is applied in selecting the interface residues for training the SVM models, which is shown to result in better discrimination between the core interface and other residues. The best SVM model trained was tested on a test set of 50 randomly selected proteins. The sensitivity, specificity, and MCC for the prediction of the core interface residues were 60.6%, 53.4%, and 0.243, respectively. Our prediction results on this test set were compared with other three binding site prediction methods and found to perform better. Furthermore, our method was tested on the 101 unbound proteins from the protein-protein interaction benchmark v2.0. The sensitivity, specificity, and MCC of this test were 57.5%, 32.5%, and 0.168, respectively. Conclusion By improving both the descriptions of the interface residues and their surrounding environment and the training strategy, better SVM models were obtained and shown to outperform previous methods. Our tests on the unbound protein structures suggest further improvement is possible.

  16. Protein kinase C interaction with calcium: a phospholipid-dependent process.

    LENUS (Irish Health Repository)

    Bazzi, M D

    1990-08-21

    The calcium-binding properties of calcium- and phospholipid-dependent protein kinase C (PKC) were investigated by equilibrium dialysis in the presence and the absence of phospholipids. Calcium binding to PKC displayed striking and unexpected behavior; the free proteins bound virtually no calcium at intracellular calcium concentrations and bound limited calcium (about 1 mol\\/mol of PKC) at 200 microM calcium. However, in the presence of membranes containing acidic phospholipids, PKC bound at least eight calcium ions per protein. The presence of 1 microM phorbol dibutyrate (PDBu) in the dialysis buffer had little effect on these calcium-binding properties. Analysis of PKC-calcium binding by gel filtration under equilibrium conditions gave similar results; only membrane-associated PKC bound significant amounts of calcium. Consequently, PKC is a member of what may be a large group of proteins that bind calcium in a phospholipid-dependent manner. The calcium concentrations needed to induce PKC-membrane binding were similar to those needed for calcium binding (about 40 microM calcium at the midpoint). However, the calcium concentration required for PKC-membrane binding was strongly influenced by the phosphatidylserine composition of the membranes. Membranes with higher percentages of phosphatidylserine required lower concentrations of calcium. These properties suggested that the calcium sites may be generated at the interface between PKC and the membrane. Calcium may function as a bridge between PKC and phospholipids. These studies also suggested that calcium-dependent PKC-membrane binding and PKC function could be regulated by a number of factors in addition to calcium levels and diacylglycerol content of the membrane.

  17. Recrystallization inhibition in ice due to ice binding protein activity detected by nuclear magnetic resonance.

    Science.gov (United States)

    Brown, Jennifer R; Seymour, Joseph D; Brox, Timothy I; Skidmore, Mark L; Wang, Chen; Christner, Brent C; Luo, Bing-Hao; Codd, Sarah L

    2014-09-01

    Liquid water present in polycrystalline ice at the interstices between ice crystals results in a network of liquid-filled veins and nodes within a solid ice matrix, making ice a low porosity porous media. Here we used nuclear magnetic resonance (NMR) relaxation and time dependent self-diffusion measurements developed for porous media applications to monitor three dimensional changes to the vein network in ices with and without a bacterial ice binding protein (IBP). Shorter effective diffusion distances were detected as a function of increased irreversible ice binding activity, indicating inhibition of ice recrystallization and persistent small crystal structure. The modification of ice structure by the IBP demonstrates a potential mechanism for the microorganism to enhance survivability in ice. These results highlight the potential of NMR techniques in evaluation of the impact of IBPs on vein network structure and recrystallization processes; information useful for continued development of ice-interacting proteins for biotechnology applications.

  18. A sequence-based dynamic ensemble learning system for protein ligand-binding site prediction

    KAUST Repository

    Chen, Peng

    2015-12-03

    Background: Proteins have the fundamental ability to selectively bind to other molecules and perform specific functions through such interactions, such as protein-ligand binding. Accurate prediction of protein residues that physically bind to ligands is important for drug design and protein docking studies. Most of the successful protein-ligand binding predictions were based on known structures. However, structural information is not largely available in practice due to the huge gap between the number of known protein sequences and that of experimentally solved structures

  19. Evolving Transcription Factor Binding Site Models From Protein Binding Microarray Data

    KAUST Repository

    Wong, Ka-Chun

    2016-02-02

    Protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner. In this paper, we describe the PBM motif model building problem. We apply several evolutionary computation methods and compare their performance with the interior point method, demonstrating their performance advantages. In addition, given the PBM domain knowledge, we propose and describe a novel method called kmerGA which makes domain-specific assumptions to exploit PBM data properties to build more accurate models than the other models built. The effectiveness and robustness of kmerGA is supported by comprehensive performance benchmarking on more than 200 datasets, time complexity analysis, convergence analysis, parameter analysis, and case studies. To demonstrate its utility further, kmerGA is applied to two real world applications: 1) PBM rotation testing and 2) ChIP-Seq peak sequence prediction. The results support the biological relevance of the models learned by kmerGA, and thus its real world applicability.

  20. Salicylate clearance, the resultant of protein binding and metabolism.

    Science.gov (United States)

    Furst, D E; Tozer, T N; Melmon, K L

    1979-09-01

    Steady-state plasma salicylate concentrations and protein binding were examined in 9 normal subjects to determine relationships among daily dose, total and unbound salicylate concentrations, and total and unbound clearances. Aspirin doses ranging from 0.66 to 4.0 mg/kg/hr were given to steady state. Free and total salicylate concentrations were measured with spectrophotometric, fluorimetric, and equilibrium dialysis techniques. Although unbound clearance decreased over the therapeutic range, total clearance was unchanged. The former is a consequence of saturable metabolism; the latter, of saturable plasma protein binding as well as saturable metabolism. The fraction unbound increased linearly with unbound concentration. Clearance determined at 1.8 mg/kg/hr was used to predict levels obtained at higher aspirin doses. Analysis of residuals was used to ascertain the accuracy of the prediction. The coefficient of variation from prediction among subjects was found to be +/- 14%. It is concluded that, in normal subjects, salicylate clearance changes relatively little over the therapeutic range because the increasing fraction unbound compensates for decreasing clearance of unbound drug.

  1. Tannin-binding salivary proteins in three captive rhinoceros species.

    Science.gov (United States)

    Clauss, Marcus; Gehrke, Janin; Hatt, Jean-Michel; Dierenfeld, Ellen S; Flach, Edmund J; Hermes, Robert; Castell, Johanna; Streich, W Juergen; Fickel, Joerns

    2005-01-01

    Tannin-binding salivary proteins (TBSP) are considered to be counter-defences acquired in the course of evolution by animals whose natural forage contains such tannins. As tannins mostly occur in browse material but not in grasses, it is assumed that grazers do not have a need for TBSP. Whereas it has been shown in several non-ungulate species that TBSP can be induced by dietary tannins, their presence or absence in ungulates has, so far, been shown to be a species-specific characteristic independent of dietary manipulations. We investigated saliva from three rhinoceros species from zoological gardens fed comparable, conventional zoo diets. As expected, saliva from white rhinoceroses (Ceratotherum simum, grazer) had lower tannin-binding capacities than that from black rhinoceroses (Diceros bicornis, browser). Surprisingly, however, Indian rhinoceroses (Rhinoceros unicornis), commonly regarded as grazers as well, displayed the highest tannin-binding capacities of the three species investigated. It is speculated that this discrepancy might be a result of an evolutionarily recent switch to a grass-dominated diet in Indian rhinoceroses, and that the black rhinoceros, which is closer related to the white rhinoceros than the Indian species, has evolved an inducible mechanism of TBSP production. In separate trials during which the tannin content of the diets of black rhinoceroses was increased by the addition of either tannic acid or quebracho, the tannin-binding capacity of black rhinoceros saliva was increased to levels within the same range as that of Indian rhinoceroses on the conventional diets. While induction trials in white and Indian rhinoceroses remain to be performed for a full understanding of salivary anti-tannin defence in rhinoceroses, these results are the first report of an induced salivary response to increased dietary tannin levels in an ungulate species.

  2. The telomere binding protein TRF2 induces chromatin compaction.

    Directory of Open Access Journals (Sweden)

    Asmaa M Baker

    2011-04-01

    Full Text Available Mammalian telomeres are specialized chromatin structures that require the telomere binding protein, TRF2, for maintaining chromosome stability. In addition to its ability to modulate DNA repair activities, TRF2 also has direct effects on DNA structure and topology. Given that mammalian telomeric chromatin includes nucleosomes, we investigated the effect of this protein on chromatin structure. TRF2 bound to reconstituted telomeric nucleosomal fibers through both its basic N-terminus and its C-terminal DNA binding domain. Analytical agarose gel electrophoresis (AAGE studies showed that TRF2 promoted the folding of nucleosomal arrays into more compact structures by neutralizing negative surface charge. A construct containing the N-terminal and TRFH domains together altered the charge and radius of nucleosomal arrays similarly to full-length TRF2 suggesting that TRF2-driven changes in global chromatin structure were largely due to these regions. However, the most compact chromatin structures were induced by the isolated basic N-terminal region, as judged by both AAGE and atomic force microscopy. Although the N-terminal region condensed nucleosomal array fibers, the TRFH domain, known to alter DNA topology, was required for stimulation of a strand invasion-like reaction with nucleosomal arrays. Optimal strand invasion also required the C-terminal DNA binding domain. Furthermore, the reaction was not stimulated on linear histone-free DNA. Our data suggest that nucleosomal chromatin has the ability to facilitate this activity of TRF2 which is thought to be involved in stabilizing looped telomere structures.

  3. Identification of DNA-Binding Proteins Using Mixed Feature Representation Methods.

    Science.gov (United States)

    Qu, Kaiyang; Han, Ke; Wu, Song; Wang, Guohua; Wei, Leyi

    2017-09-22

    DNA-binding proteins play vital roles in cellular processes, such as DNA packaging, replication, transcription, regulation, and other DNA-associated activities. The current main prediction method is based on machine learning, and its accuracy mainly depends on the features extraction method. Therefore, using an efficient feature representation method is important to enhance the classification accuracy. However, existing feature representation methods cannot efficiently distinguish DNA-binding proteins from non-DNA-binding proteins. In this paper, a multi-feature representation method, which combines three feature representation methods, namely, K-Skip-N-Grams, Information theory, and Sequential and structural features (SSF), is used to represent the protein sequences and improve feature representation ability. In addition, the classifier is a support vector machine. The mixed-feature representation method is evaluated using 10-fold cross-validation and a test set. Feature vectors, which are obtained from a combination of three feature extractions, show the best performance in 10-fold cross-validation both under non-dimensional reduction and dimensional reduction by max-relevance-max-distance. Moreover, the reduced mixed feature method performs better than the non-reduced mixed feature technique. The feature vectors, which are a combination of SSF and K-Skip-N-Grams, show the best performance in the test set. Among these methods, mixed features exhibit superiority over the single features.

  4. Identification of DNA-Binding Proteins Using Mixed Feature Representation Methods

    Directory of Open Access Journals (Sweden)

    Kaiyang Qu

    2017-09-01

    Full Text Available DNA-binding proteins play vital roles in cellular processes, such as DNA packaging, replication, transcription, regulation, and other DNA-associated activities. The current main prediction method is based on machine learning, and its accuracy mainly depends on the features extraction method. Therefore, using an efficient feature representation method is important to enhance the classification accuracy. However, existing feature representation methods cannot efficiently distinguish DNA-binding proteins from non-DNA-binding proteins. In this paper, a multi-feature representation method, which combines three feature representation methods, namely, K-Skip-N-Grams, Information theory, and Sequential and structural features (SSF, is used to represent the protein sequences and improve feature representation ability. In addition, the classifier is a support vector machine. The mixed-feature representation method is evaluated using 10-fold cross-validation and a test set. Feature vectors, which are obtained from a combination of three feature extractions, show the best performance in 10-fold cross-validation both under non-dimensional reduction and dimensional reduction by max-relevance-max-distance. Moreover, the reduced mixed feature method performs better than the non-reduced mixed feature technique. The feature vectors, which are a combination of SSF and K-Skip-N-Grams, show the best performance in the test set. Among these methods, mixed features exhibit superiority over the single features.

  5. Acyl-CoA binding protein is an essential protein in mammalian cell lines

    DEFF Research Database (Denmark)

    Knudsen, Jens; Færgeman, Nils J.

    2002-01-01

    In the present work, small interference RNA was used to knock-down acyl-CoA binding protein (ACBP) in HeLa, HepG2 and Chang cells. Transfection with ACBP-specific siRNA stopped growth, detached cells from the growth surface and blocked thymidine and acetate incorporation. The results show...... that depletion of ACBP in mammalian cells results in lethality, suggesting that ACBP is an essential protein....

  6. Plant Kinesin-Like Calmodulin Binding Protein Employs Its Regulatory Domain for Dimerization.

    Directory of Open Access Journals (Sweden)

    Maia V Vinogradova

    Full Text Available Kinesin-like calmodulin binding protein (KCBP, a Kinesin-14 family motor protein, is involved in the structural organization of microtubules during mitosis and trichome morphogenesis in plants. The molecular mechanism of microtubule bundling by KCBP remains unknown. KCBP binding to microtubules is regulated by Ca(2+-binding proteins that recognize its C-terminal regulatory domain. In this work, we have discovered a new function of the regulatory domain. We present a crystal structure of an Arabidopsis KCBP fragment showing that the C-terminal regulatory domain forms a dimerization interface for KCBP. This dimerization site is distinct from the dimerization interface within the N-terminal domain. Side chains of hydrophobic residues of the calmodulin binding helix of the regulatory domain form the C-terminal dimerization interface. Biochemical experiments show that another segment of the regulatory domain located beyond the dimerization interface, its negatively charged coil, is unexpectedly and absolutely required to stabilize the dimers. The strong microtubule bundling properties of KCBP are unaffected by deletion of the C-terminal regulatory domain. The slow minus-end directed motility of KCBP is also unchanged in vitro. Although the C-terminal domain is not essential for microtubule bundling, we suggest that KCBP may use its two independent dimerization interfaces to support different types of bundled microtubule structures in cells. Two distinct dimerization sites may provide a mechanism for microtubule rearrangement in response to Ca(2+ signaling since Ca(2+- binding proteins can disengage KCBP dimers dependent on its C-terminal dimerization interface.

  7. Localization of cellular retinol-binding protein and retinol-binding protein in cells comprising the blood-brain barrier of rat and human

    International Nuclear Information System (INIS)

    MacDonald, P.N.; Ong, D.E.; Bok, D.

    1990-01-01

    Brain is not generally recognized as an organ that requires vitamin A, perhaps because no obvious histologic lesions have been observed in severely vitamin A-deficient animals. However, brain tissue does contain cellular vitamin A-binding proteins and a nuclear receptor protein for retinoic acid. In the present study, immunohistochemical techniques were used to determine the cell-specific location of cellular retinol-binding protein in human and rat brain tissue. Cellular retinol-binding protein was localized specifically within the cuboidal epithelial cells of the choroid plexus, two primary sites of the mammalian blood-brain barrier. In addition, autoradiographic procedures demonstrated binding sites for serum retinol-binding protein in the choroidal epithelium. These observations suggest that a significant movement of retinol across the blood-brain barrier may occur

  8. Electrophilicities and Protein Covalent Binding of Demethylation Metabolites of Colchicine.

    Science.gov (United States)

    Guo, Xiucai; Lin, Dongju; Li, Weiwei; Wang, Kai; Peng, Ying; Zheng, Jiang

    2016-03-21

    Colchicine, an alkaloid existing in plants of Liliaceous colchicum, has been widely used in the treatment of gout and familial Mediterranean fever. The administration of colchicine was found to cause liver injury in humans. The mechanisms of colchicine-induced liver toxicity remain unknown. The objectives of this study were to determine the electrophilicities of demethylation metabolites of colchicine and investigate the protein adductions derived from the reactive metabolites of colchicine. Four demethylated colchicine (1-, 2-, 3-, and 10-DMCs), namely, M1-M4, were detected in colchicine-fortified microsomal incubations. Four N-acetyl cysteine (NAC) conjugates (M5-M8) derived from colchicine were detected in the microsomes in the presence of NAC. M5 and M6 were derived from 10-DMC. M7 resulted from the reaction of 2-DMC or 3-DMC with NAC, and M8 originated from 10-DMC. Microsomal protein covalent binding was observed after exposure to colchicine. Two cysteine adducts (CA-1 and CA-2) derived from 10-DMC were found in proteolytically digested microsomal protein samples after incubation with colchicine. The findings allow us to define the chemical property of demethylation metabolites of colchicine and the interaction between protein and the reactive metabolites of colchicine generated in situ.

