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Sample records for affinity binding site

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

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    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

  2. Label-free microscale thermophoresis discriminates sites and affinity of protein-ligand binding.

    Science.gov (United States)

    Seidel, Susanne A I; Wienken, Christoph J; Geissler, Sandra; Jerabek-Willemsen, Moran; Duhr, Stefan; Reiter, Alwin; Trauner, Dirk; Braun, Dieter; Baaske, Philipp

    2012-10-15

    Look, no label! Microscale thermophoresis makes use of the intrinsic fluorescence of proteins to quantify the binding affinities of ligands and discriminate between binding sites. This method is suitable for studying binding interactions of very small amounts of protein in solution. The binding of ligands to iGluR membrane receptors, small-molecule inhibitorss to kinase p38, aptamers to thrombin, and Ca(2+) ions to synaptotagmin was quantified.

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

    Science.gov (United States)

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

    2014-01-15

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

  4. Soybean. beta. -glucan binding sites display maximal affinity for a heptaglucoside phytoalexin-elicitor

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    Cosio, E.G.; Waldmueller, T.; Frey, T.; Ebel, J. (Biologisches Institut II der Universitat Freiburg (West Germany))

    1990-05-01

    The affinity of soybean {beta}-glucan-binding sites for a synthetic heptaglucan elicitor was tested in a ligand-competition assay against a {sup 125}I-labeled 1,3-1,6-{beta}-glucan preparation (avg. DP=20). Half-maximal displacement of label (IC{sub 50}) was obtained at 9nM heptaglucan, the highest affinity of all fractions tested to date. Displacement followed a uniform sigmoidal pattern and was complete at 1{mu}M indicating access of heptaglucan to all sites available to the labeled elicitor. A mathematical model was used to predict IC{sub 50} values according to the DP of glucan fragments obtained from fungal cell walls. The lowest IC{sub 50} predicted by this model is 3nM. Binding affinity of the glucans was compared with their elicitor activity in a bioassay.

  5. SELMAP - SELEX affinity landscape MAPping of transcription factor binding sites using integrated microfluidics.

    Science.gov (United States)

    Chen, Dana; Orenstein, Yaron; Golodnitsky, Rada; Pellach, Michal; Avrahami, Dorit; Wachtel, Chaim; Ovadia-Shochat, Avital; Shir-Shapira, Hila; Kedmi, Adi; Juven-Gershon, Tamar; Shamir, Ron; Gerber, Doron

    2016-09-15

    Transcription factors (TFs) alter gene expression in response to changes in the environment through sequence-specific interactions with the DNA. These interactions are best portrayed as a landscape of TF binding affinities. Current methods to study sequence-specific binding preferences suffer from limited dynamic range, sequence bias, lack of specificity and limited throughput. We have developed a microfluidic-based device for SELEX Affinity Landscape MAPping (SELMAP) of TF binding, which allows high-throughput measurement of 16 proteins in parallel. We used it to measure the relative affinities of Pho4, AtERF2 and Btd full-length proteins to millions of different DNA binding sites, and detected both high and low-affinity interactions in equilibrium conditions, generating a comprehensive landscape of the relative TF affinities to all possible DNA 6-mers, and even DNA10-mers with increased sequencing depth. Low quantities of both the TFs and DNA oligomers were sufficient for obtaining high-quality results, significantly reducing experimental costs. SELMAP allows in-depth screening of hundreds of TFs, and provides a means for better understanding of the regulatory processes that govern gene expression.

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

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    Klotz, K.L.

    1984-01-01

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

  7. Photo-Affinity Labeling of Specific Acetylcholine-Binding Sites on Membranes

    Science.gov (United States)

    Kiefer, Hansruedi; Lindstrom, Jon; Lennox, Edwin S.; Singer, S. J.

    1970-01-01

    Acetylcholinesterase of intact red blood cell membranes and the acetylcholine receptor at the neuromuscular junction of whole-frog sartorius muscle have been irreversibly inactivated by photo-affinity labeling with two quaternary ammonium aryl azides. The inactivation requires that the azides, at the time of their photolytic conversion to highly reactive nitrenes, are reversibly bound to the specific acetylcholine-binding sites. PMID:5275370

  8. Muscarinic cholinergic receptor binding sites differentiated by their affinity for pirenzepine do not interconvert

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    Gil, D.W.; Wolfe, B.B.

    1986-05-01

    Although it has been suggested by many investigators that subtypes of muscarinic cholinergic receptors exist, physical studies of solubilized receptors have indicated that only a single molecular species may exist. To test the hypothesis that the putative muscarinic receptor subtypes in rat forebrain are interconvertible states of the same receptor, the selective antagonist pirenzepine (PZ) was used to protect muscarinic receptors from blockade by the irreversible muscarinic receptor antagonist propylbenzilylcholine mustard (PBCM). If interconversion of high (M1) and low (M2) affinity binding sites for PZ occurs, incubation of cerebral cortical membranes with PBCM in the presence of PZ should not alter the proportions of M1 and M2 binding sites that are unalkylated (i.e., protected). If, on the other hand, the binding sites are not interconvertible, PZ should be able to selectively protect M1 sites and alter the proportions of unalkylated M1 and M2 binding sites. In the absence of PZ, treatment of cerebral cortical membranes with 20 nM PBCM at 4 degrees C for 50 min resulted in a 69% reduction in the density of M1 binding sites and a 55% reduction in the density of M2 binding sites with no change in the equilibrium dissociation constants of the radioligands (/sup 3/H)quinuclidinyl benzilate or (/sup 3/H)PZ. The reasons for this somewhat selective effect of PBCM are not apparent. In radioligand binding experiments using cerebral cortical membranes, PZ inhibited the binding of (/sup 3/H)quinuclidinyl benzilate in a biphasic manner.

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

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    Nye, J.S.

    1988-01-01

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

  10. Low affinity Ca2+-binding sites of calcineurin B mediate conformational changes in calcineurin A.

    Science.gov (United States)

    Yang, S A; Klee, C B

    2000-12-26

    Limited proteolysis of calcineurin in the presence of Ca(2+) suggested that its calmodulin-binding domain, readily degraded by proteases, was unfolded while calcineurin B was compactly folded [Hubbard, M. J., and Klee, C. B. (1989) Biochemistry 28, 1868-1874]. Moreover, in the crystal structure of calcineurin, with the four Ca(2+) sites of calcineurin B occupied, the calmodulin-binding domain is not visible in the electron density map [Kissinger, C. R., et al. (1995) Nature 378, 641-644]. Limited proteolysis of calcineurin in the presence of EGTA, shows that, when the low affinity sites of calcineurin B are not occupied, the calmodulin-binding domain is completely protected against proteolytic attack. Slow cleavages are, however, detected in the linker region between the calmodulin-binding and the autoinhibitory domains of calcineurin A. Upon prolonged exposure to the protease, selective cleavages in carboxyl-terminal end of the first helix and the central helix linker of calcineurin B and the calcineurin B-binding helix of calcineurin A are also detected. Thus, Ca(2+) binding to the low-affinity sites of calcineurin B affects the conformation of calcineurin B and induces a conformational change of the regulatory domain of calcineurin A, resulting in the exposure of the calmodulin-binding domain. This conformational change is needed for the partial activation of the enzyme in the absence of calmodulin and its full activation by calmodulin. A synthetic peptide corresponding to the calmodulin-binding domain is shown to interact with a peptide corresponding to the calcineurin B-binding domain, and this interaction is prevented by calcineurin B in the presence but not the absence of Ca(2+). These observations provide a mechanism to explain the dependence on Ca(2+) binding to calcineurin B for calmodulin activation and for the 10-20-fold increase in affinity of calcineurin for Ca(2+) upon removal of the regulatory domain by limited proteolysis [Stemmer, P. M., and Klee

  11. Thermodynamics of Calcium binding to the Calmodulin N-terminal domain to evaluate site-specific affinity constants and cooperativity.

    Science.gov (United States)

    Beccia, Maria Rosa; Sauge-Merle, Sandrine; Lemaire, David; Brémond, Nicolas; Pardoux, Romain; Blangy, Stéphanie; Guilbaud, Philippe; Berthomieu, Catherine

    2015-07-01

    Calmodulin (CaM) is an essential Ca(II)-dependent regulator of cell physiology. To understand its interaction with Ca(II) at a molecular level, it is essential to examine Ca(II) binding at each site of the protein, even if it is challenging to estimate the site-specific binding properties of the interdependent CaM-binding sites. In this study, we evaluated the site-specific Ca(II)-binding affinity of sites I and II of the N-terminal domain by combining site-directed mutagenesis and spectrofluorimetry. The mutations had very low impact on the protein structure and stability. We used these binding constants to evaluate the inter-site cooperativity energy and compared it with its lower limit value usually reported in the literature. We found that site I affinity for Ca(II) was 1.5 times that of site II and that cooperativity induced an approximately tenfold higher affinity for the second Ca(II)-binding event, as compared to the first one. We further showed that insertion of a tryptophan at position 7 of site II binding loop significantly increased site II affinity for Ca(II) and the intra-domain cooperativity. ΔH and ΔS parameters were studied by isothermal titration calorimetry for Ca(II) binding to site I, site II and to the entire N-terminal domain. They showed that calcium binding is mainly entropy driven for the first and second binding events. These findings provide molecular information on the structure-affinity relationship of the individual sites of the CaM N-terminal domain and new perspectives for the optimization of metal ion binding by mutating the EF-hand loops sequences.

  12. Active site - a site of binding of affinity inhibitors in baker's yeast inorganic pyrophosphatase

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    Svyato, I.E.; Sklyankina, V.A.; Avaeva, S.M.

    1986-03-20

    The interaction of the enzyme-substrate complex with methyl phosphate, O-phosphoethanolamine, O-phosphopropanolamine, N-acetylphosphoserine, and phosphoglyolic acid, as well as pyrophosphatase, modified by monoesters of phosphoric acid, with pyrophosphate and tripolyphosphate, was investigated. It was shown that the enzyme containing the substrate in the active site does not react with monophosphates, but modified pyrophosphatase entirely retains the ability to bind polyanions to the regulatory site. It is concluded that the inactivation of baker's yeast inorganic pyrophosphatase by monoesters of phosphoric acid, which are affinity inhibitors of it, is the result of modification of the active site of the enzyme.

  13. High affinity binding site-mediated prevention of chemical absorption across the gastrointestinal tract.

    Science.gov (United States)

    Rasmussen, M V; Barker, T T; Silbart, L K

    2001-12-15

    Preventing mucosal absorption of low-molecular weight compounds such as carcinogens, toxins and drugs could help prevent many diseases. To characterize the effects of dose and timing on high-affinity binding site mediated sequestration of specific chemical ligands in the gastrointestinal tract, avidin was perorally-administered to mice either prior to or mixed with 3H-biotin. Avidin enhanced fecal 3H-biotin excretion in a dose-dependent manner, consistent with the accepted mechanism of egg white-induced biotin deficiency syndrome. Avidin administration up to 4 h before 3H-biotin administration also enhanced fecal 3H-biotin excretion. Activated charcoal (AC) reduced 3H-biotin absorption when mixed with 3H-biotin before ingestion, but was ineffective when ingested prior to 3H-biotin. These studies suggest that ingestion of high-affinity protein binding sites can establish an absorptive barrier at the gastrointestinal mucosa to prevent the uptake of unwanted low molecular-weight chemicals.

  14. Purification of capping protein using the capping protein binding site of CARMIL as an affinity matrix.

    Science.gov (United States)

    Remmert, Kirsten; Uruno, Takehito; Hammer, John A

    2009-10-01

    Capping protein (CP) is a ubiquitously expressed, heterodimeric actin binding protein that is essential for normal actin dynamics in cells. The existing methods for purifying native CP from tissues and recombinant CP from bacteria are time-consuming processes that involve numerous conventional chromatographic steps and functional assays to achieve a homogeneous preparation of the protein. Here, we report the rapid purification of Acanthamoeba CP from amoeba extracts and recombinant mouse CP from E. coli extracts using as an affinity matrix GST-fusion proteins containing the CP binding site from Acanthamoeba CARMIL and mouse CARMIL-1, respectively. This improved method for CP purification should facilitate the in vitro analysis of CP structure, function, and regulation.

  15. Affinity polymers tailored for the protein A binding site of immunoglobulin G proteins.

    Science.gov (United States)

    Latza, Patricia; Gilles, Patrick; Schaller, Torsten; Schrader, Thomas

    2014-09-01

    Rational design in combination with a screening process was used to develop affinity polymers for a specific binding site on the surface of immunoglobulin G (IgG) proteins. The concept starts with the identification of critical amino acid residues on the protein interface and their topological arrangement. Appropriate binding monomers were subsequently synthesized. Together with a sugar monomer (2-5 equiv) for water solubility and a dansyl monomer (0.5 equiv) as a fluorescent label, they were subjected in aqueous solution to linear radical copolymerization in various compositions (e.g., azobisisobutyronitrile (AIBN), homogeneous water/DMF mixtures). After ultrafiltration and lyophilization, colorless dry water-soluble powders were obtained. NMR spectroscopic and gel permeation chromatography (GPC) characterization indicated molecular weights between 30 and 500 kD and confirmed retention of monomer composition as well as the absence of monomers. In a competitive enzyme-linked immunosorbent assay (ELISA) screen of the polymer libraries (20-50 members), few copolymers qualified as strong and selective binders for the protein A binding site on the Fc fragment of the antibody. Their monomer composition precisely reflected the critical amino acids found at the interface. The simple combination of a charged and a nonpolar binding monomer sufficed for selective submicromolar IgG recognition by the synthetic polymer. Affinities were confirmed by fluorescence titrations; they increased with decreasing salt load but remained largely unaltered at lowered pH. Other proteins, including those of similar size and isoelectric point (pI), were bound 10-1000 times less tightly. This example indicates that interaction domains in other proteins may also be targeted by synthetic polymers if their comonomer composition reflects the nature and arrangement of amino acid residues on the protein surface.

  16. SNP2TFBS – a database of regulatory SNPs affecting predicted transcription factor binding site affinity

    Science.gov (United States)

    Kumar, Sunil; Ambrosini, Giovanna; Bucher, Philipp

    2017-01-01

    SNP2TFBS is a computational resource intended to support researchers investigating the molecular mechanisms underlying regulatory variation in the human genome. The database essentially consists of a collection of text files providing specific annotations for human single nucleotide polymorphisms (SNPs), namely whether they are predicted to abolish, create or change the affinity of one or several transcription factor (TF) binding sites. A SNP's effect on TF binding is estimated based on a position weight matrix (PWM) model for the binding specificity of the corresponding factor. These data files are regenerated at regular intervals by an automatic procedure that takes as input a reference genome, a comprehensive SNP catalogue and a collection of PWMs. SNP2TFBS is also accessible over a web interface, enabling users to view the information provided for an individual SNP, to extract SNPs based on various search criteria, to annotate uploaded sets of SNPs or to display statistics about the frequencies of binding sites affected by selected SNPs. Homepage: http://ccg.vital-it.ch/snp2tfbs/. PMID:27899579

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

    OpenAIRE

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

    2013-01-01

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

  18. Short-term desensitization of muscarinic cholinergic receptors in mouse neuroblastoma cells: selective loss of agonist low-affinity and pirenzepine high-affinity binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Cioffi, C.L.; el-Fakahany, E.E.

    1986-09-01

    The effects of brief incubation with carbamylcholine on subsequent binding of (/sup 3/H)N-methylscopolamine were investigated in mouse neuroblastoma cells (clone N1E-115). This treatment demonstrated that the muscarinic receptors in this neuronal clone can be divided into two types; one which is readily susceptible to regulation by receptor agonists, whereas the other is resistant in this regard. In control cells, both pirenzepine and carbamylcholine interacted with high- and low-affinity subsets of muscarinic receptors. Computer-assisted analysis of the competition between pirenzepine and carbamylcholine with (/sup 3/H)N-methylscopolamine showed that the receptor sites remaining upon desensitization are composed mainly of pirenzepine low-affinity and agonist high-affinity binding sites. Furthermore, there was an excellent correlation between the ability of various muscarinic receptor agonists to induce a decrease in consequent (/sup 3/H)N-methylscopolamine binding and their efficacy in stimulating cyclic GMP synthesis in these cells. Thus, only the agonists that are known to recognize the receptor's low-affinity conformation in order to elicit increases in cyclic GMP levels were capable of diminishing ligand binding. Taken together, our present results suggest that the receptor population that is sensitive to regulation by agonists includes both the pirenzepine high-affinity and the agonist low-affinity receptor binding states. In addition, the sensitivity of these receptor subsets to rapid regulation by agonists further implicates their involvement in desensitization of muscarinic receptor-mediated cyclic GMP formation.

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

    Science.gov (United States)

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

    2013-01-01

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

  20. New synthesis and tritium labeling of a selective ligand for studying high-affinity γ-hydroxybutyrate (GHB) binding sites.

    Science.gov (United States)

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

    2013-10-24

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

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

    DEFF Research Database (Denmark)

    Vogensen, Stine B.; Marek, Ales; Bay, Tina

    2013-01-01

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

  2. Tuning the affinity of anion binding sites in porin channels with negatively charged residues: molecular details for OprP.

    Science.gov (United States)

    Modi, Niraj; Bárcena-Uribarri, Iván; Bains, Manjeet; Benz, Roland; Hancock, Robert E W; Kleinekathöfer, Ulrich

    2015-02-20

    The cell envelope of the Gram negative opportunistic pathogen Pseudomonas aeruginosa is poorly permeable to many classes of hydrophilic molecules including antibiotics due to the presence of the narrow and selective porins. Here we focused on one of the narrow-channel porins, that is, OprP, which is responsible for the high-affinity uptake of phosphate ions. Its two central binding sites for phosphate contain a number of positively charged amino acids together with a single negatively charged residue (D94). The presence of this negatively charged residue in a binding site for negatively charged phosphate ions is highly surprising due to the potentially reduced binding affinity. The goal of this study was to better understand the role of D94 in phosphate binding, selectivity, and transport using a combination of mutagenesis, electrophysiology, and free-energy calculations. The presence of a negatively charged residue in the binding site is critical for this specific porin OprP as emphasized by the evolutionary conservation of such negatively charged residue in the binding site of several anion-selective porins. Mutations of D94 in OprP to any positively charged or neutral residue increased the binding affinity of phosphate for OprP. Detailed analysis indicated that this anionic residue in the phosphate binding site of OprP, despite its negative charge, maintained energetically favorable phosphate binding sites in the central region of the channel and at the same time decreased residence time thus preventing excessively strong binding of phosphate that would oppose phosphate flux through the channel. Intriguingly mutations of D94 to positively charged residues, lysine and arginine, resulted in very different binding affinities and free energy profiles, indicating the importance of side chain conformations of these positively charged residues in phosphate binding to OprP.