  9. Metals and Neuronal Metal Binding Proteins Implicated in Alzheimer's Disease

    Science.gov (United States)

    2016-01-01

    Alzheimer's disease (AD) is the most prevalent age-related dementia affecting millions of people worldwide. Its main pathological hallmark feature is the formation of insoluble protein deposits of amyloid-β and hyperphosphorylated tau protein into extracellular plaques and intracellular neurofibrillary tangles, respectively. Many of the mechanistic details of this process remain unknown, but a well-established consequence of protein aggregation is synapse dysfunction and neuronal loss in the AD brain. Different pathways including mitochondrial dysfunction, oxidative stress, inflammation, and metal metabolism have been suggested to be implicated in this process. In particular, a body of evidence suggests that neuronal metal ions such as copper, zinc, and iron play important roles in brain function in health and disease states and altered homeostasis and distribution as a common feature across different neurodegenerative diseases and aging. In this focused review, we overview neuronal proteins that are involved in AD and whose metal binding properties may underlie important biochemical and regulatory processes occurring in the brain during the AD pathophysiological process. PMID:26881049

  10. Isolation and Purification of Thiamine Binding Protein from Mung Bean

    Directory of Open Access Journals (Sweden)

    DWIRINI RETNO GUNARTI

    2013-03-01

    Full Text Available Thiamine has fundamental role in energy metabolism. The organs mostly sensitive to the lack of thiamine levels in the body are the nervous system and the heart. Thiamine deficiency causes symptoms of polyneuritis and cardiovascular diseases. Because of its importance in the metabolism of carbohydrates, we need to measure the levels of thiamine in the body fluids by using an easy and inexpensive way without compromising the sensitivity and selectivity. An option to it is thiamine measurement based on the principle of which is analogous to ELISA, in which a thiamine binding protein (TBP act by replacing antibodies. The presence of TBP in several seeds have been reported by previous researchers, but the presence of TBP in mung beans has not been studied. This study was aimed to isolate and purify TBP from mung bean. The protein was isolated from mung bean through salting out by ammonium sulphate of 40, 70, and 90% (w/v. TBP has a negative charge as shown by cellulose acetate electrophoresis. The result obtained after salting out by ammonium sulphate was further purified bymeans of DEAE-cellulose chromatography and affinity chromatography. In precipitation of 90% of salting out method, one peak protein was obtained by using affinity chromatography. The protein was analyzed by SDS PAGE electrophoresis. The result of SDS PAGE electrophoresis showed that TBP has a molecular weight of 72.63 kDa.

  11. Perinuclear localisation of cellular retinoic acid binding protein I mRNA

    International Nuclear Information System (INIS)

    Levadoux-Martin, M.; Li, Y.; Blackburn, A.; Chabanon, H.; Hesketh, J.E.

    2006-01-01

    Retinoids are important metabolic and developmental regulators that act through nuclear receptors. The cellular retinoic acid binding protein CRABPI has been suggested to play a role in trafficking of retinoic acid but its exact functions and subcellular localisation remain unclear. Here we show that in CHO cells both exogenous CRABPI transcripts and tagged CRABPI protein have a perinuclear distribution that depends upon the 3'UTR of the mRNA. The CRABPI 3'UTR conferred perinuclear localisation on globin reporter transcripts. Deletion analysis indicated that First 123nt of CRABPI 3'UTR are necessary for localisation of both CRABPI mRNA and protein. We propose that CRABPI mRNA is localised by a signal within its 3'UTR and that this partly determines the distribution of CRABPI protein

  12. Complement-Mediated Bactericidal Activity of Anti-Factor H Binding Protein Monoclonal Antibodies against the Meningococcus Relies upon Blocking Factor H Binding

    Science.gov (United States)

    Giuntini, Serena; Reason, Donald C.; Granoff, Dan M.

    2011-01-01

    Binding of the complement-downregulating protein factor H (fH) to the surface of the meningococcus is important for survival of the organism in human serum. The meningococcal vaccine candidate factor H binding protein (fHbp) is an important ligand for human fH. While some fHbp-specific monoclonal antibodies (MAbs) block binding of fH to fHbp, the stoichiometry of blocking in the presence of high serum concentrations of fH and its effect on complement-mediated bactericidal activity are unknown. To investigate this question, we constructed chimeric antibodies in which the human IgG1 constant region was paired with three murine fHbp-specific binding domains designated JAR 3, JAR 5, and MAb502. By surface plasmon resonance, the association rates for binding of all three MAbs to immobilized fHbp were >50-fold higher than that for binding of fH to fHbp, and the MAb dissociation rates were >500-fold lower than that for fH. While all three MAbs elicited similar C1q-dependent C4b deposition on live bacteria (classical complement pathway), only those antibodies that inhibited binding of fH to fHbp (JAR 3 and JAR 5) had bactericidal activity with human complement. MAb502, which did not inhibit fH binding, had complement-mediated bactericidal activity only when tested with fH-depleted human complement. When an IgG1 anti-fHbp MAb binds to sparsely exposed fHbp on the bacterial surface, there appears to be insufficient complement activation for bacteriolysis unless fH binding also is inhibited. The ability of fHbp vaccines to elicit protective antibodies, therefore, is likely to be enhanced if the antibody repertoire is of high avidity and includes fH-blocking activity. PMID:21708990

  13. Temperature dependent conformation studies of Calmodulin Protein using Molecular Dynamics

    Science.gov (United States)

    Aneja, Sahil; Bhartiya, Vivek Kumar; Negi, Sunita

    2016-10-01

    Calmodulin (CaM) protein plays a very crucial role in the calcium signaling inside the eukaryotic cell structure [1, 2]. It can also bind to other proteins/targets and facilitate various activities inside the cell [3, 4]. Temperature dependent conformation changes in the CaM protein are studied with extensive molecular dynamics simulations. The quantitative comparison of simulation data with various forms of experimental results probing different aspects of the folding process can facilitate robust assessment of the accuracy of the calculations. It can also provide a detailed structural interpretation for the experimental observations as well as physical interpretation for theory behind different aspects of the experiment. Earlier these kinds of studies have been performed experimentally using fluorescence measurements as in [5]. The calcium bound form of CaM is observed to undergo a reversible conformation change in the range 295-301 K at calcium ion concentration 150 mM. The transition temperature was observed to depend on the calcium ion concentration of the protein. Leap-dynamics approach was used earlier to study the temperature dependent conformation change of CaM [6]. At 290 K, both the N- and C-lobes were stable, at 325 K, the C-lobe unfolds whereas at 360 both the lobes unfold [6]. In this work, we perform molecular dynamics simulations of 100 ns each for the temperatures 325 K and 375 K on the apo form of CaM, 3CLN and 1CFD. A remarkable dependence of the temperature is observed on the overall dynamics of both the forms of the protein as reported in our earlier study [7, 8]. 1CFD shows a much flexible linker as compared to 3CLN whereas the overall dynamics of the lobes mainly N-lobe is observed to be more in later case. Salt bridge formation between the residues 2 (ASP) and 148 (LYS) leads to a more compact form of 1CFD at 325 K. The unfolding of the protein is observed to increase with the increase in the temperature similar to the earlier reported

  14. Differential dissociation micromethod for the investigation of binding of metandrostenolone (Nerobol) to plasma proteins

    International Nuclear Information System (INIS)

    Bojadzsieva, Milka; Kocsar, Laszlo; Kremmer, Tibor

    1985-01-01

    A micromethod was developed to determine the binding of anabolic streoids to plasma proteins. The new procedure combines precipitation with ammonium sulphate and differential dissociation. The binding parameters (association constant, specific binding capacity) are calculated on the basis of dissociation curves of sup(3)H-metandrostenolone from the precipitated sexual binding globuline. (L.E.)

  15. Differential dissociation micromethod for the investigation of binding of metandrostenolone (Nerobol) to plasma proteins

    Energy Technology Data Exchange (ETDEWEB)

    Bojadzsieva, M.; Kocsar, L. (Orszagos Frederic Joliot-Curie Sugarbiologiai es Sugaregeszseguegyi Kutato Intezet, Budapest (Hungary)); Kremmer, T. (Orszagos Onkologiai Intezet, Budapest (Hungary))

    1985-01-01

    A micromethod was developed to determine the binding of anabolic steroids to plasma proteins. The new procedure combines precipitation with ammonium sulphate and differential dissociation. The binding parameters (association constant, specific binding capacity) are calculated on the basis of dissociation curves of sup(3)H-metandrostenolone from the precipitated sexual binding globuline.

  16. Identification of novel DNA binding proteins using DNA affinity chromatography-pulldown

    OpenAIRE

    Jutras, Brandon L; Verma, Ashutosh; Stevenson, Brian

    2012-01-01

    Methods are presented through which one may isolate and identify novel bacterial DNA-binding proteins. Briefly, the DNA sequence of interest is affixed to beads, then incubated with bacterial cytoplasmic extract. Washes with buffers containing non-specific DNA and low salt concentrations will remove non-adhering and low-specificity DNA-binding proteins, while subsequent washes with higher salt concentrations will elute more specific DNA-binding proteins. Eluted proteins may then be identified...

  17. Differentiation inducing factor-1 (DIF-1) induces gene and protein expression of the Dictyostelium nuclear calmodulin-binding protein nucleomorphin.

    Science.gov (United States)

    O'Day, Danton H; Poloz, Yekaterina; Myre, Michael A

    2009-02-01

    The nucleomorphin gene numA1 from Dictyostelium codes for a multi-domain, calmodulin binding protein that regulates nuclear number. To gain insight into the regulation of numA, we assessed the effects of the stalk cell differentiation inducing factor-1 (DIF-1), an extracellular signalling molecule, on the expression of numA1 RNA and protein. For comparison, the extracellular signalling molecules cAMP (mediates chemotaxis, prestalk and prespore differentiation) and ammonia (NH(3)/NH(4)(+); antagonizes DIF) were also studied. Starvation, which is a signal for multicellular development, results in a greater than 80% decrease in numA1 mRNA expression within 4 h. Treatment with ammonium chloride led to a greater than 90% inhibition of numA1 RNA expression within 2 h. In contrast, the addition of DIF-1 completely blocked the decrease in numA1 gene expression caused by starvation. Treatment of vegetative cells with cAMP led to decreases in numA1 RNA expression that were equivalent to those seen with starvation. Western blotting after various morphogen treatments showed that the maintenance of vegetative levels of numA1 RNA by DIF-1 in starved cells was reflected in significantly increased numA1 protein levels. Treatment with cAMP and/or ammonia led to decreased protein expression and each of these morphogens suppressed the stimulatory effects of DIF-1. Protein expression levels of CBP4a, a calcium-dependent binding partner of numA1, were regulated in the same manner as numA1 suggesting this potential co-regulation may be related to their functional relationship. NumA1 is the first calmodulin binding protein shown to be regulated by developmental morphogens in Dictyostelium being upregulated by DIF-1 and down-regulated by cAMP and ammonia.

  18. Evidence that the [3H]estradiol-binding protein in pancreas is localized in exocrine cells

    International Nuclear Information System (INIS)

    Grossman, A.; Richardson, S.B.; Altszuler, N.; Lane, B.

    1985-01-01

    Extracts of rat pancreas contain significant amounts of an [ 3 H]estradiol-binding protein. The amount of steroid-binding activity that could be measured varied considerably depending on the tonicity of the homogenizing medium. High speed supernatants of homogenates initially prepared in isotonic buffer contained about 10% of the binding activity as homogenates prepared in hypotonic buffer. Extraction with hypotonic buffer of pellets obtained by the isotonic procedure yielded most of the remaining [ 3 H]estradiol-binding activity. In an attempt to avoid errors resulting from incomplete homogenization and to detect possible changes in intracellular distribution of [ 3 H]estradiol-binding activity, pancreata were initially homogenized in isotonic buffer and centrifuged at high speed (100,000 X g; 1 hr). The pellet was then extracted with hypotonic buffer and centrifuged again at high speed, and both supernatants were analyzed for [ 3 H]estradiol-binding and amylase activities. Two or 14 days after treatment of male rats with streptozotocin, no apparent decline or redistribution of [ 3 H]estradiol-binding activity to the cytosol was noted despite extensive alteration of beta-islet cells, as determined by electron microscopic examination of sections of these pancreata and significant loss of insulin, as measured by RIA. Amylase activity was unaffected 2 days after streptozotocin treatment, but was depressed to about 1% of control levels at 14 days. Administration of insulin to the latter group of animals resulted in return of amylase to normal levels and a modest increase (approximately 50%) in [ 3 H]estradiol-binding activity

  19. Free and total insulin-like growth factor I (IGF-I), IGF-binding protein-1 (IGFBP-1), and IGFBP-3 and their relationships to the presence of diabetic retinopathy and glomerular hyperfiltration in insulin-dependent diabetes mellitus

    NARCIS (Netherlands)

    J.A.M.J.L. Janssen (Joseph); M.L. Jacobs (Marloes); F.H.M. Derkx (Frans); R.F.A. Weber (Robert); A-J. van der Lely (Aart-Jan); S.W.J. Lamberts (Steven)

    1997-01-01

    textabstractThe existing literature on serum insulin-like growth factor I (IGF-I) levels in insulin-dependent diabetes mellitus (IDDM) is conflicting. Free IGF-I may have greater physiological and clinical relevance than total IGF-I. Recently, a validated method has

  20. Binding of plasma proteins to titanium dioxide nanotubes with different diameters

    Directory of Open Access Journals (Sweden)

    Kulkarni M

    2015-02-01

    Full Text Available Mukta Kulkarni,1,* Ajda Flašker,1,* Maruša Lokar,1 Katjuša Mrak-Poljšak,2 Anca Mazare,3 Andrej Artenjak,4 Saša Čučnik,2 Slavko Kralj,5 Aljaž Velikonja,1 Patrik Schmuki,3 Veronika Kralj-Iglič,6 Snezna Sodin-Semrl,2,7 Aleš Iglič11Laboratory of Biophysics, Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia; 2Department of Rheumatology, University Medical Centre Ljubljana, Ljubljana, Slovenia; 3Department of Materials Science and Engineering, University of Erlangen Nuremberg, Erlangen, Germany; 4Sandoz Biopharmaceuticals Mengeš, Lek Pharmaceuticals dd, Menges, Slovenia; 5Department for Materials Synthesis, Institute Jožef Stefan (IJS, Ljubljana, Slovenia; 6Faculty of Health Studies, University of Ljubljana, Ljubljana, Slovenia; 7Faculty of Mathematics, Natural Science and Information Technology, University of Primorska, Koper, Slovenia *These authors contributed equally to this workAbstract: Titanium and titanium alloys are considered to be one of the most applicable materials in medical devices because of their suitable properties, most importantly high corrosion resistance and the specific combination of strength with biocompatibility. In order to improve the biocompatibility of titanium surfaces, the current report initially focuses on specifying the topography of titanium dioxide (TiO2 nanotubes (NTs by electrochemical anodization. The zeta potential (ζ-potential of NTs showed a negative value and confirmed the agreement between the measured and theoretically predicted dependence of ζ-potential on salt concentration, whereby the absolute value of ζ-potential diminished with increasing salt concentrations. We investigated binding of various plasma proteins with different sizes and charges using the bicinchoninic acid assay and immunofluorescence microscopy. Results showed effective and comparatively higher protein binding to NTs with 100 nm diameters (compared to 50 or 15 nm. We also showed a dose-dependent