  3. Role of the low-affinity binding site in electron transfer from cytochrome C to cytochrome C peroxidase.

    Science.gov (United States)

    Mei, Hongkang; Geren, Lois; Miller, Mark A; Durham, Bill; Millett, Francis

    2002-03-26

    The interaction of yeast iso-1-cytochrome c (yCc) with the high- and low-affinity binding sites on cytochrome c peroxidase compound I (CMPI) was studied by stopped-flow spectroscopy. When 3 microM reduced yCc(II) was mixed with 0.5 microM CMPI at 10 mM ionic strength, the Trp-191 radical cation was reduced from the high-affinity site with an apparent rate constant >3000 s(-1), followed by slow reduction of the oxyferryl heme with a rate constant of only 10 s(-1). In contrast, mixing 3 microM reduced yCc(II) with 0.5 microM preformed CMPI *yCc(III) complex led to reduction of the radical cation with a rate constant of 10 s(-1), followed by reduction of the oxyferryl heme in compound II with the same rate constant. The rate constants for reduction of the radical cation and the oxyferryl heme both increased with increasing concentrations of yCc(II) and remained equal to each other. These results are consistent with a mechanism in which both the Trp-191 radical cation and the oxyferryl heme are reduced by yCc(II) in the high-affinity binding site, and the reaction is rate-limited by product dissociation of yCc(III) from the high-affinity site with apparent rate constant k(d). Binding yCc(II) to the low-affinity site is proposed to increase the rate constant for dissociation of yCc(III) from the high-affinity site in a substrate-assisted product dissociation mechanism. The value of k(d) is 2000 s(-1) for the 2:1 complex at 10 mM ionic strength. The reaction of horse Cc(II) with CMPI was greatly inhibited by binding 1 equiv of yCc(III) to the high-affinity site, providing evidence that reduction of the oxyferryl heme involves electron transfer from the high-affinity binding site rather than the low-affinity site. The effects of CcP surface mutations on the dissociation rate constant indicate that the high-affinity binding site used for the reaction in solution is the same as the one identified in the yCc*CcP crystal structure.

  4. Autoradiographic imaging and quantification of the high-affinity GHB binding sites in rodent brain using (3)H-HOCPCA

    DEFF Research Database (Denmark)

    Klein, A B; Bay, T; Villumsen, I S

    2016-01-01

    analogue, 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) as a tritiated version ((3)H-HOCPCA) to radioactively label the specific GHB high-affinity binding site and gain further insight into the density, distribution and developmental profile of this protein. We show that, in low nanomolar concentrations......, (3)H-HOCPCA displays excellent signal-to-noise ratios using rodent brain autoradiography, which makes it a valuable ligand for anatomical quantification of native GHB binding site levels. Our data confirmed that (3)H-HOCPCA labels only the high-affinity specific GHB binding site, found in high...... density in cortical and hippocampal regions. The experiments revealed markedly stronger binding at pH 6.0 (Kd 73.8 nM) compared to pH 7.4 (Kd 2312 nM), as previously reported for other GHB radioligands but similar Bmax values. Using (3)H-HOCPCA we analyzed the GHB binding protein profile during mouse...

  5. Identification and properties of very high affinity brain membrane-binding sites for a neurotoxic phospholipase from the taipan venom

    Energy Technology Data Exchange (ETDEWEB)

    Lambeau, G.; Barhanin, J.; Schweitz, H.; Qar, J.; Lazdunski, M. (Centre de Biochimie, Nice (France))

    1989-07-05

    Four new monochain phospholipases were purified from the Oxyuranus scutellatus (taipan) venom. Three of them were highly toxic when injected into mice brain. One of these neurotoxic phospholipases, OS2, was iodinated and used in binding experiments to demonstrate the presence of two families of specific binding sites in rat brain synaptic membranes. The affinities were exceptionally high, Kd1 = 1.5 +/- 0.5 pM and Kd2 = 45 +/- 10 pM, and the maximal binding capacities were Bmax 1 = 1 +/- 0.4 and Bmax 2 = 3 +/- 0.5 pmol/mg of protein. Both binding sites were sensitive to proteolysis and demonstrated to be located on proteins of Mr 85,000-88,000 and 36,000-51,000 by cross-linking and photoaffinity labeling techniques. The binding of {sup 125}I-OS2 to synaptic membranes was dependent on Ca2+ ions and enhanced by Zn2+ ions which inhibit phospholipase activity. Competition experiments have shown that, except for beta-bungarotoxin, a number of known toxic snake or bee phospholipases have very high affinities for the newly identified binding sites. A good correlation (r = 0.80) was observed between toxicity and affinity but not between phospholipase activity and affinity.

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

    Energy Technology Data Exchange (ETDEWEB)

    Altar, C.A.; Burton, L.E.; Bennett, G.L.; Dugich-Djordjevic, M. (Genentech, Inc., South San Francisco, CA (USA))

    1991-01-01

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

  7. Autoradiographic imaging and quantification of the high-affinity GHB binding sites in rodent brain using (3)H-HOCPCA.

    Science.gov (United States)

    Klein, A B; Bay, T; Villumsen, I S; Falk-Petersen, C B; Marek, A; Frølund, B; Clausen, R P; Hansen, H D; Knudsen, G M; Wellendorph, P

    2016-11-01

    GHB (γ-hydroxybutyric acid) is a compound endogenous to mammalian brain with high structural resemblance to GABA. GHB possesses nanomolar-micromolar affinity for a unique population of binding sites, but the exact nature of these remains elusive. In this study we utilized the highly selective GHB analogue, 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) as a tritiated version ((3)H-HOCPCA) to radioactively label the specific GHB high-affinity binding site and gain further insight into the density, distribution and developmental profile of this protein. We show that, in low nanomolar concentrations, (3)H-HOCPCA displays excellent signal-to-noise ratios using rodent brain autoradiography, which makes it a valuable ligand for anatomical quantification of native GHB binding site levels. Our data confirmed that (3)H-HOCPCA labels only the high-affinity specific GHB binding site, found in high density in cortical and hippocampal regions. The experiments revealed markedly stronger binding at pH 6.0 (Kd 73.8 nM) compared to pH 7.4 (Kd 2312 nM), as previously reported for other GHB radioligands but similar Bmax values. Using (3)H-HOCPCA we analyzed the GHB binding protein profile during mouse brain development. Due to the high sensitivity of this radioligand, we were able to detect low levels of specific binding already at E15 in mouse brain, which increased progressively until adulthood. Collectively, we show that (3)H-HOCPCA is a highly sensitive radioligand, offering advantages over the commonly used radioligand (3)H-NCS-382, and thus a very suitable in vitro tool for qualitative and quantitative autoradiography of the GHB high-affinity site.

  8. Radiosynthesis and Evaluation of [(11)C]3-Hydroxycyclopent-1-enecarboxylic Acid as Potential PET Ligand for the High-Affinity γ-Hydroxybutyric Acid Binding Sites

    DEFF Research Database (Denmark)

    Jensen, Claus H; Hansen, Hanne D; Bay, Tina

    2017-01-01

    the (11)C-labeling and subsequent evaluation of [(11)C]HOCPCA in a domestic pig, as a PET-radioligand for visualization of the high-affinity GHB binding sites in the live pig brain. To investigate the regional binding of HOCPCA in pig brain prior to in vivo PET studies, in vitro quantitative......γ-Hydroxybutyric acid (GHB) is an endogenous neuroactive substance and proposed neurotransmitter with affinity for both low- and high-affinity binding sites. A radioligand with high and specific affinity toward the high-affinity GHB binding site would be a unique tool toward a more complete...... understanding of this population of binding sites. With its high specific affinity and monocarboxylate transporter (MCT1) mediated transport across the blood-brain barrier in pharmacological doses, 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) seems like a suitable PET radiotracer candidate. Here, we report...

  9. Novel cyclic gamma-hydroxybutyrate (GHB) analogs with high affinity and stereoselectivity of binding to GHB sites in rat brain

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Høg, Signe; Greenwood, Jeremy R

    2005-01-01

    acid [(RS)-HOCHCA] and 3-hydroxycyclopent-1-enecarboxylic acid [(RS)-HOCPCA], were found to be high-affinity GHB ligands, with IC50 values in the nanomolar range, and had 9 and 27 times, respectively, higher affinity than GHB. The stereo-selectively synthesized R,R-isomer of the trans-cyclopropyl GHB...... analog, HOCPrCA, proved to have 10-fold higher affinity than its enantiomer. Likewise, the R-enantiomers of HOCHCA and HOCPCA selectively inhibited [3H]NCS-382 binding. The best inhibitor of these, (R)-HOCPCA, has an affinity 39 times higher than GHB and is thus among the best GHB ligands reported......Gamma-hydroxybutyrate (GHB) is a psychotropic compound endogenous to the brain. Despite its potentially great physiological significance, its exact molecular mechanism of action is unknown. GHB is a weak agonist at GABA(B) receptors, but there is also evidence of specific GHB receptor sites...

  10. Novel cyclic gamma-hydroxybutyrate (GHB) analogs with high affinity and stereoselectivity of binding to GHB sites in rat brain.

    Science.gov (United States)

    Wellendorph, Petrine; Høg, Signe; Greenwood, Jeremy R; de Lichtenberg, Anne; Nielsen, Birgitte; Frølund, Bente; Brehm, Lotte; Clausen, Rasmus P; Bräuner-Osborne, Hans

    2005-10-01

    Gamma-hydroxybutyrate (GHB) is a psychotropic compound endogenous to the brain. Despite its potentially great physiological significance, its exact molecular mechanism of action is unknown. GHB is a weak agonist at GABA(B) receptors, but there is also evidence of specific GHB receptor sites, the molecular cloning of which remains a challenge. Ligands with high affinity and specificity for the reported GHB binding site are needed for pharmacological dissection of the GHB and GABA(B) effects and for mapping the structural requirements of the GHB receptor-ligand interactions. For this purpose, we have synthesized and assayed three conformationally restricted GHB analogs for binding against the GHB-specific ligand [3H]NCS-382 [(E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene-)acetic acid] in rat brain homogenate. The cyclohexene and cyclopentene analogs, 3-hydroxycyclohex-1-enecarboxylic acid [(RS)-HOCHCA] and 3-hydroxycyclopent-1-enecarboxylic acid [(RS)-HOCPCA], were found to be high-affinity GHB ligands, with IC50 values in the nanomolar range, and had 9 and 27 times, respectively, higher affinity than GHB. The stereo-selectively synthesized R,R-isomer of the trans-cyclopropyl GHB analog, HOCPrCA, proved to have 10-fold higher affinity than its enantiomer. Likewise, the R-enantiomers of HOCHCA and HOCPCA selectively inhibited [3H]NCS-382 binding. The best inhibitor of these, (R)-HOCPCA, has an affinity 39 times higher than GHB and is thus among the best GHB ligands reported to date. Neither of the cycloalkenes showed any affinity (IC50 > 1 mM) for GABA(A) or GABA(B) receptors. These compounds show excellent potential as lead structures and novel tools for studying specific GHB receptor-mediated pharmacology.

  11. The predicted 3D structure of the human D2 dopamine receptor and the binding site and binding affinities for agonists and antagonists

    Science.gov (United States)

    Kalani, M. Yashar S.; Vaidehi, Nagarajan; Hall, Spencer E.; Trabanino, Rene J.; Freddolino, Peter L.; Kalani, Maziyar A.; Floriano, Wely B.; Tak Kam, Victor Wai; Goddard, William A., III

    2004-03-01

    Dopamine neurotransmitter and its receptors play a critical role in the cell signaling process responsible for information transfer in neurons functioning in the nervous system. Development of improved therapeutics for such disorders as Parkinson's disease and schizophrenia would be significantly enhanced with the availability of the 3D structure for the dopamine receptors and of the binding site for dopamine and other agonists and antagonists. We report here the 3D structure of the long isoform of the human D2 dopamine receptor, predicted from primary sequence using first-principles theoretical and computational techniques (i.e., we did not use bioinformatic or experimental 3D structural information in predicting structures). The predicted 3D structure is validated by comparison of the predicted binding site and the relative binding affinities of dopamine, three known dopamine agonists (antiparkinsonian), and seven known antagonists (antipsychotic) in the D2 receptor to experimentally determined values. These structures correctly predict the critical residues for binding dopamine and several antagonists, identified by mutation studies, and give relative binding affinities that correlate well with experiments. The predicted binding site for dopamine and agonists is located between transmembrane (TM) helices 3, 4, 5, and 6, whereas the best antagonists bind to a site involving TM helices 2, 3, 4, 6, and 7 with minimal contacts to TM helix 5. We identify characteristic differences between the binding sites of agonists and antagonists.

  12. Irreversible blockade of the high and low affinity ( sup 3 H) naloxone binding sites by C-6 derivatives of morphinane-6-ones

    Energy Technology Data Exchange (ETDEWEB)

    Krizsan, D. (EGIS Pharmaceutical Works, Budapest (Hungary)); Varga, E.; Benyhe, S.; Szucs, M.; Borsodi, A. (Biological Research Center of the Hungarian Academy of Sciences, Szeged (Hungary)); Hosztafi, S. (Alkaloida Chemical Works, Tiszavasvari (Hungary))

    1991-01-01

    C-6 derivatives-hydrazones, phenylhydrazones, dinitrophenylhydrazones, oximes and semicarbazones - of morphinane-6-ones were synthesized and their binding characteristics were studied on rat brain membranes. The dihydromorphinone and oxymorphone derivatives compete for the ({sup 3}H)naloxone binding sites with high affinity, while the dihydrocodeinone and oxycodone derivatives are less potent. The affinity of the new compounds is decreased for the delta sites as compared to the parent ligands. The ligands bearing bulky substituents also bind with low affinity to the kappa sites. The modification decreased the Na{sup +}-index of compounds indicating their mixed agonist-antagonist character. The dihydromorphinone derivatives are all capable to block irreversibly the high affinity binding site of ({sup 3}H)naloxone, whereas the dihydrocodeinone derivatives block irreversibly the low affinity site. A possible mechanism for the inhibition is suggested.

  13. Positive allosteric modulation of the GHB high-affinity binding site by the GABAA receptor modulator monastrol and the flavonoid catechin

    DEFF Research Database (Denmark)

    Eghorn, Laura Friis; Høstgaard-Jensen, Kirsten; Kongstad, Kenneth Thermann

    2014-01-01

    conformational changes in the binding site, demonstrating a positive allosteric modulation of radioligand binding. Surprisingly, binding of [3H]GHB and the GHB high-affinity site-specific radioligands [125I]BnOPh-GHB and [3H]HOCPCA was either decreased or only weakly increased, indicating that the observed......γ-Hydroxybutyric acid (GHB) is a metabolite of γ-aminobutyric acid (GABA) and a proposed neurotransmitter in the mammalian brain. We recently identified α4βδ GABAA receptors as possible high-affinity GHB targets. GABAA receptors are highly sensitive to allosteric modulation. Thus to investigate...... whether GHB high-affinity binding sites are also sensitive to allosteric modulation, we screened both known GABAA receptor ligands and a library of natural compounds in the rat cortical membrane GHB specific high-affinity [3H]NCS-382 binding assay. Two hits were identified: Monastrol, a positive...

  14. Targeted deletion of a high-affinity GATA-binding site in the GATA-1 promoter leads to selective loss of the eosinophil lineage in vivo

    National Research Council Canada - National Science Library

    Yu, Channing; Cantor, Alan B; Yang, Haidi; Browne, Carol; Wells, Richard A; Fujiwara, Yuko; Orkin, Stuart H

    2002-01-01

    .... Here we demonstrate that deletion of a high-affinity GATA-binding site in the GATA-1 promoter, an element presumed to mediate positive autoregulation of GATA-1 expression, leads to selective loss...

  15. Insecticidal 3-benzamido-N-phenylbenzamides specifically bind with high affinity to a novel allosteric site in housefly GABA receptors.

    Science.gov (United States)

    Ozoe, Yoshihisa; Kita, Tomo; Ozoe, Fumiyo; Nakao, Toshifumi; Sato, Kazuyuki; Hirase, Kangetsu

    2013-11-01

    γ-Aminobutyric acid (GABA) receptors (GABARs) are an important target for existing insecticides such as fiproles. These insecticides act as noncompetitive antagonists (channel blockers) for insect GABARs by binding to a site within the intrinsic channel of the GABAR. Recently, a novel class of insecticides, 3-benzamido-N-phenylbenzamides (BPBs), was shown to inhibit GABARs by binding to a site distinct from the site for fiproles. We examined the binding site of BPBs in the adult housefly by means of radioligand-binding and electrophysiological experiments. 3-Benzamido-N-(2,6-dimethyl-4-perfluoroisopropylphenyl)-2-fluorobenzamide (BPB 1) (the N-demethyl BPB) was a partial, but potent, inhibitor of [(3)H]4'-ethynyl-4-n-propylbicycloorthobenzoate (GABA channel blocker) binding to housefly head membranes, whereas the 3-(N-methyl)benzamido congener (the N-methyl BPB) had low or little activity. A total of 15 BPB analogs were tested for their abilities to inhibit [(3)H]BPB 1 binding to the head membranes. The N-demethyl analogs, known to be highly effective insecticides, potently inhibited the [(3)H]BPB 1 binding, but the N-methyl analogs did not even though they, too, are considered highly effective. [(3)H]BPB 1 equally bound to the head membranes from wild-type and dieldrin-resistant (rdl mutant) houseflies. GABA allosterically inhibited [(3)H]BPB 1 binding. By contrast, channel blocker-type antagonists enhanced [(3)H]BPB 1 binding to housefly head membranes by increasing the affinity of BPB 1. Antiparasitic macrolides, such as ivermectin B1a, were potent inhibitors of [(3)H]BPB 1 binding. BPB 1 inhibited GABA-induced currents in housefly GABARs expressed in Xenopus oocytes, whereas it failed to inhibit l-glutamate-induced currents in inhibitory l-glutamate receptors. Overall, these findings indicate that BPBs act at a novel allosteric site that is different from the site for channel blocker-type antagonists and that is probably overlapped with the site for macrolides

  16. Decavanadate binding to a high affinity site near the myosin catalytic centre inhibits F-actin-stimulated myosin ATPase activity.