  1. Leishmania mexicana: LACK (Leishmania homolog of receptors for activated C-kinase) is a plasminogen binding protein.

    Science.gov (United States)

    Gómez-Arreaza, Amaranta; Acosta, Héctor; Barros-Álvarez, Ximena; Concepción, Juan L; Albericio, Fernando; Avilan, Luisana

    2011-04-01

    Leishmania mexicana is able to interact with the fibrinolytic system through its component plasminogen, the zymogenic form of the protease plasmin. In this study a new plasminogen binding protein of this parasite was identified: LACK, the Leishmania homolog of receptors for activated C-kinase. Plasminogen binds recombinant LACK with a K(d) value of 1.6±0.4 μM, and binding is lysine-dependent since it is inhibited by the lysine analog ε-aminocaproic acid. Inhibition studies with specific peptides and plasminogen binding activity of a mutated recombinant LACK have highlighted the internal motif (260)VYDLESKAV(268), similar to those found in several enolases, as involved in plasminogen binding. Recombinant LACK and secreted proteins, in medium conditioned by parasites, enhance plasminogen activation to plasmin by the tissue plasminogen activator (t-PA). In addition to its localization in the cytosol, in the microsomal fraction and as secreted protein in conditioned medium, LACK was also localized on the external surface of the membrane. The results presented here suggest that LACK might bind and enhance plasminogen activation in vivo promoting the formation of plasmin. Plasminogen binding of LACK represents a new function for this protein and might contribute to the invasiveness of the parasite. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. Profiling of drug binding proteins by monolithic affinity chromatography in combination with liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Zhang, Xuepei; Wang, Tongdan; Zhang, Hanzhi; Han, Bing; Wang, Lishun; Kang, Jingwu

    2014-09-12

    A new approach for proteome-wide profiling drug binding proteins by using monolithic capillary affinity chromatography in combination with HPLC-MS/MS is reported. Two immunosuppresive drugs, namely FK506 and cyclosporin A, were utilized as the experimental models for proof-of-concept. The monolithic capillary affinity columns were prepared through a single-step copolymerization of the drug derivatives with glycidyl methacrylate and ethylene dimethacrylate. The capillary chromatography with the affinity monolithic column facilitates the purification of the drug binding proteins from the cell lysate. By combining the capillary affinity column purification and the shot-gun proteomic analysis, totally 33 FK506- and 32 CsA-binding proteins including all the literature reported target proteins of these two drugs were identified. Among them, two proteins, namely voltage-dependent anion-selective channel protein 1 and serine/threonine-protein phosphatase PGAM5 were verified by using the recombinant proteins. The result supports that the monolithic capillary affinity chromatography is likely to become a valuable tool for profiling of binding proteins of small molecular drugs as well as bioactive compounds. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins.

    Science.gov (United States)

    Morris, Jacqueline; Jayanthi, Srinivas; Langston, Rebekah; Daily, Anna; Kight, Alicia; McNabb, David S; Henry, Ralph; Kumar, Thallapuranam Krishnaswamy Suresh

    2016-10-01

    Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms. Recombinant protein purification is achieved by fusing appropriate affinity tags to either N- or C- terminus of the target recombinant proteins. Currently available protein/peptide affinity tags have proved quite useful in the purification of recombinant proteins. However, these affinity tags suffer from specific limitations in their use under different conditions of purification. In this study, we have designed a novel 34-amino acid heparin-binding affinity tag (HB-tag) for the purification of recombinant proteins expressed in Escherichia coli (E. coli) cells. HB-tag fused recombinant proteins were overexpressed in E. coli in high yields. A one-step heparin-Sepharose-based affinity chromatography protocol was developed to purify HB-fused recombinant proteins to homogeneity using a simple sodium chloride step gradient elution. The HB-tag has also been shown to facilitate the purification of target recombinant proteins from their 8 M urea denatured state(s). The HB-tag has been demonstrated to be successfully released from the fusion protein by an appropriate protease treatment to obtain the recombinant target protein(s) in high yields. Results of the two-dimensional NMR spectroscopy experiments indicate that the purified recombinant target protein(s) exist in the native conformation. Polyclonal antibodies raised against the HB-peptide sequence, exhibited high binding specificity and sensitivity to the HB-fused recombinant proteins (∼10 ng) in different crude cell extracts obtained from diverse expression hosts. In our opinion, the HB-tag provides a

  4. Fibronectin-binding protein TDE1579 affects cytotoxicity of Treponema denticola.

    Science.gov (United States)

    Xu, Xiaoping; Steffensen, Bjorn; Robichaud, Trista K; Mikhailova, Margarita; Lai, Veronica; Montgomery, Ryan; Chu, Lianrui

    2015-12-01

    While FbpA, a family of bacterial fibronectin (FN) binding proteins has been studied in several gram-positive bacteria, the gram-negative Treponema denticola, an anaerobic periodontal pathogen, also has an overlooked fbp gene (tde1579). In this research, we confirm that recombinant Fbp protein (rFbp) of T. denticola binds human FN with a Kdapp of 1.5 × 10(-7) M and blocks the binding of T. denticola to FN in a concentration-dependent manner to a level of 42%. The fbp gene was expressed in T. denticola. To reveal the roles of fbp in T. denticola pathogenesis, an fbp isogenic mutant was constructed. The fbp mutant had 51% reduced binding ability to human gingival fibroblasts (hGF). When hGF were challenged with T. denticola, the fbp mutant caused less cell morphology change, had 50% reduced cytotoxicity to hGF, and had less influence on the growth of hGF cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Cu(II) bis(thiosemicarbazone) radiopharmaceutical binding to serum albumin: further definition of species dependence and associated substituent effects

    Energy Technology Data Exchange (ETDEWEB)

    Basken, Nathan E. [Division of Nuclear Pharmacy, Department of Industrial and Physical Pharmacy, Purdue University, West Lafayette, IN 47907 (United States); Green, Mark A. [Division of Nuclear Pharmacy, Department of Industrial and Physical Pharmacy, Purdue University, West Lafayette, IN 47907 (United States)], E-mail: magreen@purdue.edu

    2009-07-15

    Introduction: The pyruvaldehyde bis(N{sup 4}-methylthiosemicarbazonato)copper(II) (Cu-PTSM) and diacetyl bis(N{sup 4}-methylthiosemicarbazonato)copper(II) (Cu-ATSM) radiopharmaceuticals exhibit strong, species-dependent binding to the IIA site of human serum albumin (HSA), while the related ethylglyoxal bis(thiosemicarbazonato)copper(II) (Cu-ETS) radiopharmaceutical appears to exhibit only nonspecific binding to HSA and animal serum albumins. Methods: To further probe the structural basis for the species dependence of this albumin binding interaction, we examined protein binding of these three radiopharmaceuticals in solutions of albumin and/or serum from a broader array of mammalian species (rat, sheep, donkey, rabbit, cow, pig, dog, baboon, mouse, cat and elephant). We also evaluated the albumin binding of several copper(II) bis(thiosemicarbazone) chelates offering more diverse substitution of the ligand backbone. Results: Cu-PTSM and Cu-ATSM exhibit a strong interaction with HSA that is not apparent with the albumins of other species, while the binding of Cu-ETS to albumin is much less species dependent. The strong interaction of Cu-PTSM with HSA does not appear to simply correlate with variation, relative to the animal albumins, of a single amino acid lining HSA's IIA site. Those agents that selectively interact with HSA share the common feature of only methyl or hydrogen substitution at the carbon atoms of the diimine fragment of the ligand backbone. Conclusions: The interspecies variations in albumin binding of Cu-PTSM and Cu-ATSM are not simply explained by unique amino acid substitutions in the IIA binding pocket of the serum albumins. However, the specific affinity for this region of HSA is disrupted when substituents bulkier than a methyl group appear on the imine carbons of the copper bis(thiosemicarbazone) chelate.

  6. A Venom Gland Extracellular Chitin-Binding-Like Protein from Pupal Endoparasitoid Wasps, Pteromalus Puparum, Selectively Binds Chitin

    Directory of Open Access Journals (Sweden)

    Yu Zhu

    2015-11-01

    Full Text Available Chitin-binding proteins (CBPs are present in many species and they act in a variety of biological processes. We analyzed a Pteromalus puparum venom apparatus proteome and transcriptome and identified a partial gene encoding a possible CBP. Here, we report cloning a full-length cDNA of a sequence encoding a chitin-binding-like protein (PpCBP from P. puparum, a pupal endoparasitoid of Pieris rapae. The cDNA encoded a 96-amino-acid protein, including a secretory signal peptide and a chitin-binding peritrophin-A domain. Phylogenetic analysis of chitin binding domains (CBDs of cuticle proteins and peritrophic matrix proteins in selected insects revealed that the CBD of PpCBP clustered with the CBD of Nasonia vitripennis. The PpCBP is specifically expressed in the venom apparatus of P. puparum, mostly in the venom gland. PpCBP expression was highest at day one after adult eclosion and much lower for the following five days. We produced a recombinant PpCBP and binding assays showed the recombinant protein selectively binds chitin but not cellulose in vitro. We infer that PpCBP serves a structural role in the venom reservoir, or may be injected into the host to help wound healing of the host exoskeleton.

  7. Growth hormone receptor/binding protein: physiology and function.

    Science.gov (United States)

    Herington, A C; Ymer, S I; Stevenson, J L; Roupas, P

    1994-07-01

    Soluble truncated forms of the growth hormone receptor (GHR) are present in the circulation of many species and are also produced by many tissues/cell types. The major high-affinity forms of these GH-binding proteins (GHBP) are derived by alternative splicing of GHR mRNA in rodents, but probably by proteolytic cleavage in other species. Questions still remain with respect to the origins, native molecular form(s), physiology, and function of the GHBPs, however. The observation that GH induces dimerization of the soluble GHBP and membrane GHR, and that dimerization of GHR appears to be critical for GH bioactivity suggests that the presentation of GH to target cells, in an unbound form or as a monomeric or dimeric complex with GHBP, may have significant implications for the ability of GH to activate specific postreceptor signaling pathways (tyrosine kinase, protein kinase C, G-protein pathways) known to be utilized by GH for its diverse biological effects. This minireview addresses some of these aspects and highlights several new questions which have arisen as a result of recent advances in our understanding of the structure, function, and signaling mechanisms of the membrane bound GHR.

  8. Growth hormone receptor/binding protein: Physiology and function

    Energy Technology Data Exchange (ETDEWEB)

    Herington, A.C.; Ymer, S.I.; Stevenson, J.L.; Roupas, P. [Royal Children`s Hospital, Melbourne (Australia)

    1994-12-31

    Soluble truncated forms of the growth hormone receptor (GHR) are present in the circulation of many species and are also produced by many tissues/cell types. The major high-affinity forms of these GH-binding proteins (GHBP) are derived by alternative splicing of GHR mRNA in rodents, but probably by proteolytic cleavage in other species. Questions still remain with respect to the origins, native molecular forms(s), physiology, and function of the GHBPs, however. The observation that GH induces dimerization of the soluble GHBP and a membrane GHR, and that dimerization of GHR appears to be critical for GH bioactivity suggests that the presentation of GH to target cells, in an unbound form or as a monomeric or dimeric complex with GHBP, may have significant implications for the ability of GH to activate specific postreceptor signaling pathways (tyrosine kinase, protein kinase C, G-protein pathways) known to be utilized by GH for its diverse biological effects. This minireview addresses some of these aspects and highlights several new questions which have arisen as a result of recent advances in our understanding of the structure, function, and signaling mechanisms of the membrane bound GHR. 43 refs.

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

    Science.gov (United States)

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

    2016-04-06

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

  10. DNA-binding polarity of human replication protein A positions nucleases in nucleotide excision repair.

    Science.gov (United States)

    de Laat, W L; Appeldoorn, E; Sugasawa, K; Weterings, E; Jaspers, N G; Hoeijmakers, J H

    1998-08-15

    The human single-stranded DNA-binding replication A protein (RPA) is involved in various DNA-processing events. By comparing the affinity of hRPA for artificial DNA hairpin structures with 3'- or 5'-protruding single-stranded arms, we found that hRPA binds ssDNA with a defined polarity; a strong ssDNA interaction domain of hRPA is positioned at the 5' side of its binding region, a weak ssDNA-binding domain resides at the 3' side. Polarity appears crucial for positioning of the excision repair nucleases XPG and ERCC1-XPF on the DNA. With the 3'-oriented side of hRPA facing a duplex ssDNA junction, hRPA interacts with and stimulates ERCC1-XPF, whereas the 5'-oriented side of hRPA at a DNA junction allows stable binding of XPG to hRPA. Our data pinpoint hRPA to the undamaged strand during nucleotide excision repair. Polarity of hRPA on ssDNA is likely to contribute to the directionality of other hRPA-dependent processes as well.

  11. Heterodimerization of Hox proteins with Pbx1 and oncoprotein E2a-Pbx1 generates unique DNA-binding specifities at nucleotides predicted to contact the N-terminal arm of the Hox homeodomain--demonstration of Hox-dependent targeting of E2a-Pbx1 in vivo.

    Science.gov (United States)

    Lu, Q; Kamps, M P

    1997-01-09

    Hox proteins control genetic programs that orchestrate development, and a large subset of Hox proteins can bind DNA elements as heterodimers with the Pbx family of homeodomain proteins. A transcriptionally activated version of Pbx1, E2a-Pbx1, is an oncoprotein in human pre-B cell leukemia that strongly suppresses differentiation and retains its ability to heterodimerize with Hox proteins. Because monomeric Hox proteins bind very similar DNA motifs, it is unclear how they activate diverse developmental programs. Here we demonstrate that heterodimers containing different Hox proteins and a common Pbx1 or E2a-Pbx1 partner bind different DNA motifs. Structural models suggest that the specificity of the Hox protein is altered by a conformation change involving residues in the N-terminal arm of the Hox homeodomain. Mutational analysis also supported the hypothesis that unique sequences in the N-terminal arm of the Hox homeodomain are at least partially responsible for mediating this specificity. In vivo, Hox proteins directed E2a-Pbx1-mediated transactivation with moderate specificity to cognate Hox-Pbx motifs. Thus, the development specificity of individual Hox proteins may be mediated, in part, by differential targeting of cellular genes by Pbx1-Hox complexes. Likewise, through its function as a common heterodimer partner, oncoprotein E2a-Pbx1 may be able to interfere with multiple programs of development that are induced by the sequential or simultaneous expression of Hox proteins during hematopoiesis.

  12. DSCG binding protein and process for preparing same

    Energy Technology Data Exchange (ETDEWEB)

    Pecht, I.; Mazurek, N.