    Science.gov (United States)

    Tiago, Teresa; Aureliano, Manuel; Gutiérrez-Merino, Carlos

    2004-05-11

    Decameric vanadate (V(10)) inhibits the actin-stimulated myosin ATPase activity, noncompetitively with actin or with ATP upon interaction with a high-affinity binding site (K(i) = 0.27 +/- 0.05 microM) in myosin subfragment-1 (S1). The binding of V(10) to S1 can be monitored from titration with V(10) of the fluorescence of S1 labeled at Cys-707 and Cys-697 with N-iodo-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) or 5-(iodoacetamido) fluorescein, which showed the presence of only one V(10) binding site per monomer with a dissociation constant of 0.16-0.7 microM, indicating that S1 labeling with these dyes produced only a small distortion of the V(10) binding site. The large quenching of AEDANS-labeled S1 fluorescence produced by V(10) indicated that the V(10) binding site is close to Cys-697 and 707. Fluorescence studies demonstrated the following: (i) the binding of V(10) to S1 is not competitive either with actin or with ADP.V(1) or ADP.AlF(4); (ii) the affinity of V(10) for the complex S1/ADP.V(1) and S1/ADP.AlF(4) is 2- and 3-fold lower than for S1; and (iii) it is competitive with the S1 "back door" ligand P(1)P(5)-diadenosine pentaphosphate. A local conformational change in S1 upon binding of V(10) is supported by (i) a decrease of the efficiency of fluorescence energy transfer between eosin-labeled F-actin and fluorescein-labeled S1, and (ii) slower reassociation between S1 and F-actin after ATP hydrolysis. The results are consistent with binding of V(10) to the Walker A motif of ABC ATPases, which in S1 corresponds to conserved regions of the P-loop which form part of the phosphate tube.

  17. Mutational analysis of the high-affinity zinc binding site validates a refined human dopamine transporter homology model.

    Directory of Open Access Journals (Sweden)

    Thomas Stockner

    Full Text Available The high-resolution crystal structure of the leucine transporter (LeuT is frequently used as a template for homology models of the dopamine transporter (DAT. Although similar in structure, DAT differs considerably from LeuT in a number of ways: (i when compared to LeuT, DAT has very long intracellular amino and carboxyl termini; (ii LeuT and DAT share a rather low overall sequence identity (22% and (iii the extracellular loop 2 (EL2 of DAT is substantially longer than that of LeuT. Extracellular zinc binds to DAT and restricts the transporter's movement through the conformational cycle, thereby resulting in a decrease in substrate uptake. Residue H293 in EL2 praticipates in zinc binding and must be modelled correctly to allow for a full understanding of its effects. We exploited the high-affinity zinc binding site endogenously present in DAT to create a model of the complete transmemberane domain of DAT. The zinc binding site provided a DAT-specific molecular ruler for calibration of the model. Our DAT model places EL2 at the transporter lipid interface in the vicinity of the zinc binding site. Based on the model, D206 was predicted to represent a fourth co-ordinating residue, in addition to the three previously described zinc binding residues H193, H375 and E396. This prediction was confirmed by mutagenesis: substitution of D206 by lysine and cysteine affected the inhibitory potency of zinc and the maximum inhibition exerted by zinc, respectively. Conversely, the structural changes observed in the model allowed for rationalizing the zinc-dependent regulation of DAT: upon binding, zinc stabilizes the outward-facing state, because its first coordination shell can only be completed in this conformation. Thus, the model provides a validated solution to the long extracellular loop and may be useful to address other aspects of the transport cycle.

  18. Mutational analysis of the high-affinity zinc binding site validates a refined human dopamine transporter homology model.

    Science.gov (United States)

    Stockner, Thomas; Montgomery, Therese R; Kudlacek, Oliver; Weissensteiner, Rene; Ecker, Gerhard F; Freissmuth, Michael; Sitte, Harald H

    2013-01-01

    The high-resolution crystal structure of the leucine transporter (LeuT) is frequently used as a template for homology models of the dopamine transporter (DAT). Although similar in structure, DAT differs considerably from LeuT in a number of ways: (i) when compared to LeuT, DAT has very long intracellular amino and carboxyl termini; (ii) LeuT and DAT share a rather low overall sequence identity (22%) and (iii) the extracellular loop 2 (EL2) of DAT is substantially longer than that of LeuT. Extracellular zinc binds to DAT and restricts the transporter's movement through the conformational cycle, thereby resulting in a decrease in substrate uptake. Residue H293 in EL2 praticipates in zinc binding and must be modelled correctly to allow for a full understanding of its effects. We exploited the high-affinity zinc binding site endogenously present in DAT to create a model of the complete transmemberane domain of DAT. The zinc binding site provided a DAT-specific molecular ruler for calibration of the model. Our DAT model places EL2 at the transporter lipid interface in the vicinity of the zinc binding site. Based on the model, D206 was predicted to represent a fourth co-ordinating residue, in addition to the three previously described zinc binding residues H193, H375 and E396. This prediction was confirmed by mutagenesis: substitution of D206 by lysine and cysteine affected the inhibitory potency of zinc and the maximum inhibition exerted by zinc, respectively. Conversely, the structural changes observed in the model allowed for rationalizing the zinc-dependent regulation of DAT: upon binding, zinc stabilizes the outward-facing state, because its first coordination shell can only be completed in this conformation. Thus, the model provides a validated solution to the long extracellular loop and may be useful to address other aspects of the transport cycle.

  19. Substrate and Substrate-Mimetic Chaperone Binding Sites in Human α-Galactosidase A Revealed by Affinity-Mass Spectrometry

    Science.gov (United States)

    Moise, Adrian; Maeser, Stefan; Rawer, Stephan; Eggers, Frederike; Murphy, Mary; Bornheim, Jeff; Przybylski, Michael

    2016-06-01

    Fabry disease (FD) is a rare metabolic disorder of a group of lysosomal storage diseases, caused by deficiency or reduced activity of the enzyme α-galactosidase. Human α-galactosidase A (hαGAL) hydrolyses the terminal α-galactosyl moiety from glycosphingolipids, predominantly globotriaosylceramide (Gb3). Enzyme deficiency leads to incomplete or blocked breakdown and progressive accumulation of Gb3, with detrimental effects on normal organ functions. FD is successfully treated by enzyme replacement therapy (ERT) with purified recombinant hαGAL. An emerging treatment strategy, pharmacologic chaperone therapy (PCT), employs small molecules that can increase and/or reconstitute the activity of lysosomal enzyme trafficking by stabilizing misfolded isoforms. One such chaperone, 1-deoxygalactonojirimycin (DGJ), is a structural galactose analogue currently validated in clinical trials. DGJ is an active-site-chaperone that binds at the same or similar location as galactose; however, the molecular determination of chaperone binding sites in lysosomal enzymes represents a considerable challenge. Here we report the identification of the galactose and DGJ binding sites in recombinant α-galactosidase through a new affinity-mass spectrometry-based approach that employs selective proteolytic digestion of the enzyme-galactose or -inhibitor complex. Binding site peptides identified by mass spectrometry, [39-49], [83-100], and [141-168], contain the essential ligand-contacting amino acids, in agreement with the known X-ray crystal structures. The inhibitory effect of DGJ on galactose recognition was directly characterized through competitive binding experiments and mass spectrometry. The methods successfully employed in this study should have high potential for the characterization of (mutated) enzyme-substrate and -chaperone interactions, and for identifying chaperones without inhibitory effects.

  20. High affinity RGD-binding sites at the plasma membrane of Arabidopsis thaliana links the cell wall.

    Science.gov (United States)

    Canut, H; Carrasco, A; Galaud, J P; Cassan, C; Bouyssou, H; Vita, N; Ferrara, P; Pont-Lezica, R

    1998-10-01

    The heptapeptide Tyr-Gly-Arg-Gly-Asp-Ser-Pro containing the sequence Arg-Gly-Asp (RGD--the essential structure recognised by animal cells in substrate adhesion molecules) was tested on epidermal cells of onion and cultured cells of Arabidopsis upon plasmolysis. Dramatic changes were observed on both types of cells following treatment: on onion cells, Hechtian strands linking the cell wall to the membrane were lost, while Arabidopsis cells changed from concave to convex plasmolysis. A control heptapeptide Tyr-Gly-Asp-Gly-Arg-Ser-Pro had no effect on the shape of plasmolysed cells. Protoplasts isolated from Arabidopsis cells agglutinate in the presence of ProNectinF, a genetically engineered protein of 72 kDa containing 13 RGD sequences: several protoplasts may adhere to a single molecule of ProNectinF. The addition of the RGD-heptapeptide disrupted the adhesion between the protoplasts. Purified plasma membrane from Arabidopsis cells exhibits specific binding sites for the iodinated RGD-heptapeptide. The binding is saturable, reversible, and two types of high affinity sites (Kd1 approximately 1 nM, and Kd2 approximately 40 nM) can be discerned. Competitive inhibition by several structurally related peptides and proteins noted the specific requirement for the RGD sequence. Thus, the RGD-binding activity of Arabidopsis fulfils the adhesion features of integrins, i.e. peptide specificity, subcellular location, and involvement in plasma membrane-cell wall attachments.

  1. High-affinity DNA binding sites for H-NS provide a molecular basis for selective silencing within proteobacterial genomes.

    Science.gov (United States)

    Lang, Benjamin; Blot, Nicolas; Bouffartigues, Emeline; Buckle, Malcolm; Geertz, Marcel; Gualerzi, Claudio O; Mavathur, Ramesh; Muskhelishvili, Georgi; Pon, Cynthia L; Rimsky, Sylvie; Stella, Stefano; Babu, M Madan; Travers, Andrew

    2007-01-01

    The global transcriptional regulator H-NS selectively silences bacterial genes associated with pathogenicity and responses to environmental insults. Although there is ample evidence that H-NS binds preferentially to DNA containing curved regions, we show here that a major basis for this selectivity is the presence of a conserved sequence motif in H-NS target transcriptons. We further show that there is a strong tendency for the H-NS binding sites to be clustered, both within operons and in genes contained in the pathogenicity-associated islands. In accordance with previously published findings, we show that these motifs occur in AT-rich regions of DNA. On the basis of these observations, we propose that H-NS silences extensive regions of the bacterial chromosome by binding first to nucleating high-affinity sites and then spreading along AT-rich DNA. This spreading would be reinforced by the frequent occurrence of the motif in such regions. Our findings suggest that such an organization enables the silencing of extensive regions of the genetic material, thereby providing a coherent framework that unifies studies on the H-NS protein and a concrete molecular basis for the genetic control of H-NS transcriptional silencing.

  2. Radiosynthesis and Evaluation of [(11)C]3-Hydroxycyclopent-1-enecarboxylic Acid as Potential PET Ligand for the High-Affinity γ-Hydroxybutyric Acid Binding Sites.

    Science.gov (United States)

    Jensen, Claus H; Hansen, Hanne D; Bay, Tina; Vogensen, Stine B; Lehel, Szabolcs; Thiesen, Louise; Bundgaard, Christoffer; Clausen, Rasmus P; Knudsen, Gitte M; Herth, Matthias M; Wellendorph, Petrine; Frølund, Bente

    2017-01-18

    γ-Hydroxybutyric acid (GHB) is an endogenous neuroactive substance and proposed neurotransmitter with affinity for both low- and high-affinity binding sites. A radioligand with high and specific affinity toward the high-affinity GHB binding site would be a unique tool toward a more complete understanding of this population of binding sites. With its high specific affinity and monocarboxylate transporter (MCT1) mediated transport across the blood-brain barrier in pharmacological doses, 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) seems like a suitable PET radiotracer candidate. Here, we report the (11)C-labeling and subsequent evaluation of [(11)C]HOCPCA in a domestic pig, as a PET-radioligand for visualization of the high-affinity GHB binding sites in the live pig brain. To investigate the regional binding of HOCPCA in pig brain prior to in vivo PET studies, in vitro quantitative autoradiography on sections of pig brain was performed using [(3)H]HOCPCA. In vivo evaluation of [(11)C]HOCPCA showed no brain uptake, possibly due to a limited uptake of HOCPCA by the MCT1 transporter at tracer doses of [(11)C]HOCPCA.

  3. GHB receptor targets in the CNS: Focus on high-affinity binding sites

    DEFF Research Database (Denmark)

    Bay, Tina; Eghorn, Laura Friis; Klein, Anders Bue;

    2014-01-01

    γ-Hydroxybutyric acid (GHB) is an endogenous compound in the mammalian brain with both low- and high-affinity receptor targets. GHB is used clinically in the treatment of symptoms of narcolepsy and alcoholism, but also illicitly abused as the recreational drug Fantasy. Major pharmacological effects...

  4. Design, Synthesis, and in Vitro Pharmacology of New Radiolabeled γ-Hydroxybutyric Acid Analogues Including Photolabile Analogues with Irreversible Binding to the High-Affinity γ-Hydroxybutyric Acid Binding Sites

    DEFF Research Database (Denmark)

    Sabbatini, Paola; Wellendorph, Petrine; Høg, Signe;

    2010-01-01

    γ-Hydroxybutyric acid (GHB) is a psychotropic compound endogenous to the brain. Despite its potential physiological significance, the complete molecular mechanisms of action remain unexplained. To facilitate the isolation and identification of the high-affinity GHB binding site, we herein report...... the design and synthesis of the first 125I-labeled radioligands in the field, one of which contains a photoaffinity label which enables it to bind irreversibly to the high-affinity GHB binding sites....

  5. Positive allosteric modulation of the GHB high-affinity binding site by the GABAA receptor modulator monastrol and the flavonoid catechin.

    Science.gov (United States)

    Eghorn, Laura F; Hoestgaard-Jensen, Kirsten; Kongstad, Kenneth T; Bay, Tina; Higgins, David; Frølund, Bente; Wellendorph, Petrine

    2014-10-05

    γ-Hydroxybutyric acid (GHB) is a metabolite of γ-aminobutyric acid (GABA) and a proposed neurotransmitter in the mammalian brain. We recently identified α4βδ GABAA receptors as possible high-affinity GHB targets. GABAA receptors are highly sensitive to allosteric modulation. Thus to investigate whether GHB high-affinity binding sites are also sensitive to allosteric modulation, we screened both known GABAA receptor ligands and a library of natural compounds in the rat cortical membrane GHB specific high-affinity [3H]NCS-382 binding assay. Two hits were identified: Monastrol, a positive allosteric modulator of GABA function at δ-containing GABAA receptors, and the naturally occurring flavonoid catechin. These compounds increased [3H]NCS-382 binding to 185-272% in high micromolar concentrations. Monastrol and (+)-catechin significantly reduced [3H]NCS-382 dissociation rates and induced conformational changes in the binding site, demonstrating a positive allosteric modulation of radioligand binding. Surprisingly, binding of [3H]GHB and the GHB high-affinity site-specific radioligands [125I]BnOPh-GHB and [3H]HOCPCA was either decreased or only weakly increased, indicating that the observed modulation was critically probe-dependent. Both monastrol and (+)-catechin were agonists at recombinant α4β3δ receptors expressed in Xenopus laevis oocytes. When monastrol and GHB were co-applied no changes were seen compared to the individual responses. In summary, we have identified the compounds monastrol and catechin as the first allosteric modulators of GHB high-affinity binding sites. Despite their relatively weak affinity, these compounds may aid in further characterization of the GHB high-affinity sites that are likely to represent certain GABAA receptors.

  6. High- and low-affinity binding sites for Cd on the bacterial cell walls of Bacillus subtilis and Shewanella oneidensis

    Science.gov (United States)

    Mishra, Bhoopesh; Boyanov, Maxim; Bunker, Bruce A.; Kelly, Shelly D.; Kemner, Kenneth M.; Fein, Jeremy B.

    2010-08-01

    Bulk Cd adsorption isotherm experiments, thermodynamic equilibrium modeling, and Cd K edge EXAFS were used to constrain the mechanisms of proton and Cd adsorption to bacterial cells of the commonly occurring Gram-positive and Gram-negative bacteria, Bacillus subtilis and Shewanella oneidensis, respectively. Potentiometric titrations were used to characterize the functional group reactivity of the S. oneidensis cells, and we model the titration data using the same type of non-electrostatic surface complexation approach as was applied to titrations of B. subtilis suspensions by Fein et al. (2005). Similar to the results for B. subtilis, the S. oneidensis cells exhibit buffering behavior from approximately pH 3-9 that requires the presence of four distinct sites, with p Ka values of 3.3 ± 0.2, 4.8 ± 0.2, 6.7 ± 0.4, and 9.4 ± 0.5, and site concentrations of 8.9(±2.6) × 10 -5, 1.3(±0.2) × 10 -4, 5.9(±3.3) × 10 -5, and 1.1(±0.6) × 10 -4 moles/g bacteria (wet mass), respectively. The bulk Cd isotherm adsorption data for both species, conducted at pH 5.9 as a function of Cd concentration at a fixed biomass concentration, were best modeled by reactions with a Cd:site stoichiometry of 1:1. EXAFS data were collected for both bacterial species as a function of Cd concentration at pH 5.9 and 10 g/L bacteria. The EXAFS results show that the same types of binding sites are responsible for Cd sorption to both bacterial species at all Cd loadings tested (1-200 ppm). Carboxyl sites are responsible for the binding at intermediate Cd loadings. Phosphoryl ligands are more important than carboxyl ligands for Cd binding at high Cd loadings. For the lowest Cd loadings studied here, a sulfhydryl site was found to dominate the bound Cd budgets for both species, in addition to the carboxyl and phosphoryl sites that dominate the higher loadings. The EXAFS results suggest that both Gram-positive and Gram-negative bacterial cell walls have a low concentration of very high-affinity

  7. A binding-site barrier affects imaging efficiency of high affinity amyloid-reactive peptide radiotracers in vivo.

    Directory of Open Access Journals (Sweden)

    Jonathan S Wall

    Full Text Available Amyloid is a complex pathology associated with a growing number of diseases including Alzheimer's disease, type 2 diabetes, rheumatoid arthritis, and myeloma. The distribution and extent of amyloid deposition in body organs establishes the prognosis and can define treatment options; therefore, determining the amyloid load by using non-invasive molecular imaging is clinically important. We have identified a heparin-binding peptide designated p5 that, when radioiodinated, was capable of selectively imaging systemic visceral AA amyloidosis in a murine model of the disease. The p5 peptide was posited to bind effectively to amyloid deposits, relative to similarly charged polybasic heparin-reactive peptides, because it adopted a polar α helix secondary structure. We have now synthesized a variant, p5R, in which the 8 lysine amino acids of p5 have been replaced with arginine residues predisposing the peptide toward the α helical conformation in an effort to enhance the reactivity of the peptide with the amyloid substrate. The p5R peptide had higher affinity for amyloid and visualized AA amyloid in mice by using SPECT/CT imaging; however, the microdistribution, as evidenced in micro-autoradiographs, was dramatically altered relative to the p5 peptide due to its increased affinity and a resultant "binding site barrier" effect. These data suggest that radioiodinated peptide p5R may be optimal for the in vivo detection of discreet, perivascular amyloid, as found in the brain and pancreatic vasculature, by using molecular imaging techniques; however, peptide p5, due to its increased penetration, may yield more quantitative imaging of expansive tissue amyloid deposits.

  8. Specific high-affinity binding sites for a synthetic gliadin heptapeptide of human peripheral blood lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Payan, D.G.; Horvath, K.; Graf, L.

    1987-03-23

    The synthetic peptide containing residues 43-49 of ..cap alpha..-gliadin, the major protein component of gluten, has previously been shown to inhibit the production of lymphokine activities by mononuclear leukocytes. The authors demonstrate using radiolabeled ..cap alpha..-gliadin(43-49) that human peripheral blood lymphocytes express approximately 20,000-25,000 surface receptors for this peptide, with a dissociation constant (K/sub D/) of 20 nM. In addition, binding is inhibited by naloxone and an enkephalin analog, thus confirming the functional correlate which demonstrates inhibition by these agents of ..cap alpha..-gliadin(43-49) functional effects. Furthermore, B-lymphocytes bind specifically a greater amount of (/sup 125/I)..cap alpha..-gliadin(43-49) than T-lymphocytes. The lymphocyte ..cap alpha..-gliadin(43-49) receptor may play an important role in mediating the immunological response to ..cap alpha..-gliadin. 16 references, 4 figures.

  9. The location of the high- and low-affinity bilirubin-binding sites on serum albumin: ligand-competition analysis investigated by circular dichroism.

    Science.gov (United States)

    Goncharova, Iryna; Orlov, Sergey; Urbanová, Marie

    2013-01-01

    The locations of three bilirubin (BR)-binding sites with different affinities were identified as subdomains IB, IIA and IIIA for five mammalian serum albumins (SAs): human (HSA), bovine (BSA), rat, (RSA), rabbit (RbSA) and sheep (SSA). The stereoselectivity of a high-affinity BR-binding site was identified in the BR/SA=1/1 system by circular dichroism (CD) spectroscopy, the sites with low affinity to BR were analyzed using difference CD. Site-specific ligand-competition experiments with ibuprofen (marker for subdomain IIIA) and hemin (marker for subdomain IB) did not reveal any changes for the BR/SA=1/1 system and showed a decrease of the bound BR at BR/SA=3/1. Both sites were identified as sites with low affinity to BR. The correlation between stereoselectivity and the arrangement of Arg-Lys residues indicated similarity between the BR-binding sites in subdomain IIIA for all of the SAs studied. Subdomain IB in HSA, BSA, SSA and RbSA has P-stereoselectivity while in RSA it has M-selectivity toward BR. A ligand-competition experiment with gossypol shows a decrease of the CD signal of bound BR for the BR/SA=1/1 system as well as for BR/SA=3/1. Subdomain IIA was assigned as a high-affinity BR-binding site. The P-stereoselectivity of this site in HSA (and RSA, RbSA) was caused by the right-hand localization of charged residues R257/R218-R222, whereas the left-hand orientation of R257/R218-R199 led to the M-stereoselectivity of the primary binding site in BSA (and SSA). Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Novel radioiodinated {gamma}-hydroxybutyric acid analogues for radiolabeling and Photolinking of high-affinity {gamma}-hydroxybutyric acid binding sites

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Høg, Signe; Sabbatini, Paola;

    2010-01-01

    ¿-Hydroxybutyric acid (GHB) is a therapeutic drug, a drug of abuse, and an endogenous substance that binds to low- and high-affinity sites in the mammalian brain. To target the specific GHB binding sites, we have developed a (125)I-labeled GHB analog and characterized its binding in rat brain...... homogenate and slices. Our data show that [(125)I]4-hydroxy-4-[4-(2-iodobenzyloxy)phenyl]butanoate ([(125)I]BnOPh-GHB) binds to one site in rat brain cortical membranes with low nanomolar affinity (K(d), 7 nM; B(max), 61 pmol/mg protein). The binding is inhibited by GHB and selected analogs......, but not by ¿-aminobutyric acid. Autoradiography using horizontal slices from rat brain demonstrates the highest density of binding in hippocampus and cortical regions and the lowest density in the cerebellum. Altogether, the findings correlate with the labeling and brain regional distribution of high-affinity GHB sites...

  11. Novel Radioiodinated γ-Hydroxybutyric Acid Analogues for Radiolabeling and Photolinking of High-Affinity γ-Hydroxybutyric Acid Binding Sites

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Høg, Signe; Sabbatini, Paola;

    2010-01-01

    γ-Hydroxybutyric acid (GHB) is a therapeutic drug, a drug of abuse, and an endogenous substance that binds to low- and high-affinity sites in the mammalian brain. To target the specific GHB binding sites, we have developed a 125I-labeled GHB analog and characterized its binding in rat brain...... homogenate and slices. Our data show that [125I]4-hydroxy-4-[4-(2-iodobenzyloxy)phenyl]butanoate ([125I]BnOPh-GHB) binds to one site in rat brain cortical membranes with low nanomolar affinity (Kd, 7 nM; Bmax, 61 pmol/mg protein). The binding is inhibited by GHB and selected analogs, but not by γ......-aminobutyric acid. Autoradiography using horizontal slices from rat brain demonstrates the highest density of binding in hippocampus and cortical regions and the lowest density in the cerebellum. Altogether, the findings correlate with the labeling and brain regional distribution of high-affinity GHB sites or [3H...

  12. A saxitoxin-binding aptamer with higher affinity and inhibitory activity optimized by rational site-directed mutagenesis and truncation.

    Science.gov (United States)

    Zheng, X; Hu, B; Gao, S X; Liu, D J; Sun, M J; Jiao, B H; Wang, L H

    2015-07-01

    Saxitoxin (STX), a member of the family of paralytic shellfish poisoning toxins, poses toxicological and ecotoxicological risks. To develop an analytical recognition element for STX, a DNA aptamer (APT(STX1)) was previously discovered via an iterative process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX) by Handy et al. Our study focused on generating an improved aptamer based on APT(STX1) through rational site-directed mutation and truncation. In this study, we generated the aptamer, M-30f, with a 30-fold higher affinity for STX compared with APT(STX1). The Kd value for M-30f was 133 nM, which was calculated by Bio-Layer Interferometry. After optimization, we detected and compared the interaction of STX with aptamers (APT(STX1) or M-30f) through several techniques (ELISA, cell bioassay, and mouse bioassay). Both aptamers' STX-binding ability was demonstrated in all three methods. Moreover, M-30f performs better than its parent sequence with higher suppressive activity against STX. As a molecular recognition element, M-30f has good prospects for practical application. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Nordimaprit, homodimaprit, clobenpropit and imetit: affinities for H3 binding sites and potencies in a functional H3 receptor model.

    Science.gov (United States)

    Kathmann, M; Schlicker, E; Detzner, M; Timmerman, H

    1993-11-01

    We determined the affinities of nordimaprit, homodimaprit, clobenpropit and imetit for H3 binding sites (labelled by 3H-N alpha-methylhistamine) in rat brain cortex homogenates and their potencies at presynaptic H3A receptors on noradrenergic nerve endings in mouse brain cortex slices. 3H-N alpha-Methylhistamine bound saturably to rat brain cortex homogenates with a Kd of 0.70 nmol/l and a Bmax of 98 fmol/mg protein. Binding of 3H-N alpha-methylhistamine was displaced monophasically by dimaprit (pKi 6.55), nordimaprit (5.94), homodimaprit (6.44), clobenpropit (9.16), imetit (9.83), R-(-)-alpha-methylhistamine (8.87) and histamine (8.20), and biphasically by burimamide (pKi high 7.73, pKi low 5.97). In superfused mouse brain cortex slices preincubated with 3H-noradrenaline, the electrically (0.3 Hz) evoked tritium overflow was inhibited by imetit (pIC35 8.93), R-(-)-alpha-methylhistamine (7.87) and histamine (7.03). The effect of histamine was attenuated by nordimaprit, homodimaprit, clobenpropit and N-ethoxycarbonyl-2- ethoxy-1,2-dihydroquinoline (EEDQ); EEDQ (but not nordimaprit, homodimaprit and clobenpropit) attenuated the effect of histamine also in slices pre-exposed to the drug 60-30 min prior to superfusion. The concentration-response curve of histamine was shifted to the right by homodimaprit and clobenpropit; Schild plots yielded straight lines with a slope of unity for both drugs (pA2 5.94 and 9.55, respectively). Nordimaprit depressed the maximum effect of histamine (pD'2 5.55) and also slightly increased the concentration of histamine producing the half-maximum effect.(ABSTRACT TRUNCATED AT 250 WORDS)

  14. The crustacean gill (Na+,K+)-ATPase: allosteric modulation of high- and low-affinity ATP-binding sites by sodium and potassium.

    Science.gov (United States)

    Masui, D C; Silva, E C C; Mantelatto, F L M; McNamara, J C; Barrabin, H; Scofano, H M; Fontes, C F L; Furriel, R P M; Leone, F A

    2008-11-15

    The blue crab, Callinectes danae, tolerates exposure to a wide salinity range employing mechanisms of compensatory ion uptake when in dilute media. Although the gill (Na+,K+)-ATPase is vital to hyperosmoregulatory ability, the interactions occurring at the sites of ATP binding on the molecule itself are unknown. Here, we investigate the modulation by Na+ and K+ of homotropic interactions between the ATP-binding sites, and of phosphoenzyme formation of the (Na+,K+)-ATPase from the posterior gills of this euryhaline crab. The contribution of the high- and low-affinity ATP-binding sites to maximum velocity was similar for both Na+ and K+. However, in contrast to Na+, a threshold K+ concentration triggers the appearance of the high-affinity binding sites, displacing the saturation curve to lower ATP concentrations.Further, a low-affinity site for phosphorylation is present on the enzyme. These findings reveal notable differences in the catalytic mechanism of the crustacean (Na+,K+)-ATPase compared to the vertebrate enzyme.

  15. The ryanodine receptor pore blocker neomycin also inhibits channel activity via a previously undescribed high-affinity Ca(2+) binding site.

    Science.gov (United States)

    Laver, Derek R; Hamada, Tomoyo; Fessenden, James D; Ikemoto, Noriaki

    2007-12-01

    In this study, we present evidence for the mechanism of neomycin inhibition of skeletal ryanodine receptors (RyRs). In single-channel recordings, neomycin produced monophasic inhibition of RyR open probability and biphasic inhibition of [(3)H]ryanodine binding. The half-maximal inhibitory concentration (IC(50)) for channel blockade by neomycin was dependent on membrane potential and cytoplasmic [Ca(2+)], suggesting that neomycin acts both as a pore plug and as a competitive antagonist at a cytoplasmic Ca(2+) binding site that causes allosteric inhibition. This novel Ca(2+)/neomycin binding site had a neomycin affinity of 100 nM: and a Ca(2+) affinity of 35 nM,: which is 30-fold higher than that of the well-described cytoplasmic Ca(2+) activation site. Therefore, a new high-affinity class of Ca(2+) binding site(s) on the RyR exists that mediates neomycin inhibition. Neomycin plugging of the channel pore induced brief (1-2 ms) conductance substates at 30% of the fully open conductance, whereas allosteric inhibition caused complete channel closure with durations that depended on the neomycin concentration. We quantitatively account for these results using a dual inhibition model for neomycin that incorporates voltage-dependent pore plugging and Ca(2+)-dependent allosteric inhibition.

  16. Reconstitution of high-affinity binding of a beta-scorpion toxin to neurotoxin receptor site 4 on purified sodium channels.

    Science.gov (United States)

    Thomsen, W; Martin-Eauclaire, M F; Rochat, H; Catterall, W A

    1995-09-01

    Reconstitution of purified sodium channels into phospholipid vesicles restores many aspects of sodium channel function including high-affinity neurotoxin binding and action at neurotoxin receptor sites 1-3 and 5, but neurotoxin binding and action at receptor site 4 has not previously been demonstrated in purified and reconstituted preparations. Toxin IV from the venom of the American scorpion Centruroides suffusus suffusus (Css IV), a beta-scorpion toxin, shifts the voltage dependence of sodium channel activation by binding with high affinity to neurotoxin receptor site 4. Sodium channels were purified from rat brain and reconstituted into phospholipid vesicles composed of phosphatidylcholine and phosphatidylethanolamine (65:35). 125I-Css IV, purified by reversed-phase HPLC, bound rapidly and specifically to reconstituted sodium channels. Dissociation of the bound toxin was biphasic with half-times of 0.22 min-1 and 0.015 min-1. At equilibrium, the toxin bound to two classes of specific high-affinity sites, a variable minor class with KD of approximately 0.1 nM and a major class with a KD of approximately 5 nM. Approximately 0.8 mol 125I-Css IV was bound per mole of reconstituted, right-side-out sodium channels, as assessed from comparison of binding of saxitoxin and Css IV. Binding of Css IV was unaffected by membrane potential or by neurotoxins that bind at sites 1-3 or 5, consistent with the characteristics of binding of beta-scorpion toxins to sodium channels in cells and membrane preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. (/sup 3/H)dihydroergotamine as a high-affinity, slowly dissociating radioligand for 5-HT1B binding sites in rat brain membranes: evidence for guanine nucleotide regulation of agonist affinity states

    Energy Technology Data Exchange (ETDEWEB)

    Hamblin, M.W.; Ariani, K.; Adriaenssens, P.I.; Ciaranello, R.D.

    1987-12-01

    (/sup 3/H)Dihydroergotamine (DE) labels a population of binding sites in rat brain membranes with an affinity of approximately 70 pM in both hippocampus (maximal binding at saturation (Bmax) = 340 fmol/mg of protein) and cerebral cortex (Bmax = 250 fmol/mg of protein). Specific binding typically comprises about 97% of total binding at the Kd of the radioligand when nonspecific binding is determined in the presence of 100 nM unlabeled DE. Association kinetics at 37 degrees C are consistent with a uniform association rate constant for all sites labeled. Specific binding is completely reversible with addition of excess unlabeled DE, but dissociation does not proceed with simple first-order kinetics, suggesting the presence of more than one discrete binding site. Competition studies with selective drugs reveal alpha adrenergic, 5-HT1A and 5-HT1B components of (/sup 3/H)DE specific binding. When phentolamine (500 nM) is included to block alpha receptors and DPAT (100 nM) or spiroxatrine (500 nM) is included to block 5-HT1A receptors, specific binding is exclusively to sites with drug affinities characteristic of 5-HT1B receptors. Under these 5-HT1B-selective conditions, (/sup 3/H)DE binding is about 90% specific, with a Kd of about 50 to 60 pM and a Bmax of 96 fmol/mg of protein in hippocampus and 77 fmol/mg of protein in cortex. (/sup 3/H)DE binding to 5-HT1B sites is very slowly dissociable, with a T1/2 of greater than 2 h at 37 degrees C. 5-HT1B antagonists and DE itself yield competition curves at (/sup 3/H)DE-labeled 5-HT1B sites that are adequately fit assuming a single site in nonlinear regression analysis. Addition of 100 microM guanylyl 5'-imidodiphosphate appears to convert nearly all 5-HT1B sites to those having low affinity for agonists while having a much smaller effect on the binding of (/sup 3/H)DE.

  18. Sourcing the affinity of flavonoids for the glycogen phosphorylase inhibitor site via crystallography, kinetics and QM/MM-PBSA binding studies: comparison of chrysin and flavopiridol.

    Science.gov (United States)

    Tsitsanou, Katerina E; Hayes, Joseph M; Keramioti, Maria; Mamais, Michalis; Oikonomakos, Nikos G; Kato, Atsushi; Leonidas, Demetres D; Zographos, Spyros E

    2013-11-01

    Flavonoids have been discovered as novel inhibitors of glycogen phosphorylase (GP), a target to control hyperglycemia in type 2 diabetes. To elucidate the mechanism of inhibition, we have determined the crystal structure of the GPb-chrysin complex at 1.9 Å resolution. Chrysin is accommodated at the inhibitor site intercalating between the aromatic side chains of Phe285 and Tyr613 through π-stacking interactions. Chrysin binds to GPb approximately 15 times weaker (Ki=19.01 μM) than flavopiridol (Ki=1.24 μM), exclusively at the inhibitor site, and both inhibitors display similar behavior with respect to AMP. To identify the source of flavopiridols' stronger affinity, molecular docking with Glide and postdocking binding free energy calculations using QM/MM-PBSA have been performed and compared. Whereas docking failed to correctly rank inhibitor binding conformations, the QM/MM-PBSA method employing M06-2X/6-31+G to model the π-stacking interactions correctly reproduced the experimental results. Flavopiridols' greater binding affinity is sourced to favorable interactions of the cationic 4-hydroxypiperidin-1-yl substituent with GPb, with desolvation effects limited by the substituent conformation adopted in the crystallographic complex. Further successful predictions using QM/MM-PBSA for the flavonoid quercetagetin (which binds at the allosteric site) leads us to propose the methodology as a useful and inexpensive tool to predict flavonoid binding. Copyright © 2012 Elsevier Ltd. All rights reserved.