    1987-07-28

    An essentially pure protein is described consisting essentially of the protein, (CBP), present in nature in membranes of basophile cells and in mast cells, having a molecular weight of about 60,000 +- 2,000 determined by SDS polyacrylamide electrophoresis an isoelectric point of about 3.9 and an amino acid composition of about 4 units of asparagine, 3 units of threonine and serine, 3 units glycine, 2 units alanine, 2 units proline, 1 unit cysteine, 2 units valine, 1 unit methionine, 1 unit isoleucine, 2 units leucine, 1 unit tyrosine, 1 unit phenylalanine, 2 units histamine, 2 units lysine and 1 unit arginine. The protein is able to build calcium and having a calcium dependent affinity to the disodium salt of 1,2 bis(-2 carboxychromon-5-yloxy)-2-hydroxy propane (DSCG).

  13. Visualization of coupled protein folding and binding in bacteria and purification of the heterodimeric complex

    Science.gov (United States)

    Wang, Haoyong; Chong, Shaorong

    2003-01-01

    During overexpression of recombinant proteins in Escherichia coli, misfolded proteins often aggregate and form inclusion bodies. If an aggregation-prone recombinant protein is fused upstream (as an N-terminal fusion) to GFP, aggregation of the recombinant protein domain also leads to misfolding of the downstream GFP domain, resulting in a decrease or loss of fluorescence. We investigated whether the GFP domain could fold correctly if aggregation of the upstream protein domain was prevented in vivo by a coupled protein folding and binding interaction. Such interaction has been previously shown to occur between the E. coli integration host factors and , and between the domains of the general transcriptional coactivator cAMP response element binding protein (CREB)-binding protein and the activator for thyroid hormone and retinoid receptors. In this study, fusion of integration host factor or the CREB-binding protein domain upstream to GFP resulted in aggregation of the fusion protein. Coexpression of their respective partners, on the other hand, allowed soluble expression of the fusion protein and a dramatic increase in fluorescence. The study demonstrated that coupled protein folding and binding could be correlated to GFP fluorescence. A modified miniintein containing an affinity tag was inserted between the upstream protein domain and GFP to allow rapid purification and identification of the heterodimeric complex. The GFP coexpression fusion system may be used to identify novel protein-protein interactions that involve coupled folding and binding or protein partners that can solubilize aggregation-prone recombinant proteins.

  14. STRUCTURAL CHARACTERIZATION OF THE RNA BINDING DOMAIN OF HUMAN STEM LOOP BINDING PROTEIN

    Directory of Open Access Journals (Sweden)

    Maruthi Kashyap

    2013-12-01

    Full Text Available A gene encoding the RNA binding domain (RBD of human stem loop binding protein (SLBP was cloned in pET 28a vector and over-expressed in E. coli codon plus cells. The over-expressed SLBP-RBD carried no tag and aggregated as inclusion bodies in the cell lysate. Inclusion bodies were semi-purified to >85% purity by establishing a method involving detergent washing and subsequently denatured in 8 M urea. Refolding of the denatured RBD was carried out by step dialysis in decreasing concentrations of urea and L-arginine. Refolded SLBP-RBD was analyzed using size exclusion chromatography that revealed its monomeric nature and folded state. Uniformly 15N and 15N,13C labeled SLBP-RBD was prepared at concentrations for solution NMR studies. Approximately, 60% of the sequence specific backbone resonance assignments have been achieved through standard triple resonance NMR experiments. Analyses of secondary chemical shifts reveal presence of a small helical secondary structural elements and large intrinsically disordered regions.

  15. Crystal structure of Methanococcus jannaschii TATA box-binding protein.

    Science.gov (United States)

    Adachi, Naruhiko; Senda, Miki; Natsume, Ryo; Senda, Toshiya; Horikoshi, Masami

    2008-11-01

    As the archaeal transcription system consists of a eukaryotic-type transcription apparatus and bacterial-type regulatory transcription factors, analyses of the molecular interface between the transcription apparatus and regulatory transcription factors are critical to reveal the evolutionary change of the transcription system. TATA box-binding protein (TBP), the central components of the transcription apparatus are classified into three groups: eukaryotic, archaeal-I and archaeal-II TBPs. Thus, comparative functional analysis of these three groups of TBP is important for the study of the evolution of the transcription system. Here, we present the first crystal structure of an archaeal-II TBP from Methanococcus jannaschii. The highly conserved and group-specific conserved surfaces of TBP bind to DNA and TFIIB/TFB, respectively. The phylogenetic trees of TBP and TFIIB/TFB revealed that they evolved in a coupled manner. The diversified surface of TBP is negatively charged in the archaeal-II TBP, which is completely different from the case of eukaryotic and archaeal-I TBPs, which are positively charged and biphasic, respectively. This difference is responsible for the diversification of the regulatory functions of TBP during evolution.

  16. Characterization of little skate (Leucoraja erinacea) recombinant transthyretin: Zinc-dependent 3,3',5-triiodo-l-thyronine binding.

    Science.gov (United States)

    Suzuki, Shunsuke; Kasai, Kentaro; Yamauchi, Kiyoshi

    2015-01-01

    Transthyretin (TTR) diverged from an ancestral 5-hydroxyisourate hydrolase (HIUHase) by gene duplication at some early stage of chordate evolution. To clarify how TTR had participated in the thyroid system as an extracellular thyroid hormone (TH) binding protein, TH binding properties of recombinant little skate Leucoraja erinacea TTR was investigated. At the amino acid level, skate TTR showed 37-46% identities with the other vertebrate TTRs. Because the skate TTR had a unique histidine-rich segment in the N-terminal region, it could be purified by Ni-affinity chromatography. The skate TTR was a 46-kDa homotetramer of 14.5kDa subunits, and had one order of magnitude higher affinity for 3,3',5-triiodo-l-thyronine (T3) and some halogenated phenols than for l-thyroxine. However, the skate TTR had no HIUHase activity. Ethylenediaminetetraacetic acid (EDTA) treatment inhibited [(125)I]T3 binding activity whereas the addition of Zn(2+) to the EDTA-treated TTR recovered [(125)I]T3 binding activity in a Zn(2+) concentration-dependent manner. Scatchard analysis revealed the presence of two classes of binding site for T3, with dissociation constants of 0.24 and 17nM. However, the high-affinity sites were completely abolished with 1mM EDTA, whereas the remaining low-affinity sites decreased binding capacity. The number of zinc per TTR was quantified to be 4.5-6.3. Our results suggest that skate TTR has tight Zn(2+)-binding sites, which are essential for T3 binding to at least the high-affinity sites. Zn(2+) binding to the N-terminal histidine-rich segment may play an important role in acquisition or reinforcement of TH binding ability during early evolution of TTR. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Earthworm Lumbricus rubellus MT-2: Metal Binding and Protein Folding of a True Cadmium-MT

    Directory of Open Access Journals (Sweden)

    Gregory R. Kowald

    2016-01-01

    Full Text Available Earthworms express, as most animals, metallothioneins (MTs—small, cysteine-rich proteins that bind d10 metal ions (Zn(II, Cd(II, or Cu(I in clusters. Three MT homologues are known for Lumbricus rubellus, the common red earthworm, one of which, wMT-2, is strongly induced by exposure of worms to cadmium. This study concerns composition, metal binding affinity and metal-dependent protein folding of wMT-2 expressed recombinantly and purified in the presence of Cd(II and Zn(II. Crucially, whilst a single Cd7wMT-2 species was isolated from wMT-2-expressing E. coli cultures supplemented with Cd(II, expressions in the presence of Zn(II yielded mixtures. The average affinities of wMT-2 determined for either Cd(II or Zn(II are both within normal ranges for MTs; hence, differential behaviour cannot be explained on the basis of overall affinity. Therefore, the protein folding properties of Cd- and Zn-wMT-2 were compared by 1H NMR spectroscopy. This comparison revealed that the protein fold is better defined in the presence of cadmium than in the presence of zinc. These differences in folding and dynamics may be at the root of the differential behaviour of the cadmium- and zinc-bound protein in vitro, and may ultimately also help in distinguishing zinc and cadmium in the earthworm in vivo.

  18. Lanosterol metabolism and sterol regulatory element binding protein (SREBP) expression in male germ cell maturation.

    Science.gov (United States)

    Fon Tacer, Klementina; Kalanj-Bognar, Svjetlana; Waterman, Michael R; Rozman, Damjana

    2003-06-01

    Expression of genes involved in cholesterol biosynthesis in male germ cells is insensitive to the negative cholesterol feedback regulation, in contrast to cholesterol level-sensitive/sterol regulatory element binding protein (SREBP)-dependent gene regulation in somatic cells. The role of sterol regulatory element binding proteins in spermatogenic cells was an enigma until recently, when a soluble, 55kDa cholesterol-insensitive form of SREBP2 (SREBP2gc) was discovered [Mol. Cell. Endocrinol. 22 (2002) 8478], being translated from a germ cell-specific SREBP2 mRNA. Our RT-PCR results also show that SREBP2 as well as SREBP1c mRNAs are detectable in prepubertal and postpubertal male germ cells while SREBP1a is not detected. Surprisingly, three SREBP2 immunoreactive proteins (72, 63 and 55kDa), that are not present in mouse liver nuclei, reside in testis nuclei of prepubertal and adult mice. The 55kDa protein is likely SREBP2gc, the other two isoforms are novel. HPLC measurements in liver and testes of fasted prepubertal and postpubertal mice showed no significant difference in cholesterol level. However, FF-MAS and lanosterol/testis-meiosis activating sterol (T-MAS) intermediates that are detectable mainly in testes, increase in fasted postpubertal mice which coincides well with the elevated level of 68kDa SREBP2. Similar to SREBP2gc, the two novel SREBP2 immunoreactive proteins seem to be insensitive to the level of cholesterol.

  19. Methods and systems for identifying ligand-protein binding sites

    KAUST Repository

    Gao, Xin

    2016-05-06

    The invention provides a novel integrated structure and system-based approach for drug target prediction that enables the large-scale discovery of new targets for existing drugs Novel computer-readable storage media and computer systems are also provided. Methods and systems of the invention use novel sequence order-independent structure alignment, hierarchical clustering, and probabilistic sequence similarity techniques to construct a probabilistic pocket ensemble (PPE) that captures even promiscuous structural features of different binding sites for a drug on known targets. The drug\\'s PPE is combined with an approximation of the drug delivery profile to facilitate large-scale prediction of novel drug- protein interactions with several applications to biological research and drug development.

  20. Temperature dependence and GABA modulation of [3H]triazolam binding in the rat brain

    International Nuclear Information System (INIS)

    Earle, M.E.; Concas, A.; Wamsley, J.K.; Yamamura, H.I.

    1987-01-01

    The hypnotic triazolam (TZ), a triazolobenzodiazepine displays a short physiological half life and has been used for the treatment of insomnia related to anxiety states. The authors major objectives were the direct measurement of the temperature dependence and the gamma-aminobutyric acid (GABA) effect of [ 3 H]TZ binding in the rat brain. Saturation studies showed a shift to lower affinity with increasing temperatures (K/sub d/ = 0.27 +/- 08 nM at 0 0 C; K/sub d/ = 1.96 +/- 0.85 nM at 37 0 C) while the B/sub max/ values remained unchanged (1220 +/- 176 fmoles/mg protein at 0 0 C and 1160 +/- 383 fmoles/mg protein at 37 0 C). Saturation studies of [ 3 H]TZ binding in the presence or absence of GABA (100μM) showed a GABA-shift. At 0 0 C the K/sub d/ values were (K/sub d/ = 0.24 +/- 0.03 nM/-GABA; K/sub d/ = 0.16 +/- 0.04/+GABA) and at 37 0 C the K/sub d/ values were (K/sub d/ = 1.84 +/- 0.44 nM/-GABA; K/sub d/ = 0.95 +/- 0.29 nM/+GABA). In contrast to reported literature, the authors findings show that TZ interacts with benzodiazepine receptors with a temperature dependence and GABA-shift consistent with predicted behavior for benzodiazepine agonists. 20 references, 3 tables

  1. Detection of two isomeric binding configurations in a protein-aptamer complex with a biological nanopore.

    Science.gov (United States)

    Van Meervelt, Veerle; Soskine, Misha; Maglia, Giovanni

    2014-12-23

    Protein-DNA interactions play critical roles in biological systems, and they often involve complex mechanisms and dynamics that are not easily measured by ensemble experiments. Recently, we showed that folded proteins can be internalized inside ClyA nanopores and studied by ionic current recordings at the single-molecule level. Here, we use ClyA nanopores to sample the interaction between the G-quadruplex fold of the thrombin binding aptamer (TBA) and human thrombin (HT). Surprisingly, the internalization of the HT:TBA complex inside the nanopore induced two types of current blockades with distinguished residual current and lifetime. Using single nucleobase substitutions to TBA we showed that these two types of blockades originate from TBA binding to thrombin with two isomeric orientations. Voltage dependencies and the use of ClyA nanopores with two different diameters allowed assessing the effect of the applied potential and confinement and revealed that the two binding configurations of TBA to HT display different lifetimes. These results show that the ClyA nanopores can be used to probe conformational heterogeneity in protein:DNA interactions.

  2. Dengue virus capsid protein binds core histones and inhibits nucleosome formation in human liver cells.

    Directory of Open Access Journals (Sweden)

    Tonya M Colpitts

    Full Text Available Dengue virus (DENV is a member of the Flaviviridae and a globally (reemerging pathogen that causes serious human disease. There is no specific antiviral or vaccine for dengue virus infection. Flavivirus capsid (C is a structural protein responsible for gathering the viral RNA into a nucleocapsid that forms the core of a mature virus particle. Flaviviral replication is known to occur in the cytoplasm yet a large portion of capsid protein localizes to the nucleus during infection. The reasons for the nuclear presences of capsid are not completely understood. Here, we expressed mature DENV C in a tandem affinity purification assay to identify potential binding partners in human liver cells. DENV C targeted the four core histones, H2A, H2B, H3 and H4. DENV C bound recombinant histones in solution and colocalized with histones in the nucleus and cytoplasm of liver cells during DENV infection. We show that DENV C acts as a histone mimic, forming heterodimers with core histones, binding DNA and disrupting nucleosome formation. We also demonstrate that DENV infection increases the amounts of core histones in livers cells, which may be a cellular response to C binding away the histone proteins. Infection with DENV additionally alters levels of H2A phosphorylation in a time-dependent manner. The interactions of C and histones add an interesting new role for the presence of C in the nucleus during DENV infection.

  3. Dengue virus capsid protein binds core histones and inhibits nucleosome formation in human liver cells.

    Science.gov (United States)

    Colpitts, Tonya M; Barthel, Sebastian; Wang, Penghua; Fikrig, Erol

    2011-01-01

    Dengue virus (DENV) is a member of the Flaviviridae and a globally (re)emerging pathogen that causes serious human disease. There is no specific antiviral or vaccine for dengue virus infection. Flavivirus capsid (C) is a structural protein responsible for gathering the viral RNA into a nucleocapsid that forms the core of a mature virus particle. Flaviviral replication is known to occur in the cytoplasm yet a large portion of capsid protein localizes to the nucleus during infection. The reasons for the nuclear presences of capsid are not completely understood. Here, we expressed mature DENV C in a tandem affinity purification assay to identify potential binding partners in human liver cells. DENV C targeted the four core histones, H2A, H2B, H3 and H4. DENV C bound recombinant histones in solution and colocalized with histones in the nucleus and cytoplasm of liver cells during DENV infection. We show that DENV C acts as a histone mimic, forming heterodimers with core histones, binding DNA and disrupting nucleosome formation. We also demonstrate that DENV infection increases the amounts of core histones in livers cells, which may be a cellular response to C binding away the histone proteins. Infection with DENV additionally alters levels of H2A phosphorylation in a time-dependent manner. The interactions of C and histones add an interesting new role for the presence of C in the nucleus during DENV infection.