  19. The intervening domain from MeCP2 enhances the DNA affinity of the methyl binding domain and provides an independent DNA interaction site

    Science.gov (United States)

    Claveria-Gimeno, Rafael; Lanuza, Pilar M.; Morales-Chueca, Ignacio; Jorge-Torres, Olga C.; Vega, Sonia; Abian, Olga; Esteller, Manel; Velazquez-Campoy, Adrian

    2017-01-01

    Methyl-CpG binding protein 2 (MeCP2) preferentially interacts with methylated DNA and it is involved in epigenetic regulation and chromatin remodelling. Mutations in MeCP2 are linked to Rett syndrome, the leading cause of intellectual retardation in girls and causing mental, motor and growth impairment. Unstructured regions in MeCP2 provide the plasticity for establishing interactions with multiple binding partners. We present a biophysical characterization of the methyl binding domain (MBD) from MeCP2 reporting the contribution of flanking domains to its structural stability and dsDNA interaction. The flanking disordered intervening domain (ID) increased the structural stability of MBD, modified its dsDNA binding profile from an entropically-driven moderate-affinity binding to an overwhelmingly enthalpically-driven high-affinity binding. Additionally, ID provided an additional site for simultaneously and autonomously binding an independent dsDNA molecule, which is a key feature linked to the chromatin remodelling and looping activity of MeCP2, as well as its ability to interact with nucleosomes replacing histone H1. The dsDNA interaction is characterized by an unusually large heat capacity linked to a cluster of water molecules trapped within the binding interface. The dynamics of disordered regions together with extrinsic factors are key determinants of MeCP2 global structural properties and functional capabilities. PMID:28139759

  20. Anti-human IgE monoclonal antibodies recognizing epitopes related to the binding sites of high and low affinity IgE receptors.

    Science.gov (United States)

    Takemoto, H; Nishimura, S; Kosada, Y; Hata, S; Takagi, S; Hosoi, S; Ezumi, K; Ide, M; Harada, S

    1994-01-01

    Anti-human IgE monoclonal antibodies (mAbs) were produced and eight clones recognizing epitopes on native IgE were selected. Epitopes were mapped by a competitive inhibition enzyme-linked immunosorbent assay, Western blotting and a multi-pin peptide technology. Four sites (one each in the C epsilon 1, C epsilon 2, C epsilon 2/C epsilon 3 junction and C epsilon 3) were recognized by the mAbs. The relationship between the four epitopes and the binding sites of high and low affinity IgE receptors (Fc epsilon RI and Fc epsilon RII, respectively) was studied using a monovalent Fab fragment of each mAb as a binding inhibitor. The IgE-Fc epsilon RII binding was clearly inhibited by the mAb recognizing the C epsilon 2/C epsilon 3 junction, suggesting that Fc epsilon RII binds to a rather limited area around the C epsilon 2/C epsilon 3 junction. The IgE-Fc epsilon RI binding, on the other hand, was scarcely inhibited by any single mAb. However, the binding was inhibited when the epitope in C epsilon 2 was blocked simultaneously with that at the C epsilon 2/C epsilon 3 junction or with that in C epsilon 3, indicating that these three distinct epitopes are related to the Fc epsilon RI binding sites. When these three epitopes were shown in the stereograph of human IgE, the Fc epsilon RI binding area was spread largely on the groove side between C epsilon 2 and C epsilon 3 domains. These results suggest that Fc epsilon RI acquires the high affinity through multiple bindings.

  1. Specificity of Bacillus thuringiensis endotoxins is correlated with the presence of high-affinity binding sites in the brush border membrane of target insect midguts

    Energy Technology Data Exchange (ETDEWEB)

    Hofmann, C.; Vanderbruggen, H.; Hoefte, H.; Van Rie, J.; Jansens, S.; Van Mellaert, H. (J. Plateaustraat, Gent (Belgium))

    1988-11-01

    Binding studies were performed with two {sup 125}I-labeled Bacillus thuringiensis {delta}-endotoxins on brush border membrane vesicles prepared from the larval midgut of the tobacco hornworm Manduca sexta or the cabbage butterfly Pieris brassicae. One {delta}-endotoxin, Bt2-protoxin, is a 130-kDa recombinant crystalline protein from B. thuringiensis subsp. berliner. It kills larvae of both insect species. The active Bt2-toxin is a 60-kDa proteolytic fragment of the Bt2-protoxin. It binds saturably and with high affinity to brush border membrane vesicles from the midgut of both species. The other {delta}-endotoxin, Bt4412-protoxin, is a 136-kDa crystalline protein from B. thuringiensis subsp. thuringiensis, which is highly toxic for P. brassicae, but not for M. sexta larvae. Bt4412-toxin, obtained after proteolytic activation of Bt4412-protoxin, shows high-affinity saturable binding to P. brassicae vesicles but not to M. sexta vesicles. The correlation between toxicity and specific binding is further strengthened by competition studies. Other B. thuringiensis {delta}-endotoxins active against M. sexta compete for binding of {sup 125}I-labeled Bt2-toxin to M. sexta vesicles, whereas toxins active against dipteran or coleopteran larvae do not compete. Bt2-toxin and Bt4412-toxin bind to different sites on P. brassicae vesicles.

  2. Allosteric effects of R- and S-citalopram on the human 5-HT transporter: evidence for distinct high- and low-affinity binding sites

    DEFF Research Database (Denmark)

    Plenge, Per; Gether, Ulrik; Rasmussen, Søren G

    2007-01-01

    SERT and the three mutants. Further, R-citalopram previously thought of as an inactive enantiomer strongly attenuated dissociation of the wild-type [(3)H]-imipramine:hSERT complex, whereas S-citalopram had almost no effect on this complex. These results suggest that 1: The allosteric site on hSERT is distinct from...... the site to which S-citalopram binds with high affinity. 2: The allosteric effects of R-citalopram on the dissociation of [(3)H]-imipramine from hSERT indicate that R-citalopram introduces a conformational change in hSERT....

  3. Affinity labeling of the carbohydrate binding site of the lectin discoidin I using a photoactivatable radioiodinated monosaccharide

    Energy Technology Data Exchange (ETDEWEB)

    Kohnken, R.E.; Berger, E.A.

    1987-12-29

    N-(4-Azidosalicyl) galactosamine (GalNASA), a photoactivatable, radioiodinatable analog of N-acetylgalactosamine (GalNAc), has been prepared and characterized. The authors have used this reagent for labeling of the carbohydrate binding site of discoidin I, an endogenous lectin produced by Dictyostelium discoideum. GalNASA behaved as a ligand for discoidin I, as judged by its ability to compete in an assay measuring the carbohydrate binding activity of discoidin I. In this assay, it exhibited a K/sub i,app/ of 800 ..mu..M, comparable to that of GalNAc. The K/sub i,app/ of GalNASA decreased to 40 ..mu..m upon prior photolysis with ultraviolet light. In contrast, N-(4-azidosalicyl) ethanolamine produced no inhibition of carbohydrate binding regardless of photolysis. Covalent labeling of discoidin I with /sup 125/I-GalNASA was entirely dependent upon ultraviolet light. A portion of labeling, representing 40-60% of the total, was sensitive to reagents which were known to inhibit carbohydrate binding by discoidin I, including GalNAc, asialofetuin, and ethyl-enediaminetetraacetic acid. The carbohydrate-sensitive fraction of discoidin I photolabeling with /sup 125/I-GalNASA exhibited a K/sub d/ of 15-40 ..mu..M, in agreement with the K/sub i,app/ of prephotolyzed GalNASA observed in the carbohydrate binding assay. Partial proteolytic digestion of photolabeled discoidin I revealed specific fragments whose labeling was completely blocked by GalNAc. This indicated that the location of carbohydrate-sensitive labeling within the structure of discoidin I was restricted. One particular tryptic fragment, Tr1, was examined in detail. These data suggest that Tr1 is derived from the carbohydrate binding site of discoidin I.

  4. Construction of a high affinity zinc binding site in the metabotropic glutamate receptor mGluR1

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Sheppard, P O; Jensen, L B

    2001-01-01

    and the loops connecting these. The findings offer valuable insight into the mechanism of ATD closure and family C receptor activation. Furthermore, the findings demonstrate that ATD regions other than those participating in agonist binding could be potential targets for new generations of ligands......The metabotropic glutamate receptors (mGluRs) belong to family C of the G-protein-coupled receptor (GPCR) superfamily. The receptors are characterized by having unusually long amino-terminal domains (ATDs), to which agonist binding has been shown to take place. Previously, we have constructed...... of a "closed" conformation, and thus stabilizing a more or less inactive "open" form of the ATD. This study presents the first metal ion site constructed in a family C GPCR. Furthermore, it is the first time a metal ion site has been created in a region outside of the seven transmembrane regions of a GPCR...

  5. Secondary PDZ domain-binding site on class B plexins enhances the affinity for PDZ-RhoGEF.

    Science.gov (United States)

    Pascoe, Heath G; Gutowski, Stephen; Chen, Hua; Brautigam, Chad A; Chen, Zhe; Sternweis, Paul C; Zhang, Xuewu

    2015-12-01

    PDZ domains are abundant protein interaction modules and typically recognize a short motif at the C terminus of their ligands, with a few residues in the motif endowing the binding specificity. The sequence-based rules, however, cannot fully account for the specificity between the vast number of PDZ domains and ligands in the cell. Plexins are transmembrane receptors that regulate processes such as axon guidance and angiogenesis. Two related guanine nucleotide exchange factors (GEFs), PDZ-RhoGEF and leukemia-associated RhoGEF (LARG), use their PDZ domains to bind class B plexins and play critical roles in signaling. Here, we present the crystal structure of the full-length cytoplasmic region of PlexinB2 in complex with the PDZ domain of PDZ-RhoGEF. The structure reveals that, in addition to the canonical C-terminal motif/PDZ interaction, the 3D domain of PlexinB2 forms a secondary interface with the PDZ domain. Our biophysical and cell-based assays show that the secondary interface contributes to the specific interaction between plexin and PDZ-RhoGEF and to signaling by plexin in the cell. Formation of secondary interfaces may be a general mechanism for increasing affinity and specificity of modular domain-mediated interactions.

  6. Secondary PDZ domain-binding site on class B plexins enhances the affinity for PDZ–RhoGEF

    Science.gov (United States)

    Pascoe, Heath G.; Gutowski, Stephen; Chen, Hua; Brautigam, Chad A.; Chen, Zhe; Sternweis, Paul C.; Zhang, Xuewu

    2015-01-01

    PDZ domains are abundant protein interaction modules and typically recognize a short motif at the C terminus of their ligands, with a few residues in the motif endowing the binding specificity. The sequence-based rules, however, cannot fully account for the specificity between the vast number of PDZ domains and ligands in the cell. Plexins are transmembrane receptors that regulate processes such as axon guidance and angiogenesis. Two related guanine nucleotide exchange factors (GEFs), PDZ–RhoGEF and leukemia-associated RhoGEF (LARG), use their PDZ domains to bind class B plexins and play critical roles in signaling. Here, we present the crystal structure of the full-length cytoplasmic region of PlexinB2 in complex with the PDZ domain of PDZ–RhoGEF. The structure reveals that, in addition to the canonical C-terminal motif/PDZ interaction, the 3D domain of PlexinB2 forms a secondary interface with the PDZ domain. Our biophysical and cell-based assays show that the secondary interface contributes to the specific interaction between plexin and PDZ–RhoGEF and to signaling by plexin in the cell. Formation of secondary interfaces may be a general mechanism for increasing affinity and specificity of modular domain-mediated interactions. PMID:26627240

  7. Structure-function studies of DNA binding domain of response regulator KdpE reveals equal affinity interactions at DNA half-sites.

    Directory of Open Access Journals (Sweden)

    Anoop Narayanan

    Full Text Available Expression of KdpFABC, a K(+ pump that restores osmotic balance, is controlled by binding of the response regulator KdpE to a specific DNA sequence (kdpFABC(BS via the winged helix-turn-helix type DNA binding domain (KdpE(DBD. Exploration of E. coli KdpE(DBD and kdpFABC(BS interaction resulted in the identification of two conserved, AT-rich 6 bp direct repeats that form half-sites. Despite binding to these half-sites, KdpE(DBD was incapable of promoting gene expression in vivo. Structure-function studies guided by our 2.5 Å X-ray structure of KdpE(DBD revealed the importance of residues R193 and R200 in the α-8 DNA recognition helix and T215 in the wing region for DNA binding. Mutation of these residues renders KdpE incapable of inducing expression of the kdpFABC operon. Detailed biophysical analysis of interactions using analytical ultracentrifugation revealed a 2∶1 stoichiometry of protein to DNA with dissociation constants of 200±100 and 350±100 nM at half-sites. Inactivation of one half-site does not influence binding at the other, indicating that KdpE(DBD binds independently to the half-sites with approximately equal affinity and no discernable cooperativity. To our knowledge, these data are the first to describe in quantitative terms the binding at half-sites under equilibrium conditions for a member of the ubiquitous OmpR/PhoB family of proteins.

  8. Effects of heterocyclic aromatic substituents on binding affinities at two distinct sites of somatostatin receptors. Correlation with the electrostatic potential of the substituents.

    Science.gov (United States)

    Prasad, Vidya; Birzin, Elizabeth T; McVaugh, Cheryl T; Van Rijn, Rachel D; Rohrer, Susan P; Chicchi, Gary; Underwood, Dennis J; Thornton, Edward R; Smith, Amos B; Hirschmann, Ralph

    2003-05-08

    In our continuing program exploring glucose-based peptidomimetics of somatostatin (SRIF-14), we sought to improve the water solubility of our glycosides. This led to insights into the nature of the ligand binding sites at the SRIF receptor. Replacement of the C4 benzyl substituent in glucoside (+)-2 with pyridinylmethyl or pyrazin-2-ylmethyl congeners increased water solubility and enhanced affinity for the human SRIF subtype receptor 4 (sst4). We attribute this effect to hydrogen bond formation. The pyridin-3-ylmethyl substituent at C4, when combined with the imidazol-4-ylmethyl group at C2, generated (-)-19, which has the highest affinity of a glucose-based peptidomimetic at a human SRIF receptor to date (K(i) 53 +/- 23 nM, n = 6 at sst4). The C4 heterocyclic congeners of glucosides bearing a 1-methoxy substituent rather than an indole side chain at the anomeric carbon, such as (+)-16, also provided information about the Trp(8) binding pocket. We correlated the SARs at both the C4 and the Trp(8) binding pockets with calculations of the electrostatic potentials of the diverse C4 aromatic substituents using Spartan 3-21G(*) MO analysis. These calculations provide an approximate analysis of a molecule's ability to interact within a receptor binding site. Our binding studies show that benzene and indole rings, but not pyridinylmethyl nor pyrazin-2-ylmethyl rings, can bind the hydrophobic Trp(8) binding pocket of sst4. The Spartan 3-21G(*) MO analysis reveals significant negative electrostatic potential in the region of the pi-clouds for the benzene and indole rings but not for the pyridinylmethyl or pyrazin-2-ylmethyl congeners. Our data further demonstrate that the replacement of benzene or indole side chains by heterocyclic aromatic rings typified by pyridine and pyrazine not only enhances water solubility and hydrogen bonding capacity as expected, but can also profoundly diminish the ability of the pi-cloud of the aromatic substituent to interact with side chains

  9. Variation in one residue associated with the metal ion-dependent adhesion site regulates αIIbβ3 integrin ligand binding affinity.

    Directory of Open Access Journals (Sweden)

    Joel Raborn

    Full Text Available The Asp of the RGD motif of the ligand coordinates with the β I domain metal ion dependent adhesion site (MIDAS divalent cation, emphasizing the importance of the MIDAS in ligand binding. There appears to be two distinct groups of integrins that differ in their ligand binding affinity and adhesion ability. These differences may be due to a specific residue associated with the MIDAS, particularly the β3 residue Ala(252 and corresponding Ala in the β1 integrin compared to the analogous Asp residue in the β2 and β7 integrins. Interestingly, mutations in the adjacent to MIDAS (ADMIDAS of integrins α4β7 and αLβ2 increased the binding and adhesion abilities compared to the wild-type, while the same mutations in the α2β1, α5β1, αVβ3, and αIIbβ3 integrins demonstrated decreased ligand binding and adhesion. We introduced a mutation in the αIIbβ3 to convert this MIDAS associated Ala(252 to Asp. By combination of this mutant with mutations of one or two ADMIDAS residues, we studied the effects of this residue on ligand binding and adhesion. Then, we performed molecular dynamics simulations on the wild-type and mutant αIIbβ3 integrin β I domains, and investigated the dynamics of metal ion binding sites in different integrin-RGD complexes. We found that the tendency of calculated binding free energies was in excellent agreement with the experimental results, suggesting that the variation in this MIDAS associated residue accounts for the differences in ligand binding and adhesion among different integrins, and it accounts for the conflicting results of ADMIDAS mutations within different integrins. This study sheds more light on the role of the MIDAS associated residue pertaining to ligand binding and adhesion and suggests that this residue may play a pivotal role in integrin-mediated cell rolling and firm adhesion.

  10. Isolation of a high affinity neutralizing monoclonal antibody against 2009 pandemic H1N1 virus that binds at the 'Sa' antigenic site.

    Directory of Open Access Journals (Sweden)

    Nachiket Shembekar

    Full Text Available Influenza virus evades host immunity through antigenic drift and shift, and continues to circulate in the human population causing periodic outbreaks including the recent 2009 pandemic. A large segment of the population was potentially susceptible to this novel strain of virus. Historically, monoclonal antibodies (MAbs have been fundamental tools for diagnosis and epitope mapping of influenza viruses and their importance as an alternate treatment option is also being realized. The current study describes isolation of a high affinity (K(D = 2.1±0.4 pM murine MAb, MA2077 that binds specifically to the hemagglutinin (HA surface glycoprotein of the pandemic virus. The antibody neutralized the 2009 pandemic H1N1 virus in an in vitro microneutralization assay (IC(50 = 0.08 µg/ml. MA2077 also showed hemagglutination inhibition activity (HI titre of 0.50 µg/ml against the pandemic virus. In a competition ELISA, MA2077 competed with the binding site of the human MAb, 2D1 (isolated from a survivor of the 1918 Spanish flu pandemic on pandemic H1N1 HA. Epitope mapping studies using yeast cell-surface display of a stable HA1 fragment, wherein 'Sa' and 'Sb' sites were independently mutated, localized the binding site of MA2077 within the 'Sa' antigenic site. These studies will facilitate our understanding of antigen antibody interaction in the context of neutralization of the pandemic influenza virus.

  11. High Throughput Screening of High-Affinity Ligands for Proteins with Anion-Binding Sites using Desorption Electrospray Ionization (DESI) Mass Spectrometry

    Science.gov (United States)

    Lu, Xin; Ning, Baoming; He, Dacheng; Huang, Lingyun; Yue, Xiangjun; Zhang, Qiming; Huang, Haiwei; Liu, Yang; He, Lan; Ouyang, Jin

    2014-03-01

    A high throughput screening system involving a linear ion trap (LTQ) analyzer, a house-made platform and a desorption electrospray ionization (DESI) source was established to screen ligands with a high affinity for proteins with anion-binding sites. The complexes were analyzed after incubation, ultrafiltration, washing, and displacement. A new anionic region inhibited dissociation (ARID) mechanism that was suitable for a protein with anion-binding site was proposed. We utilized the differences in detectable dissociation of protein-ligand complexes, combined with displacement experiments, to distinguish free ligands displaced from anion-binding sites from liberated ligands dissociated from nonspecific interactions. The method was validated by α1-acid glycoprotein (AGP) and (R), (S)-amlodipine. Site-specific enantioselectivity shown in our experiments was consistent with earlier studies. Obtaining all of the qualitative information of 15*3 samples in 2.3 min indicates that the analysis process is no longer the time-limiting step in the initial stage of drug discovery. Quantitative information verified that our method was at least a semiquantitative method.

  12. Enzymic synthesis of steroid sulphates. XI. Study of the oestrogen binding site of oestrogen sulphotransferase by affinity labelling with 4-mercuri-17beta-oestradiol.

    Science.gov (United States)

    Dodsworth, A I; Jackson, D E

    1975-04-19

    Oistrogen sulphotransferase (3"-phosphoadenylylsulphate: oestrone sulphotransferase, EC 2.8.2.4) contains asingle sulphydryl group thought to be at, or near, the oestrogen-binding site. 4-mercuri-17beta-oestradiol, the activity of the enzyme decreased with increasing concentration of the oestrogen derivative. However, some 40% of the activity remained when all the sulphydryl had reacted to form mercaptide. Formation of mercaptide was only marginally decreased in the presence of the substrate 17beta-oestradiol. Other steroids, such as 11-deoxycorticosterone and testosterone, which are non-substrates for the enzyme, were more effective than 17beta-oestradiol in inhibiting mercaptide formation. Bovine serum albumin also reacted with 4-mercure-17beta-oestradiol and the effects of various steroids on mercaptide formation by the affinity label closely paralleled those found for the enzyme. 2t is concluded that the single sulphydryl group in the enzyme is not directly involved in the binding of oestrogen at the active site but is perhaps in closer proximity to a second site capable of binding certain non-substrate steroids.

  13. Site-specific conjugation of an antibody-binding protein catalyzed by horseradish peroxidase creates a multivalent protein conjugate with high affinity to IgG.

    Science.gov (United States)

    Minamihata, Kosuke; Goto, Masahiro; Kamiya, Noriho

    2015-01-01

    Cross-linking proteins offers an approach to enhance the distinct function of proteins due to the multivalent effect. In this study, we demonstrated the preparation of a multivalent antibody-binding protein possessing high affinity to IgG by conjugating a number of antibody-binding proteins using the horseradish peroxidase (HRP)-mediated protein conjugation method. By introducing a peptide tag containing a tyrosine (Y-tag) to the C-terminus of the model protein, a chimera protein of protein G and protein A (pG2 pA), the Tyr residue in the Y-tag was efficiently recognized by HRP and cross-linked with each other to yield a pG2 pA conjugate, composed of mainly two to three units of pG2 pA. The cross-linking occurred site specifically at the Tyr residue in the Y-tag and introduction of the Y-tag showed no effect on the function of pG2 pA. The affinity of the Y-tagged pG2 pA conjugate against IgG clearly increased because of the multivalent effect, demonstrating the benefit of this protein cross-linking reaction, which yields functional protein oligomers. Such multivalent protein conjugates created by this reaction should have potential to be used in ELISA and Western blotting applications in which highly sensitive detection of target molecules is desired.

  14. Affinity purification of sequence-specific DNA binding proteins.

    OpenAIRE

    1986-01-01

    We describe a method for affinity purification of sequence-specific DNA binding proteins that is fast and effective. Complementary chemically synthesized oligodeoxynucleotides that contain a recognition site for a sequence-specific DNA binding protein are annealed and ligated to give oligomers. This DNA is then covalently coupled to Sepharose CL-2B with cyanogen bromide to yield the affinity resin. A partially purified protein fraction is combined with competitor DNA and subsequently passed t...

  15. Crystal structure of the high-affinity Na+,K+-ATPase–ouabain complex with Mg2+ bound in the cation binding site

    DEFF Research Database (Denmark)

    Laursen, Mette; Yatime, Laure; Nissen, Poul

    2013-01-01

    we describe a crystal structure of the phosphorylated pig kidney Na+,K+-ATPase in complex with the CTS representative ouabain, extending to 3.4 Å resolution. The structure provides key details on CTS binding, revealing an extensive hydrogen bonding network formed by the β-surface of the steroid core......The Na+,K+-ATPase maintains electrochemical gradients for Na+ and K+ that are critical for animal cells. Cardiotonic steroids (CTSs), widely used in the clinic and recently assigned a role as endogenous regulators of intracellular processes, are highly specific inhibitors of the Na+,K+-ATPase. Here...... of ouabain and the side chains of αM1, αM2, and αM6. Furthermore, the structure reveals that cation transport site II is occupied by Mg2+, and crystallographic studies indicate that Rb+ and Mn2+, but not Na+, bind to this site. Comparison with the low-affinity [K2]E2–MgFx–ouabain structure [Ogawa et al...

  16. Genetically encoded photocrosslinkers locate the high-affinity binding site of antidepressant drugs in the human serotonin transporter

    DEFF Research Database (Denmark)

    Rannversson, Hafsteinn; Andersen, Jacob; Hall, Lena Sørensen;

    2016-01-01

    Despite the well-established role of the human serotonin transporter (hSERT) in the treatment of depression, the molecular details of antidepressant drug binding are still not fully understood. Here we utilize amber codon suppression in a membrane-bound transporter protein to encode photocrosslin......Despite the well-established role of the human serotonin transporter (hSERT) in the treatment of depression, the molecular details of antidepressant drug binding are still not fully understood. Here we utilize amber codon suppression in a membrane-bound transporter protein to encode...

  17. Selective affinity of the benzodiazepines quazepam and 2-oxo-quazepam for BZ1 binding site and demonstration of H-2-oxo-quazepam as a BZ1 selective radioligand

    Energy Technology Data Exchange (ETDEWEB)

    Billard, W.; Crosby, G.; Iorio, L.; Chipkin, R.; Barnett, A.

    1988-01-01

    Quazepam and 2-oxo-quazepam are novel benzodiazepines containing a trifluoroethyl substituent on the ring nitrogen at position number1. Detailed competition binding experiments (25 to 30 concs.) at 4/sup 0/C were undertaken with these compounds versus /sup 3/H-flunitrazepam using synaptic membranes from rat cortex or cerebellum. Unlike other benzodiazepines, both quazepam and 2-oxo-quazepam distinguished two populations of /sup 3/H-flunitrazepam binding sites in rat cortex which were present in roughly equal proportions and for which the compounds displayed a greater than 20-fold difference in affinity. In cerebellum, no such discrimination of sites was noted for 2-oxo-quazepam, but quazepam did distinguish a small, low affinity population of sites. /sup 3/H-2-oxo-quazepam was prepared and used in competition studies to substantiate the conclusion that these compounds discriminate two populations of benzodiazepine sites in rat cortex. This new radioligand was shown to specifically label BZ binding sites with high affinity in a saturable manner. The competition experiments were then conducted using /sup 3/H-2-oxo-quazepam at a radioligand concentration sufficiently low to ensure that only the higher affinity binding sites which 2-oxo-quazepam discriminates would be occupied. 15 references, 3 figures, 4 tables.

  18. Structural arrangement of tRNA binding sites on Escherichia coli ribosomes, as revealed from data on affinity labelling with photoactivatable tRNA derivatives.

    Science.gov (United States)

    Graifer, D M; Babkina, G T; Matasova, N B; Vladimirov, S N; Karpova, G G; Vlassov, V V

    1989-07-01

    A systematic study of protein environment of tRNA in ribosomes in model complexes representing different translation steps was carried out using the affinity labelling of the ribosomes with tRNA derivatives bearing aryl azide groups scattered statistically over tRNA guanine residues. Analysis of the proteins crosslinked to tRNA derivatives showed that the location of the derivatives in the aminoacyl (A) site led to the labelling of the proteins S5 and S7 in all complexes studied, whereas the labelling of the proteins S2, S8, S9, S11, S14, S16, S17, S18, S19, S21 as well as L9, L11, L14, L15, L21, L23, L24, L29 depended on the state of tRNA in A site. Similarly, the location of tRNA derivatives in the peptidyl (P) site resulted in the labelling of the proteins L27, S11, S13 and S19 in all states, whereas the labelling of the proteins S5, S7, S9, S12, S14, S20, S21 as well as L2, L13, L14, L17, L24, L27, L31, L32, L33 depended on the type of complex. The derivatives of tRNA(fMet) were found to crosslink to S1, S3, S5, S7, S9, S14 and L1, L2, L7/L12, L27. Based on the data obtained, a general principle of the dynamic functioning of ribosomes has been proposed: (i) the formation of each type of ribosomal complex is accompanied by changes in mutual arrangement of proteins - 'conformational adjustment' of the ribosome - and (ii) a ribosome can dynamically change its internal structure at each step of initiation and elongation; on the 70 S ribosome there are no rigidly fixed structures forming tRNA-binding sites (primarily A and P sites).

  19. Receptor-associated protein (RAP) has two high-affinity binding sites for the low-density lipoprotein receptor-related protein (LRP): consequences for the chaperone functions of RAP.

    Science.gov (United States)

    Jensen, Jan K; Dolmer, Klavs; Schar, Christine; Gettins, Peter G W

    2009-06-26

    RAP (receptor-associated protein) is a three domain 38 kDa ER (endoplasmic reticulum)-resident protein that is a chaperone for the LRP (low-density lipoprotein receptor-related protein). Whereas RAP is known to compete for binding of all known LRP ligands, neither the location, the number of binding sites on LRP, nor the domains of RAP involved in binding is known with certainty. We have systematically examined the binding of each of the three RAP domains (D1, D2 and D3) to tandem and triple CRs (complement-like repeats) that span the principal ligand-binding region, cluster II, of LRP. We found that D3 binds with low nanomolar affinity to all (CR)2 species examined. Addition of a third CR domain increases the affinity for D3 slightly. A pH change from 7.4 to 5.5 gave only a 6-fold increase in Kd for D3 at 37 degrees C, whereas temperature change from 22 degrees C to 37 degrees C has a similar small effect on affinity, raising questions about the recently proposed D3-destabilization mechanism of RAP release from LRP. Surprisingly, and in contrast to literature suggestions, D1 and D2 also bind to most (CR)2 and (CR)3 constructs with nanomolar affinity. Although this suggested that there might be three high-affinity binding sites in RAP for LRP, studies with intact RAP showed that only two binding sites are available in the intact chaperone. These findings suggest a new model for RAP to function as a folding chaperone and also for the involvement of YWTD domains in RAP release from LRP in the Golgi.

  20. Pyrrolidine analogues of lobelane: relationship of affinity for the dihydrotetrabenazine binding site with function of the vesicular monoamine transporter 2 (VMAT2).

    Science.gov (United States)

    Vartak, Ashish P; Nickell, Justin R; Chagkutip, Jaturaporn; Dwoskin, Linda P; Crooks, Peter A

    2009-12-10

    Ring size reduction of the central piperidine ring of lobelane yielded pyrrolidine analogues that showed marked inconsistencies in their ability to bind to the dihydrotetrabenazine (DTBZ) binding site on the vesicular monoamine transporter-2 (VMAT2) and their ability to inhibit VMAT2 function. The structure-activity relationships indicate that structural modification within the pyrrolidine series resulted in analogues that interact with two different sites, i.e., the DTBZ binding site and an alternative site on VMAT2 to inhibit transporter function.

  1. Pyrrolidine analogs of lobelane: Relationship of affinity for the dihydrotetrabenazine binding site with function of the vesicular monoamine transporter 2 (VMAT2)

    Science.gov (United States)

    Vartak, Ashish P.; Nickell, Justin R.; Chagkutip, Jaturaporn; Dwoskin, Linda P.; Crooks, Peter A.

    2013-01-01

    Ring size reduction of the central piperidine ring of lobelane yielded pyrrolidine analogs that showed marked inconsistencies in their ability to bind to the dihydrotetrabenazine (DTBZ) binding site on the vesicular monoamine transporter-2 (VMAT2) and their ability to inhibit VMAT2 function. The structure activity relationships indicate that structural modification within the pyrrolidine series resulted in analogs that interact with two different sites, i.e., the DTBZ binding site and an alternative site on VMAT2 to inhibit transporter function. PMID:19691331

  2. High-affinity binding of (/sup 3/H)acetylcholine to muscarinic cholinergic receptors

    Energy Technology Data Exchange (ETDEWEB)

    Kellar, K.J.; Martino, A.M.; Hall, D.P. Jr.; Schwartz, R.D.; Taylor, R.L.

    1985-06-01

    High-affinity binding of (/sup 3/H)acetylcholine to muscarinic cholinergic sites in rat CNS and peripheral tissues was measured in the presence of cytisin, which occupies nicotinic cholinergic receptors. The muscarinic sites were characterized with regard to binding kinetics, pharmacology, anatomical distribution, and regulation by guanyl nucleotides. These binding sites have characteristics of high-affinity muscarinic cholinergic receptors with a Kd of approximately 30 nM. Most of the muscarinic agonist and antagonist drugs tested have high affinity for the (/sup 3/H)acetylcholine binding site, but pirenzepine, an antagonist which is selective for M-1 receptors, has relatively low affinity. The ratio of high-affinity (/sup 3/H)acetylcholine binding sites to total muscarinic binding sites labeled by (/sup 3/H)quinuclidinyl benzilate varies from 9 to 90% in different tissues, with the highest ratios in the pons, medulla, and heart atrium. In the presence of guanyl nucleotides, (/sup 3/H) acetylcholine binding is decreased, but the extent of decrease varies from 40 to 90% in different tissues, with the largest decreases being found in the pons, medulla, cerebellum, and heart atrium. The results indicate that (/sup 3/H)acetylcholine binds to high-affinity M-1 and M-2 muscarinic receptors, and they suggest that most M-2 sites have high affinity for acetylcholine but that only a small fraction of M-1 sites have such high affinity.

  3. Reactions of a fluorescent ATP analog, 2'-(5-dimethyl-aminonaphthalene-1-sulfonyl) amino-2'-deoxyATP, with E. coli F1-ATPase and its subunits: the roles of the high affinity binding site in the alpha subunit and the low affinity binding site in the beta subunit.

    Science.gov (United States)

    Matsuoka, I; Takeda, K; Futai, M; Tonomura, Y

    1982-11-01

    . The kinetic properties of the fluorescence change of DNS-ATP in the reaction with the reconstituted EF1-ATPase were quite similar to those of native EF1. Most of our findings are consistent with a simple mechanism that the high affinity catalytic site and low affinity regulatory site exist in the alpha subunit and beta subunit, respectively. However, the findings mentioned in (4) suggest that the binding of the alpha and beta subunit, which is mediated by the gamma subunit, induces conformational change(s) in the ATP binding site located probably in the alpha subunit, and that the conformational change(s) is essential to exert the full hydrolyzing activity.

  4. The High Affinity Binding Site on Plasminogen Activator Inhibitor-1 (PAI-1) for the Low Density Lipoprotein Receptor-related Protein (LRP1) Is Composed of Four Basic Residues.

    Science.gov (United States)

    Gettins, Peter G W; Dolmer, Klavs

    2016-01-08

    Plasminogen activator inhibitor 1 (PAI-1) is a serpin inhibitor of the plasminogen activators urokinase-type plasminogen activator (uPA) and tissue plasminogen activator, which binds tightly to the clearance and signaling receptor low density lipoprotein receptor-related protein 1 (LRP1) in both proteinase-complexed and uncomplexed forms. Binding sites for PAI-1 within LRP1 have been localized to CR clusters II and IV. Within cluster II, there is a strong preference for the triple CR domain fragment CR456. Previous mutagenesis studies to identify the binding site on PAI-1 for LRP1 have given conflicting results or implied small binding contributions incompatible with the high affinity PAI-1/LRP1 interaction. Using a highly sensitive solution fluorescence assay, we have examined binding of CR456 to arginine and lysine variants of PAI-1 and definitively identified the binding site as composed of four basic residues, Lys-69, Arg-76, Lys-80, and Lys-88. These are highly conserved among mammalian PAI-1s. Individual mutations result in a 13-800-fold increase in Kd values. We present evidence that binding involves engagement of CR4 by Lys-88, CR5 by Arg-76 and Lys-80, and CR6 by Lys-69, with the strongest interactions to CR5 and CR6. Collectively, the individual binding contributions account quantitatively for the overall PAI-1/LRP1 affinity. We propose that the greater efficiency of PAI-1·uPA complex binding and clearance by LRP1, compared with PAI-1 alone, is due solely to simultaneous binding of the uPA moiety in the complex to its receptor, thereby making binding of the PAI-1 moiety to LRP1 a two-dimensional surface-localized association.

  5. A High-Affinity Binding Site for the AVR9 Peptide Elicitor of Cladosporium fulvum Is Present on Plasma Membranes of Tomato and Other Solanaceous Plants.

    Science.gov (United States)

    Kooman-Gersmann, M.; Honee, G.; Bonnema, G.; De Wit, PJGM.

    1996-05-01

    The race-specific Cladosporium fulvum peptide elicitor AVR9, which specifically induces a hypersensitive response in tomato genotypes carrying the Cf-9 resistance gene, was labeled with iodine-125 at the N-terminal tyrosine residue and used in binding studies. 125I-AVR9 showed specific, saturable, and reversible binding to plasma membranes isolated from leaves of tomato cultivar Moneymaker without Cf resistance genes (MM-Cf0) or from a near-isogenic genotype with the Cf-9 resistance gene (MM-Cf9). The dissociation constant was found to be 0.07 nM, and the receptor concentration was 0.8 pmol/mg microsomal protein. Binding was highly influenced by pH and the ionic strength of the binding buffer and by temperature, indicating the involvement of both electrostatic and hydrophobic interactions. Binding kinetics and binding capacity were similar for membranes of the MM-Cf0 and MM-Cf9 genotypes. In all solanaceous plant species tested, an AVR9 binding site was present, whereas in the nonsolanaceous species that were analyzed, such a binding site could not be identified. The ability of membranes isolated from different solanaceous plant species to bind AVR9 seems to correlate with the presence of members of the Cf-9 gene family, but whether this correlation is functional remains to be determined.