  4. Dengue Virus Capsid Protein Binds Core Histones and Inhibits Nucleosome Formation in Human Liver Cells

    Science.gov (United States)

    Colpitts, Tonya M.; Barthel, Sebastian; Wang, Penghua; Fikrig, Erol

    2011-01-01

    Dengue virus (DENV) is a member of the Flaviviridae and a globally (re)emerging pathogen that causes serious human disease. There is no specific antiviral or vaccine for dengue virus infection. Flavivirus capsid (C) is a structural protein responsible for gathering the viral RNA into a nucleocapsid that forms the core of a mature virus particle. Flaviviral replication is known to occur in the cytoplasm yet a large portion of capsid protein localizes to the nucleus during infection. The reasons for the nuclear presences of capsid are not completely understood. Here, we expressed mature DENV C in a tandem affinity purification assay to identify potential binding partners in human liver cells. DENV C targeted the four core histones, H2A, H2B, H3 and H4. DENV C bound recombinant histones in solution and colocalized with histones in the nucleus and cytoplasm of liver cells during DENV infection. We show that DENV C acts as a histone mimic, forming heterodimers with core histones, binding DNA and disrupting nucleosome formation. We also demonstrate that DENV infection increases the amounts of core histones in livers cells, which may be a cellular response to C binding away the histone proteins. Infection with DENV additionally alters levels of H2A phosphorylation in a time-dependent manner. The interactions of C and histones add an interesting new role for the presence of C in the nucleus during DENV infection. PMID:21909430

  5. Protein-salt binding data from potentiometric titrations of lysozyme in aqueous solutions containing KCl

    Energy Technology Data Exchange (ETDEWEB)

    Engmann, J.; Blanch, H.W.; Prausnitz, J.M. [Univ. of California, Berkeley, CA (United States). Dept. of Chemical Engineering]|[Lawrence Berkeley National Lab., CA (United States). Chemical Sciences Div.

    1997-03-01

    An existing method for potentiometric titrations of proteins was improved, tested and applied to titrations of the enzyme hen-egg-white lysozyme in aqueous solutions containing KCl at ionic strengths from 0.1 M to 2.0 M at 25 C. Information about the protein`s net charge dependence on pH and ionic strength were obtained and salt binding numbers for the system were calculated using a linkage concept. For the pH range 2.5--11.5, the net charge slightly but distinctly increases with increasing ionic strength between 0.1 M and 2.0 M. The differences are most distinct in the pH region below 5. Above pH 11.35, the net charge decreases with increasing ionic strength. Preliminary calculation of binding numbers from titration curves at 0.1 M and 1.0 M showed selective association of chloride anions and expulsion of potassium ions at low pH. Ion-binding numbers from this work will be used to evaluate thermodynamic properties and to correlate crystallization or precipitation phase-equilibrium data in terms of a model based on the integral-equation theory of fluids which is currently under development.

  6. Reduced Fluorescent Protein Switching Fatigue by Binding-Induced Emissive State Stabilization

    Directory of Open Access Journals (Sweden)

    Thijs Roebroek

    2017-09-01

    Full Text Available Reversibly switchable fluorescent proteins (RSFPs enable advanced fluorescence imaging, though the performance of this imaging crucially depends on the properties of the labels. We report on the use of an existing small binding peptide, named Enhancer, to modulate the spectroscopic properties of the recently developed rsGreen series of RSFPs. Fusion constructs of Enhancer with rsGreen1 and rsGreenF revealed an increased molecular brightness and pH stability, although expression in living E. coli or HeLa cells resulted in a decrease of the overall emission. Surprisingly, Enhancer binding also increased off-switching speed and resistance to switching fatigue. Further investigation suggested that the RSFPs can interconvert between fast- and slow-switching emissive states, with the overall protein population gradually converting to the slow-switching state through irradiation. The Enhancer modulates the spectroscopic properties of both states, but also preferentially stabilizes the fast-switching state, supporting the increased fatigue resistance. This work demonstrates how the photo-physical properties of RSFPs can be influenced by their binding to other small proteins, which opens up new horizons for applications that may require such modulation. Furthermore, we provide new insights into the photoswitching kinetics that should be of general consideration when developing new RSFPs with improved or different photochromic properties.

  7. Ligand size is a major determinant of specificity in periplasmic oxyanion-binding proteins: the 1.2 A resolution crystal structure of Azotobacter vinelandii ModA.

    Science.gov (United States)

    Lawson, D M; Williams, C E; Mitchenall, L A; Pau, R N

    1998-12-15

    . Periplasmic receptors constitute a diverse class of binding proteins that differ widely in size, sequence and ligand specificity. Nevertheless, almost all of them display a common beta/alpha folding motif and have similar tertiary structures consisting of two globular domains. The ligand is bound at the bottom of a deep cleft, which lies at the interface between these two domains. The oxyanion-binding proteins are notable in that they can discriminate between very similar ligands. . Azotobacter vinelandii is unusual in that it possesses two periplasmic molybdate-binding proteins. The crystal structure of one of these with bound ligand has been determined at 1.2 A resolution. It superficially resembles the structure of sulphate-binding protein (SBP) from Salmonella typhimurium and uses a similar constellation of hydrogen-bonding interactions to bind its ligand. However, the detailed interactions are distinct from those of SBP and the more closely related molybdate-binding protein of Escherichia coli. . Despite differences in the residues involved in binding, the volumes of the binding pockets in the A. vinelandii and E. coli molybdate-binding proteins are similar and are significantly larger than that of SBP. We conclude that the discrimination between molybdate and sulphate shown by these binding proteins is largely dependent upon small differences in the sizes of these two oxyanions.

  8. Acanthamoeba castellanii contains a ribosomal RNA enhancer binding protein which stimulates TIF-IB binding and transcription under stringent conditions.

    Science.gov (United States)

    Yang, Q; Radebaugh, C A; Kubaska, W; Geiss, G K; Paule, M R

    1995-01-01

    The intergenic spacer (IGS) of Acanthamoeba castellanii rRNA genes contains repeated elements which are weak enhancers for transcription by RNA polymerase I. A protein, EBF, was identified and partially purified which binds to the enhancers and to several other sequences within the IGS, but not to other DNA fragments, including the rRNA core promoter. No consensus binding sequence could be discerned in these fragments and bound factor is in rapid equilibrium with unbound. EBF has functional characteristics similar to vertebrate upstream binding factors (UBF). Not only does it bind to the enhancer and other IGS elements, but it also stimulates binding of TIF-IB, the fundamental transcription initiation factor, to the core promoter and stimulates transcription from the promoter. Attempts to identify polypeptides with epitopes similar to rat or Xenopus laevis UBF suggest that structurally the protein from A.castellanii is not closely related to vertebrate UBF. Images PMID:7501455

  9. Ail proteins of Yersinia pestis and Y. pseudotuberculosis have different cell binding and invasion activities.

    Science.gov (United States)

    Tsang, Tiffany M; Wiese, Jeffrey S; Felek, Suleyman; Kronshage, Malte; Krukonis, Eric S

    2013-01-01

    The Yersinia pestis adhesin Ail mediates host cell binding and facilitates delivery of cytotoxic Yop proteins. Ail from Y. pestis and Y. pseudotuberculosis is identical except for one or two amino acids at positions 43 and 126 depending on the Y. pseudotuberculosis strain. Ail from Y. pseudotuberculosis strain YPIII has been reported to lack host cell binding ability, thus we sought to determine which amino acid difference(s) are responsible for the difference in cell adhesion. Y. pseudotuberculosis YPIII Ail expressed in Escherichia coli bound host cells, albeit at ~50% the capacity of Y. pestis Ail. Y. pestis Ail single mutants, Ail-E43D and Ail-F126V, both have decreased adhesion and invasion in E. coli when compared to wild-type Y. pestis Ail. Y. pseudotuberculosis YPIII Ail also had decreased binding to the Ail substrate fibronectin, relative to Y. pestis Ail in E. coli. When expressed in Y. pestis, there was a 30-50% decrease in adhesion and invasion depending on the substitution. Ail-mediated Yop delivery by both Y. pestis Ail and Y. pseudotuberculosis Ail were similar when expressed in Y. pestis, with only Ail-F126V giving a statistically significant reduction in Yop delivery of 25%. In contrast to results in E. coli and Y. pestis, expression of Ail in Y. pseudotuberculosis led to no measurable adhesion or invasion, suggesting the longer LPS of Y. pseudotuberculosis interferes with Ail cell-binding activity. Thus, host context affects the binding activities of Ail and both Y. pestis and Y. pseudotuberculosis Ail can mediate cell binding, cell invasion and facilitate Yop delivery.

  10. Peptide microarrays to probe for competition for binding sites in a protein interaction network

    NARCIS (Netherlands)

    Sinzinger, M.D.S.; Ruttekolk, I.R.R.; Gloerich, J.; Wessels, H.; Chung, Y.D.; Adjobo-Hermans, M.J.W.; Brock, R.E.

    2013-01-01

    Cellular protein interaction networks are a result of the binding preferences of a particular protein and the entirety of interactors that mutually compete for binding sites. Therefore, the reconstruction of interaction networks by the accumulation of interaction networks for individual proteins

  11. Characterization of the retinoblastoma binding proteins RBP1 and RBP2

    DEFF Research Database (Denmark)

    Fattaey, A R; Helin, K; Dembski, M S

    1993-01-01

    The retinoblastoma gene product, pRB, regulates cell proliferation by binding to and inhibiting the activity of key growth promoting proteins. Several cellular proteins have been shown to bind directly to pRB and the genes encoding a number of them have been isolated. The protein product of one...

  12. Liposome-binding assays to assess specificity and affinity of phospholipid-protein interactions

    NARCIS (Netherlands)

    Julkowska, M.M.; Rankenberg, J.M.; Testerink, C.

    2013-01-01

    Protein-lipid interactions play an important role in cellular protein relocation, activation and signal transduction. The liposome-binding assay is a simple and inexpensive method to examine protein-lipid binding in vitro. The phospholipids used for liposome production are dried and hydrated.

  13. Novel Bat Influenza Virus NS1 Proteins Bind Double-Stranded RNA and Antagonize Host Innate Immunity.

    Science.gov (United States)

    Turkington, Hannah L; Juozapaitis, Mindaugas; Kerry, Philip S; Aydillo, Teresa; Ayllon, Juan; García-Sastre, Adolfo; Schwemmle, Martin; Hale, Benjamin G

    2015-10-01

    We demonstrate that novel bat HL17NL10 and HL18NL11 influenza virus NS1 proteins are effective interferon antagonists but do not block general host gene expression. Solving the RNA-binding domain structures revealed the canonical NS1 symmetrical homodimer, and RNA binding required conserved basic residues in this domain. Interferon antagonism was strictly dependent on RNA binding, and chimeric bat influenza viruses expressing NS1s defective in this activity were highly attenuated in interferon-competent cells but not in cells unable to establish antiviral immunity. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  14. [Determination of plasma protein binding rate of arctiin and arctigenin with ultrafiltration].

    Science.gov (United States)

    Han, Xue-Ying; Wang, Wei; Tan, Ri-Qiu; Dou, De-Qiang

    2013-02-01

    To determine the plasma protein binding rate of arctiin and arctigenin. The ultrafiltration combined with HPLC was employed to determine the plasma protein binding rate of arctiin and arctigenin as well as rat plasma and healthy human plasma proteins. The plasma protein binding rate of arctiin with rat plasma at the concentrations of 64. 29, 32.14, 16.07 mg x L(-1) were (71.2 +/- 2.0)%, (73.4 +/- 0.61)%, (78.2 +/- 1.9)%, respectively; while the plasma protein binding rate of arctiin with healthy human plasma at the above concentrations were (64.8 +/- 3.1)%, (64.5 +/- 2.5)%, (77.5 +/- 1.7)%, respectively. The plasma protein binding rate of arctigenin with rat plasma at the concentrations of 77.42, 38.71, 19.36 mg x L(-1) were (96.7 +/- 0.41)%, (96.8 +/- 1.6)%, (97.3 +/- 0.46)%, respectively; while the plasma protein binding rate of arctigenin with normal human plasma at the above concentrations were (94.7 +/- 3.1)%, (96.8 +/- 1.6)%, (97.9 +/- 1.3)%, respectively. The binding rate of arctiin with rat plasma protein was moderate, which is slightly higher than the binding rate of arctiin with healthy human plasma protein. The plasma protein binding rates of arctigenin with both rat plasma and healthy human plasma are very high.

  15. Binding of monoclonal antibody to protein antigen in fluid phase or bound to solid supports

    Energy Technology Data Exchange (ETDEWEB)

    Kennel, S.J.

    1982-01-01

    Rat monoclonal antibody (MoAb) to fragment D (FgD) of human fibrinogen was used to characterize the direct binding of antibody to protein in solution or bound to solid supports. Purified IgG, F(ab')/sub 2/ and Fab' were prepared from ascites fluid of hybridoma 104-14B which is a fusion product of spleen cells from a rat immunized with FgD and the mouse myeloma cell line, P3-X63-Ag8. Two-dimensional electrophoresis of radioiodinated antibody preparations demonstrated the presence of hybrid immunoglobulin molecules, but only structures having rat heavy and rat light chains had active antibody combinig sites. The affinity constant for IgG as well as F(ab')/sub 2/ and Fab', 6x10/sup 9/ M/sup -1/, was identical when tested using fluid phase antigen (/sup 125/I-labeled FgD). Affinity constants determined for direct binding of iodinated IgG using FgD immobilized on solid supports showed a slight dependence on the antigen concentration used in the measurement. These values ranged from 0.5x10/sup 9/ M/sup -1/ at high antigen concentrations (1.3x10/sup -7/ M) to 9x10/sup 9/ M/sup -1/ at low antigen concentration (1.3x10/sup -10/ M). Binding constants for F(ab')/sub 2/ and Fab' gave similar results indicating that binding was homogeneous and univalent. The capacity of solid state antigen to bind antibody varied with the method used to bind FgD to the solid support. FgD bound directly to polystyrene plates was least efficient at binding labeled antibody; FgD bound to plates through intermediate carriers poly(L-lysine) was only slightly more efficient, while antigen bound to Sepharose beads by cyanogen bromide activation was the most active.

  16. A calmodulin-binding/CGCG box DNA-binding protein family involved in multiple signaling pathways in plants

    Science.gov (United States)

    Yang, Tianbao; Poovaiah, B. W.

    2002-01-01

    We reported earlier that the tobacco early ethylene-responsive gene NtER1 encodes a calmodulin-binding protein (Yang, T., and Poovaiah, B. W. (2000) J. Biol. Chem. 275, 38467-38473). Here we demonstrate that there is one NtER1 homolog as well as five related genes in Arabidopsis. These six genes are rapidly and differentially induced by environmental signals such as temperature extremes, UVB, salt, and wounding; hormones such as ethylene and abscisic acid; and signal molecules such as methyl jasmonate, H(2)O(2), and salicylic acid. Hence, they were designated as AtSR1-6 (Arabidopsis thaliana signal-responsive genes). Ca(2+)/calmodulin binds to all AtSRs, and their calmodulin-binding regions are located on a conserved basic amphiphilic alpha-helical motif in the C terminus. AtSR1 targets the nucleus and specifically recognizes a novel 6-bp CGCG box (A/C/G)CGCG(G/T/C). The multiple CGCG cis-elements are found in promoters of genes such as those involved in ethylene signaling, abscisic acid signaling, and light signal perception. The DNA-binding domain in AtSR1 is located on the N-terminal 146 bp where all AtSR1-related proteins share high similarity but have no similarity to other known DNA-binding proteins. The calmodulin-binding nuclear proteins isolated from wounded leaves exhibit specific CGCG box DNA binding activities. These results suggest that the AtSR gene family encodes a family of calmodulin-binding/DNA-binding proteins involved in multiple signal transduction pathways in plants.