  6. Affinity chromatography purification of cytochrome c binding enzymes.

    OpenAIRE

    Azzi, A; Bill, K; Broger, C

    1982-01-01

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

  7. Regulation of streptokinase expression by CovR/S in Streptococcus pyogenes: CovR acts through a single high-affinity binding site.

    Science.gov (United States)

    Churchward, Gordon; Bates, Christopher; Gusa, Asiya A; Stringer, Virginia; Scott, June R

    2009-02-01

    The important human pathogen Streptococcus pyogenes (the group A streptococcus or GAS) produces many virulence factors that are regulated by the two-component signal transduction system CovRS (CsrRS). Dissemination of GAS infection originating at the skin has been shown to require production of streptokinase, whose transcription is repressed by CovR. In this work we have studied the interaction of CovR and phosphorylated CovR (CovR-P) with the promoter for streptokinase, Pska. We found that, in contrast to the other CovR-repressed promoters, Pska regulation by CovR occurs through binding at a single ATTARA consensus binding sequence (CB) that overlaps the -10 region of the promoter. Binding of CovR to other nearby consensus sequences occurs upon phosphorylation of the protein, but these other CBs do not contribute to the regulation of Pska by CovR. Thus, binding at a specific site does not necessarily indicate that the site is involved in regulation by CovR. In addition, at Pska, CovR binding to the different sites does not appear to involve cooperative interactions, which simplifies the analysis of CovR binding and gives us insight into the modes of interaction that occur between CovR and its specific DNA-binding sites. Finally, the observation that regulation of transcription from Pska occurs at a very low concentration of phosphorylated CovR may have important implications for the regulation of virulence gene expression during GAS infection.

  8. HIV protease inhibitors act as competitive inhibitors of the cytoplasmic glucose binding site of GLUTs with differing affinities for GLUT1 and GLUT4.

    Directory of Open Access Journals (Sweden)

    Richard C Hresko

    Full Text Available The clinical use of several first generation HIV protease inhibitors (PIs is associated with the development of insulin resistance. Indinavir has been shown to act as a potent reversible noncompetitive inhibitor of zero-trans glucose influx via direct interaction with the insulin responsive facilitative glucose transporter GLUT4. Newer drugs within this class have differing effects on insulin sensitivity in treated patients. GLUTs are known to contain two distinct glucose-binding sites that are located on opposite sides of the lipid bilayer. To determine whether interference with the cytoplasmic glucose binding site is responsible for differential effects of PIs on glucose transport, intact intracellular membrane vesicles containing GLUT1 and GLUT4, which have an inverted transporter orientation relative to the plasma membrane, were isolated from 3T3-L1 adipocytes. The binding of biotinylated ATB-BMPA, a membrane impermeable bis-mannose containing photolabel, was determined in the presence of indinavir, ritonavir, atazanavir, tipranavir, and cytochalasin b. Zero-trans 2-deoxyglucose transport was measured in both 3T3-L1 fibroblasts and primary rat adipocytes acutely exposed to these compounds. PI inhibition of glucose transport correlated strongly with the PI inhibition of ATB-BMPA/transporter binding. At therapeutically relevant concentrations, ritonavir was not selective for GLUT4 over GLUT1. Indinavir was found to act as a competitive inhibitor of the cytoplasmic glucose binding site of GLUT4 with a K(I of 8.2 µM. These data establish biotinylated ATB-BMPA as an effective probe to quantify accessibility of the endofacial glucose-binding site in GLUTs and reveal that the ability of PIs to block this site differs among drugs within this class. This provides mechanistic insight into the basis for the clinical variation in drug-related metabolic toxicity.

  9. Quantitative autoradiography of the binding sites for ( sup 125 I) iodoglyburide, a novel high-affinity ligand for ATP-sensitive potassium channels in rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Gehlert, D.R.; Gackenheimer, S.L.; Mais, D.E.; Robertson, D.W. (Eli Lilly and Co., Indianapolis, IN (USA))

    1991-05-01

    We have developed a high specific activity ligand for localization of ATP-sensitive potassium channels in the brain. When brain sections were incubated with ({sup 125}I)iodoglyburide (N-(2-((((cyclohexylamino)carbonyl)amino)sulfonyl)ethyl)-5-{sup 125}I-2- methoxybenzamide), the ligand bound to a single site with a KD of 495 pM and a maximum binding site density of 176 fmol/mg of tissue. Glyburide was the most potent inhibitor of specific ({sup 125}I)iodoglyburide binding to rat forebrain sections whereas iodoglyburide and glipizide were slightly less potent. The binding was also sensitive to ATP which completely inhibited binding at concentrations of 10 mM. Autoradiographic localization of ({sup 125}I)iodoglyburide binding indicated a broad distribution of the ATP-sensitive potassium channel in the brain. The highest levels of binding were seen in the globus pallidus and ventral pallidum followed by the septohippocampal nucleus, anterior pituitary, the CA2 and CA3 region of the hippocampus, ventral pallidum, the molecular layer of the cerebellum and substantia nigra zona reticulata. The hilus and dorsal subiculum of the hippocampus, molecular layer of the dentate gyrus, cerebral cortex, lateral olfactory tract nucleus, olfactory tubercle and the zona incerta contained relatively high levels of binding. A lower level of binding (approximately 3- to 4-fold) was found throughout the remainder of the brain. These results indicate that the ATP-sensitive potassium channel has a broad presence in the rat brain and that a few select brain regions are enriched in this subtype of neuronal potassium channels.

  10. Anion-induced increases in the affinity of colcemid binding to tubulin.

    Science.gov (United States)

    Ray, K; Bhattacharyya, B; Biswas, B B

    1984-08-01

    Colcemid binds tubulin rapidly and reversibly in contrast to colchicine which binds tubulin relatively slowly and essentially irreversibly. At 37 degrees C the association rate constant for colcemid binding is 1.88 X 10(6) M-1 h-1, about 10 times higher than that for colchicine; this is reflected in the activation energies for binding which are 51.4 kJ/mol for colcemid and 84.8 kJ/mol for colchicine. Scatchard analysis indicates two binding sites on tubulin having different affinities for colcemid. The high-affinity site (Ka = 0.7 X 10(5) M-1 at 37 degrees C) is sensitive to temperature and binds both colchicine and colcemid and hence they are mutually competitive inhibitors. The low-affinity site (Kb = 1.2 X 10(4) M-1) is rather insensitive to temperature and binds only colcemid. Like colchicine, 0.6 mol of colcemid are bound/mol of tubulin dimer (at the high-affinity site) and the reaction is entropy driven (163 J K-1 mol-1). Similar to colchicine, colcemid binding to tubulin is stimulated by certain anions (viz. sulfate and tartrate) but by a different mechanism. Colcemid binding affinity at the lower-affinity site of tubulin is increased in the presence of ammonium sulfate. Interestingly, the lower-affinity site on tubulin for colcemid, even when converted to higher affinity in presence of ammonium sulfate, is not recognized by colchicine. We conclude that tubulin possesses two binding sites, one of which specifically recognized the groups present on the B-ring of colchicine molecule and is effected by the ammonium sulfate, whereas the higher-affinity site, which could accommodate both colchicine and colcemid, possibly recognized the A and C ring of colchicine.

  11. Peptide Nucleic Acids Having Enhanced Binding Affinity and Sequence Specificity

    DEFF Research Database (Denmark)

    1998-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary DNA and RNA strands more strongly than a corresponding DNA strand, and exhibit increased sequence specificity and binding affinity. Methods of increasing binding affinity and sequence specificity of peptide nucleic aci...

  12. Base-sequence specificity of Hoechst 33258 and DAPI binding to five (A/T)4 DNA sites with kinetic evidence for more than one high-affinity Hoechst 33258-AATT complex.

    Science.gov (United States)

    Breusegem, Sophia Y; Clegg, Robert M; Loontiens, Frank G

    2002-02-01

    The binding of Hoechst 33258 and DAPI to five different (A/T)4 sequences in a stable DNA hairpin was studied exploiting the substantial increase in dye fluorescence upon binding. The two dyes have comparable affinities for the AATT site (e.g. association constant K(a)=5.5 x 10(8) M(-1) for DAPI), and their affinities decrease in the series AATT > TAAT approximately equal to ATAT > TATA approximately equal to TTAA. The extreme values of K(a) differ by a factor of 200 for Hoechst 33258 but only 30 for DAPI. The binding kinetics of Hoechst 33258 were measured by stopped-flow under pseudo-first order conditions with an (A/T)4 site in excess. The lower-resolution experiments can be well represented by single exponential processes, corresponding to a single-step binding mechanism. The calculated association-rate parameters for the five (A/T)4 sites are similar (2.46 x 10(8) M(-1) s(-1) to 0.86 x 10(8) M(-1) s(-1)) and nearly diffusion-controlled, while the dissociation-rate parameters vary from 0.42 s(-1) to 96 s(-1). Thus the association constants are kinetically controlled and are close to their equilibrium-determined values. However, when obtained with increased signal-to-noise ratio, the kinetic traces for Hoechst 33258 binding at the AATT site reveal two components. The concentration dependencies of the two time constants and amplitudes are consistent with two different kinetically equivalent two-step models. In the first model, fast bimolecular binding is followed by an isomerization of the initial complex. In the second model, two single-step associations form two complexes that mutually exclude each other. For both models the four reaction-rate parameters are calculated. Finally, specific dissociation kinetics, using poly[d(A-5BrU)], show that the kinetics are even more complex than either two-step model. We correlate our results with the different binding orientations and locations of Hoechst 33258 in the DNA minor groove found in several structural studies in

  13. Calcium binding to the low affinity sites in troponin C induces conformational changes in the high affinity domain. A possible route of information transfer in activation of muscle contraction.

    Science.gov (United States)

    Grabarek, Z; Leavis, P C; Gergely, J

    1986-01-15

    Residues 89-100 of troponin C (C89-100) and 96-116 of troponin I (I96-116) interact with each other in the troponin complex (Dalgarno, D.C., Grand, R.J.A., Levine, B.A. Moir, A., J.G., Scott, G.M.M., and Perry, S.V. (1982) FEBS Lett. 150, 54-58) and are necessary for the Ca2+ sensitivity of actomyosin ATPase (Syska, H., Wilkinson, J.M., Grand, R.J.A., and Perry, S.V. (1976) Biochem. J. 153, 375-387 and Grabarek, Z., Drabikowski, W., Leavis, P.C., Rosenfeld, S.S., and Gergely, J. (1981) J. Biol. Chem. 256, 13121-13127). We have studied Ca2+-induced changes in the region C89-100 by monitoring the fluorescence of troponin C (TnC) labeled at Cys-98 with 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid. Equilibrium titration of the labeled TnC with Ca2+ indicates that the probe is sensitive to binding to both classes of sites in free TnC as well as in its complex with TnI. When Mg2 X TnC is mixed with Ca2+ in a stopped flow apparatus, there is a rapid fluorescence increase related to Ca2+ binding to the unoccupied sites I and II followed by a slower increase (k = 9.9 s-1) that represents Mg2+-Ca2+ exchange at sites III and IV. In the TnC X TnI complex, the fast phase is much larger and the Mg2+-Ca2+ exchange at sites III and IV results in a small decrease rather than an increase in the fluorescence of the probe. The possibility is discussed that the fast change in the environment of Cys-98 upon Ca2+ binding to sites I and II may be instrumental in triggering activation of the thin filament by facilitating a contact between C89-100 and I96-116.

  14. Cystatin M/E is a high affinity inhibitor of cathepsin V and cathepsin L by a reactive site that is distinct from the legumain-binding site. A novel clue for the role of cystatin M/E in epidermal cornification.

    NARCIS (Netherlands)

    Cheng, T.; Hitomi, K.; Vlijmen-Willems, I.M.J.J. van; Jongh, G.J. de; Yamamoto, K.; Nishi, K.; Watts, C.; Reinheckel, T.; Schalkwijk, J.; Zeeuwen, P.L.J.M.

    2006-01-01

    Cystatin M/E is a high affinity inhibitor of the asparaginyl endopeptidase legumain, and we have previously reported that both proteins are likely to be involved in the regulation of stratum corneum formation in skin. Although cystatin M/E contains a predicted binding site for papain-like cysteine p

  15. Relative binding affinity of steroids for the corticosterone receptor system in rat hippocampus

    NARCIS (Netherlands)

    De Kloet, E R; Veldhuis, H D; Wagenaars, J L; Bergink, E W

    1984-01-01

    In cytosol of the hippocampus corticosterone displays highest affinity for the sites that remain available for binding in the presence of excess RU 26988, which is shown to be a "pure" glucocorticoid. A rather high affinity (greater than or equal to 25%) was found for 11 beta-hydroxyprogesterone, 21

  16. The extrinsic PsbO protein modulates the oxidation/reduction rate of the exogenous Mn cation at the high-affinity Mn-binding site of Mn-depleted PSII membranes.

    Science.gov (United States)

    Semin, Boris K; Podkovirina, Tatiana E; Davletshina, Lira N; Timofeev, Kirill N; Ivanov, Il'ya I; Rubin, Andrei B

    2015-08-01

    The oxidation of exogenous Mn(II) cations at the high-affinity (HA) Mn-binding site in Mn-depleted photosystem II (PSII) membranes with or without the presence of the extrinsic PsbO polypeptide was studied by EPR. The six-lines EPR spectrum of Mn(II) cation disappears in the absence of the PsbO protein in membranes under illumination, but there was no effect when PSII preparations bound the PsbO protein. Our study demonstrates that such effect is determined by significant influence of the PsbO protein on the ratio between the rates of Mn oxidation and reduction at the HA site when the membranes are illuminated.

  17. Integrated microfluidic approach for quantitative high-throughput measurements of transcription factor binding affinities.

    Science.gov (United States)

    Glick, Yair; Orenstein, Yaron; Chen, Dana; Avrahami, Dorit; Zor, Tsaffrir; Shamir, Ron; Gerber, Doron

    2016-04-07

    Protein binding to DNA is a fundamental process in gene regulation. Methodologies such as ChIP-Seq and mapping of DNase I hypersensitive sites provide global information on this regulation in vivo In vitro methodologies provide valuable complementary information on protein-DNA specificities. However, current methods still do not measure absolute binding affinities. There is a real need for large-scale quantitative protein-DNA affinity measurements. We developed QPID, a microfluidic application for measuring protein-DNA affinities. A single run is equivalent to 4096 gel-shift experiments. Using QPID, we characterized the different affinities of ATF1, c-Jun, c-Fos and AP-1 to the CRE consensus motif and CRE half-site in two different genomic sequences on a single device. We discovered that binding of ATF1, but not of AP-1, to the CRE half-site is highly affected by its genomic context. This effect was highly correlated with ATF1 ChIP-seq and PBM experiments. Next, we characterized the affinities of ATF1 and ATF3 to 128 genomic CRE and CRE half-site sequences. Our affinity measurements explained that in vivo binding differences between ATF1 and ATF3 to CRE and CRE half-sites are partially mediated by differences in the minor groove width. We believe that QPID would become a central tool for quantitative characterization of biophysical aspects affecting protein-DNA binding.

  18. Crystal structure of an affinity-matured prolactin complexed to its dimerized receptor reveals the topology of hormone binding site 2

    DEFF Research Database (Denmark)

    Broutin, Isabelle; Jomain, Jean-Baptiste; Tallet, Estelle;

    2010-01-01

    We report the first crystal structure of a 1:2 hormone.receptor complex that involves prolactin (PRL) as the ligand, at 3.8-A resolution. Stable ternary complexes were obtained by generating affinity-matured PRL variants harboring an N-terminal tail from ovine placental lactogen, a closely relate...

  19. Binding Affinity and Capacity for the Uremic Toxin Indoxyl Sulfate

    Directory of Open Access Journals (Sweden)

    Eric Devine

    2014-01-01

    Full Text Available Protein binding prevents uremic toxins from removal by conventional extracorporeal therapies leading to accumulation in maintenance dialysis patients. Weakening of the protein binding may enhance the dialytic elimination of these toxins. In ultrafiltration and equilibrium dialysis experiments, different measures to modify the plasma binding affinity and capacity were tested: (i, increasing the sodium chloride (NaCl concentration to achieve a higher ionic strength; (ii, increasing the temperature; and (iii, dilution. The effects on the dissociation constant KD and the protein bound fraction of the prototypical uremic toxin indoxyl sulfate (IS in plasma of healthy and uremic individuals were studied. Binding of IS corresponded to one site binding in normal plasma. KD increased linearly with the NaCl concentration between 0.15 (KD = 13.2 ± 3.7 µM and 0.75 M (KD = 56.2 ± 2.0 µM. Plasma dilution further reduced the protein bound toxin fraction by lowering the protein binding capacity of the plasma. Higher temperatures also decreased the protein bound fraction of IS in human plasma. Increasing the NaCl concentration was effective to weaken the binding of IS also in uremic plasma: the protein bound fraction decreased from 89% ± 3% to 81% ± 3% at 0.15 and 0.75 M NaCl, respectively. Dilution and increasing the ionic strength and temperature enhance the free fraction of IS allowing better removal of the substance during dialysis. Applied during clinical dialysis, this may have beneficial effects on the long-term outcome of maintenance dialysis patients.

  20. Purification of proteins specifically binding human endogenous retrovirus K long terminal repeat by affinity elution chromatography.

    Science.gov (United States)

    Trubetskoy, D O; Zavalova, L L; Akopov, S B; Nikolaev, L G

    2002-11-01

    A novel affinity elution procedure for purification of DNA-binding proteins was developed and employed to purify to near homogeneity the proteins recognizing a 21 base pair sequence within the long terminal repeat of human endogenous retroviruses K. The approach involves loading the initial protein mixture on a heparin-agarose column and elution of protein(s) of interest with a solution of double-stranded oligonucleotide containing binding sites of the protein(s). The affinity elution has several advantages over conventional DNA-affinity chromatography: (i) it is easier and faster, permitting to isolate proteins in a 1 day-one stage procedure; (ii) yield of a target protein is severalfold higher than that in DNA-affinity chromatography; (iii) it is not necessary to prepare a special affinity support for each factor to be isolated. Theaffinity elution could be a useful alternative to conventional DNA-affinity chromatography.