  17. G-protein mediates voltage regulation of agonist binding to muscarinic receptors: effects on receptor-Na/sup +/ channel interaction

    Energy Technology Data Exchange (ETDEWEB)

    Cohen-Armon, M.; Garty, H.; Sokolovsky, M.

    1988-01-12

    The authors previous experiments in membranes prepared from rat heart and brain led them to suggest that the binding of agonist to the muscarinic receptors and to the Na/sup +/ channels is a coupled event mediated by guanine nucleotide binding protein(s) (G-protein(s)). These in vitro findings prompted us to employ synaptoneurosomes from brain stem tissue to examine (i) the binding properties of (/sup 3/H) acetylcholine at resting potential and under depolarization conditions in the absence and presence of pertussis toxin; (ii) the binding of (/sup 3/H)batrachotoxin to Na/sup +/ channel(s) in the presence of the muscarinic agonists; and (iii) muscarinically induced /sup 22/Na/sup +/ uptake in the presence and absence of tetrodotoxin, which blocks Na/sup +/ channels. The findings indicate that agonist binding to muscarinic receptors is voltage dependent, that this process is mediated by G-protein(s), and that muscarinic agonists induce opening of Na/sup +/channels. The latter process persists even after pertussis toxin treatment, indicating that it is not likely to be mediated by pertussis toxin sensitive G-protein(s). The system with its three interacting components-receptor, G-protein, and Na/sup +/ channel-is such that at resting potential the muscarinic receptor induces opening of Na/sup +/ channels; this property may provide a possible physiological mechanism for the depolarization stimulus necessary for autoexcitation or repetitive firing in heart or brain tissues.

  18. G-protein mediates voltage regulation of agonist binding to muscarinic receptors: effects on receptor-Na+ channel interaction

    International Nuclear Information System (INIS)

    Cohen-Armon, M.; Garty, H.; Sokolovsky, M.

    1988-01-01

    The authors previous experiments in membranes prepared from rat heart and brain led them to suggest that the binding of agonist to the muscarinic receptors and to the Na + channels is a coupled event mediated by guanine nucleotide binding protein(s) [G-protein(s)]. These in vitro findings prompted us to employ synaptoneurosomes from brain stem tissue to examine (i) the binding properties of [ 3 H] acetylcholine at resting potential and under depolarization conditions in the absence and presence of pertussis toxin; (ii) the binding of [ 3 H]batrachotoxin to Na + channel(s) in the presence of the muscarinic agonists; and (iii) muscarinically induced 22 Na + uptake in the presence and absence of tetrodotoxin, which blocks Na + channels. The findings indicate that agonist binding to muscarinic receptors is voltage dependent, that this process is mediated by G-protein(s), and that muscarinic agonists induce opening of Na + channels. The latter process persists even after pertussis toxin treatment, indicating that it is not likely to be mediated by pertussis toxin sensitive G-protein(s). The system with its three interacting components-receptor, G-protein, and Na + channel-is such that at resting potential the muscarinic receptor induces opening of Na + channels; this property may provide a possible physiological mechanism for the depolarization stimulus necessary for autoexcitation or repetitive firing in heart or brain tissues

  19. A tool for calculating binding-site residues on proteins from PDB structures

    Directory of Open Access Journals (Sweden)

    Hu Jing

    2009-08-01

    Full Text Available Abstract Background In the research on protein functional sites, researchers often need to identify binding-site residues on a protein. A commonly used strategy is to find a complex structure from the Protein Data Bank (PDB that consists of the protein of interest and its interacting partner(s and calculate binding-site residues based on the complex structure. However, since a protein may participate in multiple interactions, the binding-site residues calculated based on one complex structure usually do not reveal all binding sites on a protein. Thus, this requires researchers to find all PDB complexes that contain the protein of interest and combine the binding-site information gleaned from them. This process is very time-consuming. Especially, combing binding-site information obtained from different PDB structures requires tedious work to align protein sequences. The process becomes overwhelmingly difficult when researchers have a large set of proteins to analyze, which is usually the case in practice. Results In this study, we have developed a tool for calculating binding-site residues on proteins, TCBRP http://yanbioinformatics.cs.usu.edu:8080/ppbindingsubmit. For an input protein, TCBRP can quickly find all binding-site residues on the protein by automatically combining the information obtained from all PDB structures that consist of the protein of interest. Additionally, TCBRP presents the binding-site residues in different categories according to the interaction type. TCBRP also allows researchers to set the definition of binding-site residues. Conclusion The developed tool is very useful for the research on protein binding site analysis and prediction.

  20. Unified understanding of folding and binding mechanisms of globular and intrinsically disordered proteins.

    Science.gov (United States)

    Arai, Munehito

    2018-01-06

    Extensive experimental and theoretical studies have advanced our understanding of the mechanisms of folding and binding of globular proteins, and coupled folding and binding of intrinsically disordered proteins (IDPs). The forces responsible for conformational changes and binding are common in both proteins; however, these mechanisms have been separately discussed. Here, we attempt to integrate the mechanisms of coupled folding and binding of IDPs, folding of small and multi-subdomain proteins, folding of multimeric proteins, and ligand binding of globular proteins in terms of conformational selection and induced-fit mechanisms as well as the nucleation-condensation mechanism that is intermediate between them. Accumulating evidence has shown that both the rate of conformational change and apparent rate of binding between interacting elements can determine reaction mechanisms. Coupled folding and binding of IDPs occurs mainly by induced-fit because of the slow folding in the free form, while ligand binding of globular proteins occurs mainly by conformational selection because of rapid conformational change. Protein folding can be regarded as the binding of intramolecular segments accompanied by secondary structure formation. Multi-subdomain proteins fold mainly by the induced-fit (hydrophobic collapse) mechanism, as the connection of interacting segments enhances the binding (compaction) rate. Fewer hydrophobic residues in small proteins reduce the intramolecular binding rate, resulting in the nucleation-condensation mechanism. Thus, the folding and binding of globular proteins and IDPs obey the same general principle, suggesting that the coarse-grained, statistical mechanical model of protein folding is promising for a unified theoretical description of all mechanisms.

  1. cAMP-binding proteins in medullary tubules from rat kidney: effect of ADH

    International Nuclear Information System (INIS)

    Gapstur, S.M.; Homma, S.; Dousa, T.P.

    1988-01-01

    Little is known of the regulatory steps in the cellular action of vasopressin (AVP) on the renal epithelium, subsequent to the cAMP generation. We studied cAMP-binding proteins in the medullary collecting tubule (MCT) and the thick ascending limb of Henle's loop (MTAL) microdissected from the rat kidney by use of photoaffinity labeling. Microdissected tubules were homogenized and photoaffinity labeled by incubation with 1 microM 32P-labeled 8-azido-adenosine 3',5'-cyclic monophosphate (N3-8-[32P]-cAMP); the incorporated 32P was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Both in MCT and MTAL preparations, the analyses showed incorporation of N3-8-[32P]cAMP into two bands (Mr = 49,000 and Mr = 55,000) that comigrated with standards of the cAMP-dependent protein kinase regulatory subunits RI and RII. In MCT, most of the 32P (80%) was incorporated into RI, whereas in MTAL the 32P incorporated into RI and RII was equivalent. When freshly dissected MCT segments were incubated with 10(-12)-10(-6) M AVP, the subsequent photoaffinity labeling of RI with N3-8-[32P]cAMP was markedly diminished in a dose-dependent manner compared with controls. Our results suggest that cAMP binds in MCT and MTAL to regulatory subunits RI and RII of cAMP-dependent protein kinase. However, in MCT the dominant type of cAMP-dependent protein kinase appears to be type I. The outlined procedure is suitable to indirectly measure the occupancy of RI by endogenous cAMP generated in MCT cells in response to physiological levels (10(-12) M) of AVP

  2. The effects of GH and hormone replacement therapy on serum concentrations of mannan-binding lectin, surfactant protein D and vitamin D binding protein in Turner syndrome

    DEFF Research Database (Denmark)

    Gravholt, Claus Højbjerg; Leth-Larsen, Rikke; Lauridsen, Anna Lis

    2004-01-01

    function. In the present study we examined whether GH or hormone replacement therapy (HRT) in Turner syndrome (TS) influence the serum concentrations of MBL and two other proteins partaking in the innate immune defence, surfactant protein D (SP-D) and vitamin D binding protein (DBP). DESIGN: Study 1...

  3. Identification of a chloroplast coenzyme A-binding protein related to the peroxisomal thiolases.

    Science.gov (United States)

    Yang, L M; Lamppa, G

    1996-12-01

    A 30-kD coenzyme A (CoA)-binding protein was isolated from spinach (Spinacea oleracea) chloroplast soluble extracts using affinity chromatography under conditions in which 95% of the total protein was excluded. The 30-kD protein contains an eight-amino-acid sequence, DVRLYYGA, that is identical to a region in a 36-kD protein of unknown function that is encoded by a kiwifruit (Actinidia deliciosa) cDNA. Southern blotting also detected a spinach gene that is related to the kiwifruit cDNA. The kiwifruit 36-kD protein that was synthesized in Escherichia coli was imported into chloroplasts and cleaved to a 30-kD form; it was processed to the same size in an organelle-free assay. Furthermore, the kiwifruit protein specifically bound to CoA. The kiwifruit protein contains a single cysteine within a domain that is related to the peroxisomal beta-ketoacyl-CoA thiolases, which catalyze the CoA-dependent degradative step of fatty acid beta-oxidation. Within 50 amino acids surrounding the cysteine, considered to be part of the thiolase active site, the kiwifruit protein shows approximately 26% sequence identity with the mango, cucumber, and rat peroxisomal thiolases. N-terminal alignment with these enzymes, relative to the cysteine, indicates that the 36-kD protein is cleaved after serine-58 during import, agreeing with the estimated size (approximately 6 kD) of a transit peptide. The 30-kD protein is also related to the E. coli and mitochondrial thiolases, as well as to the acetoacetyl-CoA thiolases of prokaryotes. Features distinguish it from members of the thiolase family, suggesting that it carries out a related but novel function. The protein is more distantly related to chloroplast beta-ketoacyl-acyl carrier protein synthase III, the initial condensing enzyme of fatty acid synthetase that utilizes acetyl-CoA.

  4. Sodium-dependent reorganization of the sugar-binding site of SGLT1

    DEFF Research Database (Denmark)

    Hirayama, Bruce A; Loo, Donald D F; Díez-Sampedro, Ana

    2007-01-01

    The sodium-dependent glucose cotransporter SGLT1 undergoes a series of voltage- and ligand-induced conformational changes that underlie the cotransport mechanism. In this study we describe how the binding of external Na changes the conformation of the sugar-binding domain, exposing residues that ...

  5. Is vitamin D binding protein a novel predictor of labour?

    Directory of Open Access Journals (Sweden)

    Stella Liong

    Full Text Available Vitamin D binding protein (VDBP has previously been identified in the amniotic fluid and cervicovaginal fluid (CVF of pregnant women. The biological functions of VDBP include acting as a carrier protein for vitamin D metabolites, the clearance of actin that is released during tissue injury and the augmentation of the pro-inflammatory response. This longitudinal observational study was conducted on 221 healthy pregnant women who spontaneously laboured and delivered either at term or preterm. Serial CVF samples were collected and VDBP was measured by ELISA. Binary logistic regression analysis was performed to assess the utility of VDBP as a predictor of labour. VDBP in the CVF did not change between 20 and 35 weeks' gestation. VDBP measured in-labour was significantly increased 4.2 to 7.4-fold compared to 4-7, 8-14 and 15-28 days before labour (P<0.05. VDBP concentration was 4.3-fold significantly higher at 0-3 days compared to 15-28 days pre-labour (P<0.05. The efficacy of VDBP to predict spontaneous labour onset within 3 days provided a positive and negative predictive value of 82.8% and 95.3% respectively (area under receiver operator characteristic curve  = 0.974. This longitudinal study of pregnant women suggests that VDBP in the CVF may be a useful predictor of labour.

  6. QM/MM Molecular Dynamics Studies of Metal Binding Proteins

    Directory of Open Access Journals (Sweden)

    Pietro Vidossich

    2014-07-01

    Full Text Available Mixed quantum-classical (quantum mechanical/molecular mechanical (QM/MM simulations have strongly contributed to providing insights into the understanding of several structural and mechanistic aspects of biological molecules. They played a particularly important role in metal binding proteins, where the electronic effects of transition metals have to be explicitly taken into account for the correct representation of the underlying biochemical process. In this review, after a brief description of the basic concepts of the QM/MM method, we provide an overview of its capabilities using selected examples taken from our work. Specifically, we will focus on heme peroxidases, metallo-β-lactamases, α-synuclein and ligase ribozymes to show how this approach is capable of describing the catalytic and/or structural role played by transition (Fe, Zn or Cu and main group (Mg metals. Applications will reveal how metal ions influence the formation and reduction of high redox intermediates in catalytic cycles and enhance drug metabolism, amyloidogenic aggregate formation and nucleic acid synthesis. In turn, it will become manifest that the protein frame directs and modulates the properties and reactivity of the metal ions.

  7. Identification of pheromone components and their binding affinity to the odorant binding protein CcapOBP83a-2 of the Mediterranean fruit fly, Ceratitis capitata

    Czech Academy of Sciences Publication Activity Database

    Siciliano, P.; He, X. L.; Woodcock, C.; Pickett, J. A.; Field, L. M.; Birkett, M. A.; Kalinová, Blanka; Gomulski, L. M.; Scolari, F.; Gasperi, G.; Malacrida, A. R.; Zhou, J. J.

    2014-01-01

    Roč. 48, May (2014), s. 51-62 ISSN 0965-1748 Institutional support: RVO:61388963 Keywords : medfly * Ceratitis capitata * olfaction * odorant binding protein * pheromone binding protein * pheromone * binding studies * protein expression * electroantennography * GC-EAG * fluorescence displacement Subject RIV: CE - Biochemistry Impact factor: 3.450, year: 2014

  8. Sequential dimerization of human zipcode-binding protein IMP1 on RNA: a cooperative mechanism providing RNP stability

    DEFF Research Database (Denmark)

    Nielsen, J.; Kristensen, M. A.; Willemoes, Martin

    2004-01-01

    of low stability, whereas the second step was the discriminatory event that converted a putative RNA target into a ‘locked' stable RNP. The ability to dimerize was also observed between members of the IMP family of zipcode-binding proteins, providing a multitude of further interaction possibilities......Active cytoplasmic RNA localization depends on the attachment of RNA-binding proteins that dictate the destination of the RNA molecule. In this study, we used an electrophoretic mobility-shift assay in combination with equilibrium and kinetic analyses to characterize the assembly of the human...

  9. Adsorption of DNA binding proteins to functionalized carbon nanotube surfaces with and without DNA wrapping.

    Science.gov (United States)

    Ishibashi, Yu; Oura, Shusuke; Umemura, Kazuo

    2017-09-01

    We examined the adsorption of DNA binding proteins on functionalized, single-walled carbon nanotubes (SWNTs). When SWNTs were functionalized with polyethylene glycol (PEG-SWNT), moderate adsorption of protein molecules was observed. In contrast, nanotubes functionalized with CONH 2 groups (CONH 2 -SWNT) exhibited very strong interactions between the CONH 2 -SWNT and DNA binding proteins. Instead, when these SWNT surfaces were wrapped with DNA molecules (thymine 30-mers), protein binding was a little decreased. Our results revealed that DNA wrapped PEG-SWNT was one of the most promising candidates to realize DNA nanodevices involving protein reactions on DNA-SWNT surfaces. In addition, the DNA binding protein RecA was more adhesive than single-stranded DNA binding proteins to the functionalized SWNT surfaces.

  10. G1/S-regulated E2F-containing protein complexes bind to the mouse thymidine kinase gene promoter

    DEFF Research Database (Denmark)

    Dou, Q P; Zhao, S; Levin, A H

    1994-01-01

    report that MT2 includes an E2F-like binding site (GTTCGCGGGCAAA), as shown by the following evidence. (i) MT2 bound specifically to an affinity-purified fusion human E2F protein. (ii) Both MT2 and an authentic E2F site (TTTCGCGCGCTTT) bound specifically to similar or identical nuclear protein complexes......, a candidate repressor, from the MT2 site in late G1 may be essential for S phase-dependent transcription of the mouse TK gene....

  11. Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev-export function

    Directory of Open Access Journals (Sweden)

    St-Laurent Georges

    2006-11-01

    Full Text Available Abstract Background The HIV Rev protein is known to facilitate export of incompletely spliced and unspliced viral transcripts to the cytoplasm, a necessary step in virus life cycle. The Rev-mediated nucleo-cytoplasmic transport of nascent viral transcripts, dependents on interaction of Rev with the RRE RNA structural element present in the target RNAs. The C-terminal variant of dsRNA-binding nuclear protein 90 (NF90ctv has been shown to markedly attenuate viral replication in stably transduced HIV-1 target cell line. Here we examined a mechanism of interference of viral life cycle involving Rev-NF90ctv interaction. Results Since Rev:RRE complex formations depend on protein:RNA and protein:protein interactions, we investigated whether the expression of NF90ctv might interfere with Rev-mediated export of RRE-containing transcripts. When HeLa cells expressed both NF90ctv and Rev protein, we observed that NF90ctv inhibited the Rev-mediated RNA transport. In particular, three regions of NF90ctv protein are involved in blocking Rev function. Moreover, interaction of NF90ctv with the RRE RNA resulted in the expression of a reporter protein coding sequences linked to the RRE structure. Moreover, Rev influenced the subcellular localization of NF90ctv, and this process is leptomycin B sensitive. Conclusion The dsRNA binding protein, NF90ctv competes with HIV Rev function at two levels, by competitive protein:protein interaction involving Rev binding to specific domains of NF90ctv, as well as by its binding to the RRE-RNA structure. Our results are consistent with a model of Rev-mediated HIV-1 RNA export that envisions Rev-multimerization, a process interrupted by NF90ctv.

  12. Structures of apicomplexan calcium-dependent protein kinases reveal mechanism of activation by calcium

    Energy Technology Data Exchange (ETDEWEB)

    Wernimont, Amy K; Artz, Jennifer D.; Jr, Patrick Finerty; Lin, Yu-Hui; Amani, Mehrnaz; Allali-Hassani, Abdellah; Senisterra, Guillermo; Vedadi, Masoud; Tempel, Wolfram; Mackenzie, Farrell; Chau, Irene; Lourido, Sebastian; Sibley, L. David; Hui, Raymond (Toronto); (WU-MED)

    2010-09-21

    Calcium-dependent protein kinases (CDPKs) have pivotal roles in the calcium-signaling pathway in plants, ciliates and apicomplexan parasites and comprise a calmodulin-dependent kinase (CaMK)-like kinase domain regulated by a calcium-binding domain in the C terminus. To understand this intramolecular mechanism of activation, we solved the structures of the autoinhibited (apo) and activated (calcium-bound) conformations of CDPKs from the apicomplexan parasites Toxoplasma gondii and Cryptosporidium parvum. In the apo form, the C-terminal CDPK activation domain (CAD) resembles a calmodulin protein with an unexpected long helix in the N terminus that inhibits the kinase domain in the same manner as CaMKII. Calcium binding triggers the reorganization of the CAD into a highly intricate fold, leading to its relocation around the base of the kinase domain to a site remote from the substrate binding site. This large conformational change constitutes a distinct mechanism in calcium signal-transduction pathways.

  13. Metal binding is critical for the folding and function of laminin binding protein, Lmb of Streptococcus agalactiae.

    Directory of Open Access Journals (Sweden)

    Preethi Ragunathan

    Full Text Available Lmb is a 34 kDa laminin binding surface adhesin of Streptococcus agalactiae. The structure of Lmb reported by us recently has shown that it consists of a metal binding crevice, in which a zinc ion is coordinated to three highly conserved histidines. To elucidate the structural and functional significance of the metal ion in Lmb, these histidines have been mutated to alanine and single, double and triple mutants were generated. These mutations resulted in insolubility of the protein and revealed altered secondary and tertiary structures, as evidenced by circular dichroism and fluorescence spectroscopy studies. The mutations also significantly decreased the binding affinity of Lmb to laminin, implicating the role played by the metal binding residues in maintaining the correct conformation of the protein for its binding to laminin. A highly disordered loop, proposed to be crucial for metal acquisition in homologous structures, was deleted in Lmb by mutation (ΔLmb and its crystal structure was solved at 2.6 Å. The ΔLmb structure was identical to the native Lmb structure with a bound zinc ion and exhibited laminin binding activity similar to wild type protein, suggesting that the loop might not have an important role in metal acquisition or adhesion in Lmb. Targeted mutations of histidine residues confirmed the importance of the zinc binding crevice for the structure and function of the Lmb adhesin.

  14. GenProBiS: web server for mapping of sequence variants to protein binding sites.

    Science.gov (United States)

    Konc, Janez; Skrlj, Blaz; Erzen, Nika; Kunej, Tanja; Janezic, Dusanka

    2017-07-03

    Discovery of potentially deleterious sequence variants is important and has wide implications for research and generation of new hypotheses in human and veterinary medicine, and drug discovery. The GenProBiS web server maps sequence variants to protein structures from the Protein Data Bank (PDB), and further to protein-protein, protein-nucleic acid, protein-compound, and protein-metal ion binding sites. The concept of a protein-compound binding site is understood in the broadest sense, which includes glycosylation and other post-translational modification sites. Binding sites were defined by local structural comparisons of whole protein structures using the Protein Binding Sites (ProBiS) algorithm and transposition of ligands from the similar binding sites found to the query protein using the ProBiS-ligands approach with new improvements introduced in GenProBiS. Binding site surfaces were generated as three-dimensional grids encompassing the space occupied by predicted ligands. The server allows intuitive visual exploration of comprehensively mapped variants, such as human somatic mis-sense mutations related to cancer and non-synonymous single nucleotide polymorphisms from 21 species, within the predicted binding sites regions for about 80 000 PDB protein structures using fast WebGL graphics. The GenProBiS web server is open and free to all users at http://genprobis.insilab.org. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. Inhibition of E2F-1 transactivation by direct binding of the retinoblastoma protein

    DEFF Research Database (Denmark)

    Helin, K; Harlow, E; Fattaey, A

    1993-01-01

    to transcription factor E2F has provided a model for the mechanism of pRB-mediated growth regulation. Since adenovirus E1A proteins dissociate the pRB-E2F complexes and stimulate E2F-dependent transcription, it has been suggested that pRB inhibits E2F transactivation. Although some evidence for this hypothesis has...... been provided, it has not been possible to determine the mechanism of pRB-mediated inhibition of E2F transactivation. In this study, we constructed mutants of E2F-1 that do not bind to pRB yet retain the ability to transactivate the adenovirus E2 promoter through E2F DNA-binding sites. We demonstrated...

  16. Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides

    Directory of Open Access Journals (Sweden)

    Weonu Choe

    2016-12-01

    Full Text Available The rapidly increasing application of antibodies has inspired the development of several novel methods to isolate and target antibodies using smart biomaterials that mimic the binding of Fc-receptors to antibodies. The Fc-binding domain of antibodies is the primary binding site for e.g., effector proteins and secondary antibodies, whereas antigens bind to the Fab region. Protein A, G, and L, surface proteins expressed by pathogenic bacteria, are well known to bind immunoglobulin and have been widely exploited in antibody purification strategies. Several difficulties are encountered when bacterial proteins are used in antibody research and application. One of the major obstacles hampering the use of bacterial proteins is sample contamination with trace amounts of these proteins, which can invoke an immune response in the host. Many research groups actively develop synthetic ligands that are able to selectively and strongly bind to antibodies. Among the reported ligands, peptides that bind to the Fc-domain of antibodies are attractive tools in antibody research. Besides their use as high affinity ligands in antibody purification chromatography, Fc-binding peptides are applied e.g., to localize antibodies on nanomaterials and to increase the half-life of proteins in serum. In this review, recent developments of Fc-binding peptides are presented and their binding characteristics and diverse applications are discussed.

  17. Identification and quantification of calcium-binding proteins in squid axoplasm

    International Nuclear Information System (INIS)

    Krinks, M.H.; Klee, C.B.; Pant, H.C.; Gainer, H.

    1988-01-01

    The identities and quantities of calcium-binding proteins were determined in axoplasm isolated from the squid giant axon. 45 Ca-binding assays on nitrocellulose filters containing axoplasm proteins separated by SDS-polyacrylamide electrophoresis revealed 4 major calcium-binding bands. These included the high-molecular-weight (Mr greater than 330 and 220 X 10(3] neurofilament proteins, an unidentified protein band that migrated around Mr 55,000, and a diverse group of proteins that migrated together around Mr 17,000. The low-molecular-weight (Mr 17,000) calcium-binding proteins could be resolved into calmodulin (ca. 120 mumol/kg axoplasm), 2 other Mr 17,000 calcium-binding proteins, and a small amount of calcineurin B. It is estimated that these calcium-binding proteins in squid axoplasm could theoretically bind about 1 mmol Ca 2+ /kg axoplasm. 125 I-Calmodulin overlay and Western blot analyses disclosed a number of calmodulin-binding proteins in axoplasm. These included fodrin, calcineurin A, and Ca 2+ /CaM protein kinase II subunits

  18. Interspecies In Vitro Evaluation of Stereoselective Protein Binding for 3,4-Methylenedioxymethamphetamine

    Directory of Open Access Journals (Sweden)

    Wan Raihana Wan Aasim

    2017-01-01

    Full Text Available Abuse of 3,4-methylenedioxymethamphetamine (MDMA is becoming more common worldwide. To date, there is no information available on stereoselectivity of MDMA protein binding in humans, rats, and mice. Since stereoselectivity plays an important role in MDMA’s pharmacokinetics and pharmacodynamics, in this study we investigated its stereoselectivity in protein binding. The stereoselective protein binding of rac-MDMA was investigated using two different concentrations (20 and 200 ng/mL in human plasma and mouse and rat sera using an ultrafiltration technique. No significant stereoselectivity in protein binding was observed in both human plasma and rat serum; however, a significant stereoselective binding (p<0.05 was observed in mouse serum. Since the protein binding of MDMA in mouse serum is considerably lower than in humans and rats, caution should be exercised when using mice for in vitro studies involving MDMA.

  19. Muscle Lim Protein and myosin binding protein C form a complex regulating muscle differentiation.

    Science.gov (United States)

    Arvanitis, Demetrios A; Vafiadaki, Elizabeth; Papalouka, Vasiliki; Sanoudou, Despina

    2017-12-01

    Muscle Lim Protein (MLP) is a protein with multiple functional roles in striated muscle physiology and pathophysiology. Herein, we demonstrate that MLP directly binds to slow, fast, and cardiac myosin-binding protein C (MyBP-C) during myogenesis, as shown by yeast two-hybrid and a range of protein-protein interaction assays. The minimal interacting domains involve MLP inter-LIM and MyBP-C [C4]. The interaction is sensitive to cytosolic Ca 2+ concentrations changes and to MyBP-C phosphorylation by PKA or CaMKII. Confocal microscopy of differentiating myoblasts showed MLP and MyBP-C colocalization during myoblast differentiation. Suppression of the complex formation with recombinant MyBP-C [C4] peptide overexpression, inhibited myoblast differentiation by 65%. Suppression of both MLP and MyBP-C expression in myoblasts by siRNA revealed negative synergistic effects on differentiation. The MLP/MyBP-C complex modulates the actin activated myosin II ATPase activity in vitro, which could interfere with sarcomerogenesis and myofilaments assembly during differentiation. Our data demonstrate a critical role of the MLP/MyBP-C complex during early myoblast differentiation. Its absence in muscles with mutations or aberrant expression of MLP or MyBP-C could be directly implicated in the development of cardiac and skeletal myopathies. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Assessing protein-ligand docking for the binding of organometallic compounds to proteins.

    Science.gov (United States)

    Ortega-Carrasco, Elisabeth; Lledós, Agusti; Maréchal, Jean-Didier

    2014-01-30

    Organometallic compounds are increasingly used as molecular scaffolds in drug development projects; their structural and electronic properties offering novel opportunities in protein-ligand complementarities. Interestingly, while protein-ligand dockings have long become a spearhead in computer assisted drug design, no benchmarking nor optimization have been done for their use with organometallic compounds. Pursuing our efforts to model metal mediated recognition processes, we herein present a systematic study of the capabilities of the program GOLD to predict the interactions of protein with organometallic compounds. The study focuses on inert systems for which no alteration of the first coordination sphere of the metal occurs upon binding. Several scaffolds are used as test systems with different docking schemes and scoring functions. We conclude that ChemScore is the most robust scoring function with ASP and ChemPLP providing with good results too and GoldScore slightly underperforming. This study shows that current state-of-the-art protein-ligand docking techniques are reliable for the docking of inert organometallic compounds binding to protein. Copyright © 2013 Wiley Periodicals, Inc.

  1. Cost Function Network-based Design of Protein-Protein Interactions: predicting changes in binding affinity.

    Science.gov (United States)

    Viricel, Clément; de Givry, Simon; Schiex, Thomas; Barbe, Sophie

    2018-02-20

    Accurate and economic methods to predict change in protein binding free energy upon mutation are imperative to accelerate the design of proteins for a wide range of applications. Free energy is defined by enthalpic and entropic contributions. Following the recent progresses of Artificial Intelligence-based algorithms for guaranteed NP-hard energy optimization and partition function computation, it becomes possible to quickly compute minimum energy conformations and to reliably estimate the entropic contribution of side-chains in the change of free energy of large protein interfaces. Using guaranteed Cost Function Network algorithms, Rosetta energy functions and Dunbrack's rotamer library, we developed and assessed EasyE and JayZ, two methods for binding affinity estimation that ignore or include conformational entropic contributions on a large benchmark of binding affinity experimental measures. If both approaches outperform most established tools, we observe that side-chain conformational entropy brings little or no improvement on most systems but becomes crucial in some rare cases. as open-source Python/C ++ code at sourcesup.renater.fr/projects/easy-jayz. thomas.schiex@inra.fr and sophie.barbe@insa-toulouse.fr. Supplementary data are available at Bioinformatics online.

  2. New Parameters for Higher Accuracy in the Computation of Binding Free Energy Differences upon Alanine Scanning Mutagenesis on Protein-Protein Interfaces.

    Science.gov (United States)

    Simões, Inês C M; Costa, Inês P D; Coimbra, João T S; Ramos, Maria J; Fernandes, Pedro A

    2017-01-23

    Knowing how proteins make stable complexes enables the development of inhibitors to preclude protein-protein (P:P) binding. The identification of the specific interfacial residues that mostly contribute to protein binding, denominated as hot spots, is thus critical. Here, we refine an in silico alanine scanning mutagenesis protocol, based on a residue-dependent dielectric constant version of the Molecular Mechanics/Poisson-Boltzmann Surface Area method. We have used a large data set of structurally diverse P:P complexes to redefine the residue-dependent dielectric constants used in the determination of binding free energies. The accuracy of the method was validated through comparison with experimental data, considering the per-residue P:P binding free energy (ΔΔG binding ) differences upon alanine mutation. Different protocols were tested, i.e., a geometry optimization protocol and three molecular dynamics (MD) protocols: (1) one using explicit water molecules, (2) another with an implicit solvation model, and (3) a third where we have carried out an accelerated MD with explicit water molecules. Using a set of protein dielectric constants (within the range from 1 to 20) we showed that the dielectric constants of 7 for nonpolar and polar residues and 11 for charged residues (and histidine) provide optimal ΔΔG binding predictions. An overall mean unsigned error (MUE) of 1.4 kcal mol -1 relative to the experiment was achieved in 210 mutations only with geometry optimization, which was further reduced with MD simulations (MUE of 1.1 kcal mol -1 for the MD employing explicit solvent). This recalibrated method allows for a better computational identification of hot spots, avoiding expensive and time-consuming experiments or thermodynamic integration/ free energy perturbation/ uBAR calculations, and will hopefully help new drug discovery campaigns in their quest of searching spots of interest for binding small drug-like molecules at P:P interfaces.

  3. Efficient identification of phosphatidylserine-binding proteins by ORF phage display

    International Nuclear Information System (INIS)

    Caberoy, Nora B.; Zhou, Yixiong; Alvarado, Gabriela; Fan, Xianqun; Li, Wei

    2009-01-01

    To efficiently elucidate the biological roles of phosphatidylserine (PS), we developed open-reading-frame (ORF) phage display to identify PS-binding proteins. The procedure of phage panning was optimized with a phage clone expressing MFG-E8, a well-known PS-binding protein. Three rounds of phage panning with ORF phage display cDNA library resulted in ∼300-fold enrichment in PS-binding activity. A total of 17 PS-binding phage clones were identified. Unlike phage display with conventional cDNA libraries, all 17 PS-binding clones were ORFs encoding 13 real proteins. Sequence analysis revealed that all identified PS-specific phage clones had dimeric basic amino acid residues. GST fusion proteins were expressed for 3 PS-binding proteins and verified for their binding activity to PS liposomes, but not phosphatidylcholine liposomes. These results elucidated previously unknown PS-binding proteins and demonstrated that ORF phage display is a versatile technology capable of efficiently identifying binding proteins for non-protein molecules like PS.

  4. Influence of binding pH and protein solubility on the dynamic binding capacity in hydrophobic interaction chromatography.

    Science.gov (United States)

    Baumann, Pascal; Baumgartner, Kai; Hubbuch, Jürgen

    2015-05-29

    Hydrophobic interaction chromatography (HIC) is one of the most frequently used purification methods in biopharmaceutical industry. A major drawback of HIC, however, is the rather low dynamic binding capacity (DBC) obtained when compared to e.g. ion exchange chromatography (IEX). The typical purification procedure for HIC includes binding at neutral pH, independently of the proteins nature and isoelectric point. Most approaches to process intensification are based on resin and salt screenings. In this paper a combination of protein solubility data and varying binding pH leads to a clear enhancement of dynamic binding capacity. This is shown for three proteins of acidic, neutral, and alkaline isoelectric points. High-throughput solubility screenings as well as miniaturized and parallelized breakthrough curves on Media Scout RoboColumns (Atoll, Germany) were conducted at pH 3-10 on a fully automated robotic workstation. The screening results show a correlation between the DBC and the operational pH, the protein's isoelectric point and the overall solubility. Also, an inverse relationship of DBC in HIC and the binding kinetics was observed. By changing the operational pH, the DBC could be increased up to 30% compared to the standard purification procedure performed at neutral pH. As structural changes of the protein are reported during HIC processes, the applied samples and the elution fractions were proven not to be irreversibly unfolded. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. The interaction between anticoagulant protein S and complement regulatory C4b-binding protein (C4BP)

    NARCIS (Netherlands)

    van de Poel, R. H.; Meijers, J. C.; Bouma, B. N.

    2000-01-01

    An important mechanism of regulation of blood coagulation is the anticoagulant protein C pathway. In this pathway, the anticoagulant activity of activated protein C is increased by its cofactor protein S. The cofactor activity of protein S can be regulated by binding to complement regulatory

  6. Crystal structure of bacterial cell-surface alginate-binding protein with an M75 peptidase motif

    International Nuclear Information System (INIS)

    Maruyama, Yukie; Ochiai, Akihito; Mikami, Bunzo; Hashimoto, Wataru; Murata, Kousaku

    2011-01-01

    Research highlights: → Bacterial alginate-binding Algp7 is similar to component EfeO of Fe 2+ transporter. → We determined the crystal structure of Algp7 with a metal-binding motif. → Algp7 consists of two helical bundles formed through duplication of a single bundle. → A deep cleft involved in alginate binding locates around the metal-binding site. → Algp7 may function as a Fe 2+ -chelated alginate-binding protein. -- Abstract: A gram-negative Sphingomonas sp. A1 directly incorporates alginate polysaccharide into the cytoplasm via the cell-surface pit and ABC transporter. A cell-surface alginate-binding protein, Algp7, functions as a concentrator of the polysaccharide in the pit. Based on the primary structure and genetic organization in the bacterial genome, Algp7 was found to be homologous to an M75 peptidase motif-containing EfeO, a component of a ferrous ion transporter. Despite the presence of an M75 peptidase motif with high similarity, the Algp7 protein purified from recombinant Escherichia coli cells was inert on insulin B chain and N-benzoyl-Phe-Val-Arg-p-nitroanilide, both of which are substrates for a typical M75 peptidase, imelysin, from Pseudomonas aeruginosa. The X-ray crystallographic structure of Algp7 was determined at 2.10 A resolution by single-wavelength anomalous diffraction. Although a metal-binding motif, HxxE, conserved in zinc ion-dependent M75 peptidases is also found in Algp7, the crystal structure of Algp7 contains no metal even at the motif. The protein consists of two structurally similar up-and-down helical bundles as the basic scaffold. A deep cleft between the bundles is sufficiently large to accommodate macromolecules such as alginate polysaccharide. This is the first structural report on a bacterial cell-surface alginate-binding protein with an M75 peptidase motif.

  7. Crystal structure of bacterial cell-surface alginate-binding protein with an M75 peptidase motif

    Energy Technology Data Exchange (ETDEWEB)

    Maruyama, Yukie; Ochiai, Akihito [Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Mikami, Bunzo [Laboratory of Applied Structural Biology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Hashimoto, Wataru [Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Murata, Kousaku, E-mail: kmurata@kais.kyoto-u.ac.jp [Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan)

    2011-02-18

    Research highlights: {yields} Bacterial alginate-binding Algp7 is similar to component EfeO of Fe{sup 2+} transporter. {yields} We determined the crystal structure of Algp7 with a metal-binding motif. {yields} Algp7 consists of two helical bundles formed through duplication of a single bundle. {yields} A deep cleft involved in alginate binding locates around the metal-binding site. {yields} Algp7 may function as a Fe{sup 2+}-chelated alginate-binding protein. -- Abstract: A gram-negative Sphingomonas sp. A1 directly incorporates alginate polysaccharide into the cytoplasm via the cell-surface pit and ABC transporter. A cell-surface alginate-binding protein, Algp7, functions as a concentrator of the polysaccharide in the pit. Based on the primary structure and genetic organization in the bacterial genome, Algp7 was found to be homologous to an M75 peptidase motif-containing EfeO, a component of a ferrous ion transporter. Despite the presence of an M75 peptidase motif with high similarity, the Algp7 protein purified from recombinant Escherichia coli cells was inert on insulin B chain and N-benzoyl-Phe-Val-Arg-p-nitroanilide, both of which are substrates for a typical M75 peptidase, imelysin, from Pseudomonas aeruginosa. The X-ray crystallographic structure of Algp7 was determined at 2.10 A resolution by single-wavelength anomalous diffraction. Although a metal-binding motif, HxxE, conserved in zinc ion-dependent M75 peptidases is also found in Algp7, the crystal structure of Algp7 contains no metal even at the motif. The protein consists of two structurally similar up-and-down helical bundles as the basic scaffold. A deep cleft between the bundles is sufficiently large to accommodate macromolecules such as alginate polysaccharide. This is the first structural report on a bacterial cell-surface alginate-binding protein with an M75 peptidase motif.

  8. Regulatory pathways for ATP-binding cassette transport proteins in kidney proximal tubules

    NARCIS (Netherlands)

    Masereeuw, R.; Russel, F.G.M.

    2012-01-01

    The ATP-binding cassette transport proteins (ABC transporters) represent important determinants of drug excretion. Protective or excretory tissues where these transporters mediate substrate efflux include the kidney proximal tubule. Regulation of the transport proteins in this tissue requires

  9. Standardization for cortisol determination in human blood by competitive protein-binding

    International Nuclear Information System (INIS)

    Okada, H.

    1978-01-01

    Standardization for determination of cortisol from human plasma (17-hydroxycorticosteroids) using competitive protein-binding method is presented. Activated carbon coated with dextrans is used for separation of the hormone-protein complexe and hormone labelled free [pt

  10. Environmental xenobiotics may disrupt normal endocrine function by interfering with the binding of physiological ligands to steroid receptors and binding proteins.

    Science.gov (United States)

    Danzo, B J

    1997-01-01

    The disruption of the reproductive system of male and female animals in the wild has been attributed to environmental chemicals (xenobiotics). The effects seen mirror alterations one might anticipate if the steroid hormone-dependent processes that regulate these systems were impaired. To determine whether xenobiotics (present at a concentration of 100 microM) exert their action through steroid-mediated pathways, we examined their ability to inhibit the binding of [3H]physiological ligands (present at a concentration of 7 nM) to the androgen and estrogen receptors, rat androgen-binding protein (ABP), and human sex hormone-binding globulin (hSHBG). The gamma- and delta-isomers of hexachlorocyclohexane, congeners of dichlorodiphenyl-trichloroethane (DDT; p,p'-DDT; p,p'-DDE; o,p'-DDT), dieldrin, atrazine, and pentachlorophenol, caused a statistically significant inhibition of specific binding of [3H]5 alpha-DHT to the androgen receptor that ranged from 100% (p,p'-DDE) to 25% (dieldrin). Methoxychlor, o,p'-DDT1, pentachlorophenol, and nonylphenol significantly reduced [3H]17 beta-estradiol binding to the estrogen receptor by 10, 60, 20, and 75%, respectively. The binding of [3H]5 alpha-DHT to ABP was inhibited 70% by the delta-isomer of hexachlorocyclohexane, but the gamma-isomer did not reduce binding significantly. Methoxychlor, p,p'-DDT, atrazine, and nonylphenol reduced [3H]5 alpha-DHT binding to ABP by approximately 40%. Nonylphenol reduced the binding of [3H]5 alpha-DHT to hSHBG by 70%. Hexachlorocyclohexane reduced [3H]5 alpha-DHT binding to hSHBG by 20%, but the stereospecific effects observed with ABP did not occur. o,p'-DDT and pentachlorophenol resulted in a statistically significant 20% inhibition of [3H]5 alpha-DHT binding to hSHBG. Some xenobiotics resulted in dissociation of [3H]ligands from their binding proteins that was statistically identical to that caused by the unlabeled natural ligand, whereas others resulted in slower or more rapid dissociation

  11. Binding the mammalian high mobility group protein AT-hook 2 to AT-rich deoxyoligonucleotides: enthalpy-entropy compensation.

    Science.gov (United States)

    Joynt, Suzanne; Morillo, Victor; Leng, Fenfei

    2009-05-20

    HMGA2 is a DNA minor-groove binding protein. We previously demonstrated that HMGA2 binds to AT-rich DNA with very high binding affinity where the binding of HMGA2 to poly(dA-dT)(2) is enthalpy-driven and to poly(dA)poly(dT) is entropy-driven. This is a typical example of enthalpy-entropy compensation. To further study enthalpy-entropy compensation of HMGA2, we used isothermal-titration-calorimetry to examine the interactions of HMGA2 with two AT-rich DNA hairpins: 5'-CCAAAAAAAAAAAAAAAGCCCCCGCTTTTTTTTTTTTTTTGG-3' (FL-AT-1) and 5'-CCATATATATATATATAGCCCCCGCTATATATATATATATGG-3' (FL-AT-2). Surprisingly, we observed an atypical isothermal-titration-calorimetry-binding curve at low-salt aqueous solutions whereby the apparent binding-enthalpy decreased dramatically as the titration approached the end. This unusual behavior can be attributed to the DNA-annealing coupled to the ligand DNA-binding and is eliminated by increasing the salt concentration to approximately 200 mM. At this condition, HMGA2 binding to FL-AT-1 is entropy-driven and to FL-AT-2 is enthalpy-driven. Interestingly, the DNA-binding free energies for HMGA2 binding to both hairpins are almost temperature independent; however, the enthalpy-entropy changes are dependent on temperature, which is another aspect of enthalpy-entropy compensation. The heat capacity change for HMGA2 binding to FL-AT-1 and FL-AT-2 are almost identical, indicating that the solvent displacement and charge-charge interaction in the coupled folding/binding processes for both binding reactions are similar.

  12. Clumping factor A, von Willebrand factor-binding protein and von Willebrand factor anchor Staphylococcus aureus to the vessel wall.

    Science.gov (United States)

    Claes, J; Liesenborghs, L; Peetermans, M; Veloso, T R; Missiakas, D; Schneewind, O; Mancini, S; Entenza, J M; Hoylaerts, M F; Heying, R; Verhamme, P; Vanassche, T

    2017-05-01

    Essentials Staphylococcus aureus (S. aureus) binds to endothelium via von Willebrand factor (VWF). Secreted VWF-binding protein (vWbp) mediates S. aureus adhesion to VWF under shear stress. vWbp interacts with VWF and the Sortase A-dependent surface protein Clumping factor A (ClfA). VWF-vWbp-ClfA anchor S. aureus to vascular endothelium under shear stress. Objective When establishing endovascular infections, Staphylococcus aureus (S. aureus) overcomes shear forces of flowing blood by binding to von Willebrand factor (VWF). Staphylococcal VWF-binding protein (vWbp) interacts with VWF, but it is unknown how this secreted protein binds to the bacterial cell wall. We hypothesized that vWbp interacts with a staphylococcal surface protein, mediating the adhesion of S. aureus to VWF and vascular endothelium under shear stress. Methods We studied the binding of S. aureus to vWbp, VWF and endothelial cells in a micro-parallel flow chamber using various mutants deficient in Sortase A (SrtA) and SrtA-dependent surface proteins, and Lactococcus lactis expressing single staphylococcal surface proteins. In vivo adhesion of bacteria was evaluated in the murine mesenteric circulation using real-time intravital vascular microscopy. Results vWbp bridges the bacterial cell wall and VWF, allowing shear-resistant binding of S. aureus to inflamed or damaged endothelium. Absence of SrtA and Clumping factor A (ClfA) reduced adhesion of S. aureus to vWbp, VWF and activated endothelial cells. ADAMTS-13 and an anti-VWF A1 domain antibody, when combined, reduced S. aureus adhesion to activated endothelial cells by 90%. Selective overexpression of ClfA in the membrane of Lactococcus lactis enabled these bacteria to bind to VWF and activated endothelial cells but only in the presence of vWbp. Absence of ClfA abolished bacterial adhesion to the activated murine vessel wall. Conclusions vWbp interacts with VWF and with the SrtA-dependent staphylococcal surface protein ClfA. The complex formed by

  13. Extreme sequence divergence but conserved ligand-binding specificity in Streptococcus pyogenes M protein.

    Directory of Open Access Journals (Sweden)

    2006-05-01

    Full Text Available Many pathogenic microorganisms evade host immunity through extensive sequence variability in a protein region targeted by protective antibodies. In spite of the sequence variability, a variable region commonly retains an important ligand-binding function, reflected in the prese