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

    Directory of Open Access Journals (Sweden)

    Graham S Baldwin

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

  2. Triazoloquinazolinediones as novel high affinity ligands for the benzodiazepine site of GABA(A) receptors

    DEFF Research Database (Denmark)

    Nilsson, Jakob; Gidlöf, Ritha; Nielsen, Elsebet Østergaard

    2011-01-01

    Based on a pharmacophore model of the benzodiazepine-binding site of GABA(A) receptors, a series of 2-aryl-2,6-dihydro[1,2,4]triazolo[4,3-c]quinazoline-3,5-diones (structure type I) were designed, synthesized, and identified as high-affinity ligands of the binding site. For several compounds, K...

  3. Influence of azide incorporation on binding affinity by small papain inhibitors.

    Science.gov (United States)

    Wammes, Angelique E M; Hendriks, Tom G; Amatdjais-Groenen, Helene I V; Wijdeven, Marloes A; van Hest, Jan C M; van Delft, Floris L; Ritschel, Tina; Rutjes, Floris P J T

    2014-10-15

    In order to develop affinity-based biosensor platforms, appropriate ligands with a functional handle for immobilization onto a biosensor surface are required. To this end, a library of papain inhibitors was designed and synthesized, containing different azide linkers for subsequent immobilization by 'click' chemistry, in this particular case by copper-free, strain-promoted azide-alkyne cycloaddition (SPAAC). Furthermore, a molecular docking study was performed to obtain a better insight as to at which position such azide handles could be tolerated without affecting binding affinity. Although the azide moiety is small, in some cases its introduction strongly influenced the binding affinity. For one class of inhibitors a swapped binding mode was proposed to explain the results. In addition, a specific site for linker introduction was identified, which did not significantly affect the binding affinity.

  4. Supervised Machine Learning Methods Applied to Predict Ligand- Binding Affinity.

    Science.gov (United States)

    Heck, Gabriela S; Pintro, Val O; Pereira, Richard R; de Ávila, Mauricio B; Levin, Nayara M B; de Azevedo, Walter F

    2017-01-01

    Calculation of ligand-binding affinity is an open problem in computational medicinal chemistry. The ability to computationally predict affinities has a beneficial impact in the early stages of drug development, since it allows a mathematical model to assess protein-ligand interactions. Due to the availability of structural and binding information, machine learning methods have been applied to generate scoring functions with good predictive power. Our goal here is to review recent developments in the application of machine learning methods to predict ligand-binding affinity. We focus our review on the application of computational methods to predict binding affinity for protein targets. In addition, we also describe the major available databases for experimental binding constants and protein structures. Furthermore, we explain the most successful methods to evaluate the predictive power of scoring functions. Association of structural information with ligand-binding affinity makes it possible to generate scoring functions targeted to a specific biological system. Through regression analysis, this data can be used as a base to generate mathematical models to predict ligandbinding affinities, such as inhibition constant, dissociation constant and binding energy. Experimental biophysical techniques were able to determine the structures of over 120,000 macromolecules. Considering also the evolution of binding affinity information, we may say that we have a promising scenario for development of scoring functions, making use of machine learning techniques. Recent developments in this area indicate that building scoring functions targeted to the biological systems of interest shows superior predictive performance, when compared with other approaches. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  5. A High-Affinity Metal-Binding Peptide From Escherichia Coli Hypb

    Energy Technology Data Exchange (ETDEWEB)

    Chung, K.C.Chan; Cao, L.; Dias, A.V.; Pickering, I.J.; George, G.N.; Zamble, D.B.

    2009-05-12

    The high-affinity nickel-binding site of the Escherichia coli [NiFe]-hydrogenase accessory protein HypB was localized to residues at the immediate N-terminus of the protein. Modification of a metal-binding fusion protein, site-directed mutagenesis experiments, and DFT calculations were used to identify the N-terminal amine as a ligand as well as the three cysteine residues in the CXXCGCXXX motif. This sequence can be removed from the protein and both a synthesized peptide and a protein fusion bind nickel with a similar affinity and the same structure as the parent metalloprotein, indicating the self-sufficiency of this high-affinity nickel-binding sequence.

  6. Domain interplay in the urokinase receptor. Requirement for the third domain in high affinity ligand binding and demonstration of ligand contact sites in distinct receptor domains

    DEFF Research Database (Denmark)

    Behrendt, N; Ronne, E; Dano, K

    1996-01-01

    The urokinase plasminogen activator receptor (uPAR) is a membrane protein comprised of three extracellular domains. In order to study the importance of this domain organization in the ligand-binding process of the receptor we subjected a recombinant, soluble uPAR (suPAR) to specific proteolytic c...

  7. A 3D-QSAR-driven approach to binding mode and affinity prediction

    DEFF Research Database (Denmark)

    Tosco, Paolo; Balle, Thomas

    2012-01-01

    A method for predicting the binding mode of a series of ligands is proposed. The procedure relies on three-dimensional quantitative structure-activity relationships (3D-QSAR) and does not require structural knowledge of the binding site. Candidate alignments are automatically built and ranked...... according to a consensus scoring function. 3D-QSAR analysis based on the selected binding mode enables affinity prediction of new drug candidates having less than 10 rotatable bonds....

  8. Domain interplay in the urokinase receptor. Requirement for the third domain in high affinity ligand binding and demonstration of ligand contact sites in distinct receptor domains

    DEFF Research Database (Denmark)

    Behrendt, N; Ronne, E; Dano, K

    1996-01-01

    . The purified suPAR was cross-linked to the radiolabeled amino-terminal fragment (ATF) of urokinase, followed by cleavage with chymotrypsin. In accordance with the cleavage pattern found for the uncomplexed receptor, this treatment led to cleavage between D1 and D(2 + 3). Analysis of the radiolabeled fragments...... revealed the expected ligand labeling of D1 but a clear labeling of D(2 + 3) was also found, indicating that this part of the molecule is also situated in close contact with ATF in the receptor-ligand complex. The latter contact site may contribute to the role of molecular regions outside D1 in high...

  9. Computational protocol for predicting the binding affinities of zinc containing metalloprotein-ligand complexes.

    Science.gov (United States)

    Jain, Tarun; Jayaram, B

    2007-06-01

    Zinc is one of the most important metal ions found in proteins performing specific functions associated with life processes. Coordination geometry of the zinc ion in the active site of the metalloprotein-ligand complexes poses a challenge in determining ligand binding affinities accurately in structure-based drug design. We report here an all atom force field based computational protocol for estimating rapidly the binding affinities of zinc containing metalloprotein-ligand complexes, considering electrostatics, van der Waals, hydrophobicity, and loss in conformational entropy of protein side chains upon ligand binding along with a nonbonded approach to model the interactions of the zinc ion with all the other atoms of the complex. We examined the sensitivity of the binding affinity predictions to the choice of Lennard-Jones parameters, partial atomic charges, and dielectric treatments adopted for system preparation and scoring. The highest correlation obtained was R2 = 0.77 (r = 0.88) for the predicted binding affinity against the experiment on a heterogenous dataset of 90 zinc containing metalloprotein-ligand complexes consisting of five unique protein targets. Model validation and parameter analysis studies underscore the robustness and predictive ability of the scoring function. The high correlation obtained suggests the potential applicability of the methodology in designing novel ligands for zinc-metalloproteins. The scoring function has been web enabled for free access at www.scfbio-iitd.res.in/software/drugdesign/bapplz.jsp as BAPPL-Z server (Binding Affinity Prediction of Protein-Ligand complexes containing Zinc metal ions).

  10. Rolling adhesion of alphaL I domain mutants decorrelated from binding affinity.

    Science.gov (United States)

    Pepper, Lauren R; Hammer, Daniel A; Boder, Eric T

    2006-06-30

    Activated lymphocyte function-associated antigen-1 (LFA-1, alphaLbeta2 integrin) found on leukocytes facilitates firm adhesion to endothelial cell layers by binding to intercellular adhesion molecule-1 (ICAM-1), which is up-regulated on endothelial cells at sites of inflammation. Recent work has shown that LFA-1 in a pre-activation, low-affinity state may also be involved in the initial tethering and rolling phase of the adhesion cascade. The inserted (I) domain of LFA-1 contains the ligand-binding epitope of the molecule, and a conformational change in this region during activation increases ligand affinity. We have displayed wild-type I domain on the surface of yeast and validated expression using I domain specific antibodies and flow cytometry. Surface display of I domain supports yeast rolling on ICAM-1-coated surfaces under shear flow. Expression of a locked open, high-affinity I domain mutant supports firm adhesion of yeast, while yeast displaying intermediate-affinity I domain mutants exhibit a range of rolling phenotypes. We find that rolling behavior for these mutants fails to correlate with ligand binding affinity. These results indicate that unstressed binding affinity is not the only molecular property that determines adhesive behavior under shear flow.

  11. Neurotransmitter/sodium symporter orthologue LeuT has a single high-affinity substrate site.

    Science.gov (United States)

    Piscitelli, Chayne L; Krishnamurthy, Harini; Gouaux, Eric

    2010-12-23

    Neurotransmitter/sodium symporters (NSSs) couple the uptake of neurotransmitter with one or more sodium ions, removing neurotransmitter from the synaptic cleft. NSSs are essential to the function of chemical synapses, are associated with multiple neurological diseases and disorders, and are the targets of therapeutic and illicit drugs. LeuT, a prokaryotic orthologue of the NSS family, is a model transporter for understanding the relationships between molecular mechanism and atomic structure in a broad range of sodium-dependent and sodium-independent secondary transporters. At present there is a controversy over whether there are one or two high-affinity substrate binding sites in LeuT. The first-reported crystal structure of LeuT, together with subsequent functional and structural studies, provided direct evidence for a single, high-affinity, centrally located substrate-binding site, defined as the S1 site. Recent binding, flux and molecular simulation studies, however, have been interpreted in terms of a model where there are two high-affinity binding sites: the central, S1, site and a second, the S2 site, located within the extracellular vestibule. Furthermore, it was proposed that the S1 and S2 sites are allosterically coupled such that occupancy of the S2 site is required for the cytoplasmic release of substrate from the S1 site. Here we address this controversy by performing direct measurement of substrate binding to wild-type LeuT and to S2 site mutants using isothermal titration calorimetry, equilibrium dialysis and scintillation proximity assays. In addition, we perform uptake experiments to determine whether the proposed allosteric coupling between the putative S2 site and the S1 site manifests itself in the kinetics of substrate flux. We conclude that LeuT harbours a single, centrally located, high-affinity substrate-binding site and that transport is well described by a simple, single-substrate kinetic mechanism.

  12. Tissue specificity of endothelin binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Bolger, G.T.; Liard, F.; Krogsrud, R.; Thibeault, D.; Jaramillo, J. (BioMega, Inc., Laval, Quebec (Canada))

    1990-09-01

    A measurement was made of the binding of 125I-labeled endothelin (125I-ET) to crude membrane fractions prepared from rat aorta, atrium, ventricle, portal vein, trachea, lung parenchyma, vas deferens, ileum, bladder, and guinea-pig taenia coli and lung parenchyma. Scatchard analysis of 125I-ET binding in all tissues indicated binding to a single class of saturable sites. The affinity and density of 125I-ET binding sites varied between tissues. The Kd of 125I-ET binding was approximately 0.5 nM for rat aorta, trachea, lung parenchyma, ventricle, bladder, and vas deferens, and guinea-pig taenia coli and lung parenchyma, 1.8 nM for rat portal vein and atrium, and 3.3 nM for ileum. The Bmax of 125I-ET binding had the following rank order of density in rat tissues: trachea greater than lung parenchyma = vas deferens much greater than aorta = portal vein = atrium greater than bladder greater than ventricle = ileum. The properties of 125I-ET endothelin binding were characterized in rat ventricular membranes. 125I-ET binding was time dependent, reaching a maximum within 45-60 min at 25 degrees C. The calculated microassociation constant was 9.67 x 10(5) s-1 M-1. Only 15-20% of 125I-ET dissociated from its binding site even when dissociation was studied as long as 3 h. Preincubation of ventricular membranes with ET prevented binding of 125I-ET. 125I-ET binding was destroyed by boiling of ventricular membranes and was temperature, pH, and cation (Ca2+, Mg2+, and Na+) dependent.

  13. (/sup 3/H)desipramine binding to rat brain tissue: binding to both noradrenaline uptake sites and sites not related to noradrenaline neurons

    Energy Technology Data Exchange (ETDEWEB)

    Baeckstroem, I.T.Ro.; Ross, S.B.; Marcusson, J.O.

    1989-04-01

    The pharmacological and biochemical characteristics of (3H)desipramine binding to rat brain tissue were investigated. Competition studies with noradrenaline, nisoxetine, nortriptyline, and desipramine suggested the presence of more than one (3H)desipramine binding site. Most of the noradrenaline-sensitive binding represented a high-affinity site, and this site appeared to be the same as the high-affinity site of nisoxetine-sensitive binding. The (3H)desipramine binding sites were abolished by protease treatment, a result suggesting that the binding sites are protein in nature. When specific binding was defined by 0.1 microM nisoxetine, the binding was saturable and fitted a single-site binding model with a binding affinity of approximately 1 nM. This binding fraction was abolished by lesioning of the noradrenaline neurons with the noradrenaline neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4). In contrast, when 10 microM nisoxetine was used to define the specific binding, the binding was not saturable over the nanomolar range, but the binding fitted a two-site binding model with KD values of 0.5 and greater than 100 nM for the high- and low-affinity components, respectively. The high-affinity site was abolished after DSP4 lesioning, whereas the low-affinity site remained. The binding capacity (Bmax) for binding defined by 0.1 microM nisoxetine varied between brain regions, with very low density in the striatum (Bmax not possible to determine), 60-90 fmol/mg of protein in cortical areas and cerebellum, and 120 fmol/mg of protein in the hypothalamus. The binding capacities of these high-affinity sites correlated significantly with the regional distribution of (3H)noradrenaline uptake but not with 5-(3H)hydroxytryptamine uptake.

  14. DBD2BS: connecting a DNA-binding protein with its binding sites

    OpenAIRE

    2012-01-01

    By binding to short and highly conserved DNA sequences in genomes, DNA-binding proteins initiate, enhance or repress biological processes. Accurately identifying such binding sites, often represented by position weight matrices (PWMs), is an important step in understanding the control mechanisms of cells. When given coordinates of a DNA-binding domain (DBD) bound with DNA, a potential function can be used to estimate the change of binding affinity after base substitutions, where the changes c...

  15. High- and low-affinity binding of S-citalopram to the human serotonin transporter mutated at 20 putatively important amino acid positions

    DEFF Research Database (Denmark)

    Plenge, Per; Wiborg, Ove

    2005-01-01

    of presumed importance. Binding of S-citalopram, both to the high-affinity-binding site and to the allosteric binding site, was measured in these mutants with the purpose of investigating the connection between the two binding sites. The amino acid substitutions did not introduce large changes in the two...

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

    Energy Technology Data Exchange (ETDEWEB)

    Remmers, A.E.; Medzihradsky, F. (Univ. of Michigan Medical School, Ann Arbor (United States))

    1991-03-15

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

  17. Affinity of 3-acyl substituted 4-quinolones at the benzodiazepine site of GABAA receptors

    DEFF Research Database (Denmark)

    Lager, Erik; Nilsson, Jakob; Nielsen, Elsebet Østergaard

    2008-01-01

    The finding that alkyl 1,4-dihydro-4-oxoquinoline-3-carboxylate and N-alkyl-1,4-dihydro-4-oxoquinoline-3-carboxamide derivatives may be high-affinity ligands at the benzodiazepine binding site of the GABA(A) receptor, prompted a study of 3-acyl-1,4-dihydro-4-oxoquinoline (3-acyl-4-quinolones......). In general, the affinity of the 3-acyl derivatives was found to be comparable with the 3-carboxylate and the 3-carboxamide derivatives, and certain substituents (e.g., benzyl) in position 6 were again shown to be important. As it is believed that the benzodiazepine binding site is situated between an alpha...

  18. Combining transcription factor binding affinities with open-chromatin data for accurate gene expression prediction.

    Science.gov (United States)

    Schmidt, Florian; Gasparoni, Nina; Gasparoni, Gilles; Gianmoena, Kathrin; Cadenas, Cristina; Polansky, Julia K; Ebert, Peter; Nordström, Karl; Barann, Matthias; Sinha, Anupam; Fröhler, Sebastian; Xiong, Jieyi; Dehghani Amirabad, Azim; Behjati Ardakani, Fatemeh; Hutter, Barbara; Zipprich, Gideon; Felder, Bärbel; Eils, Jürgen; Brors, Benedikt; Chen, Wei; Hengstler, Jan G; Hamann, Alf; Lengauer, Thomas; Rosenstiel, Philip; Walter, Jörn; Schulz, Marcel H

    2017-01-09

    The binding and contribution of transcription factors (TF) to cell specific gene expression is often deduced from open-chromatin measurements to avoid costly TF ChIP-seq assays. Thus, it is important to develop computational methods for accurate TF binding prediction in open-chromatin regions (OCRs). Here, we report a novel segmentation-based method, TEPIC, to predict TF binding by combining sets of OCRs with position weight matrices. TEPIC can be applied to various open-chromatin data, e.g. DNaseI-seq and NOMe-seq. Additionally, Histone-Marks (HMs) can be used to identify candidate TF binding sites. TEPIC computes TF affinities and uses open-chromatin/HM signal intensity as quantitative measures of TF binding strength. Using machine learning, we find low affinity binding sites to improve our ability to explain gene expression variability compared to the standard presence/absence classification of binding sites. Further, we show that both footprints and peaks capture essential TF binding events and lead to a good prediction performance. In our application, gene-based scores computed by TEPIC with one open-chromatin assay nearly reach the quality of several TF ChIP-seq data sets. Finally, these scores correctly predict known transcriptional regulators as illustrated by the application to novel DNaseI-seq and NOMe-seq data for primary human hepatocytes and CD4+ T-cells, respectively.

  19. Combining transcription factor binding affinities with open-chromatin data for accurate gene expression prediction

    Science.gov (United States)

    Schmidt, Florian; Gasparoni, Nina; Gasparoni, Gilles; Gianmoena, Kathrin; Cadenas, Cristina; Polansky, Julia K.; Ebert, Peter; Nordström, Karl; Barann, Matthias; Sinha, Anupam; Fröhler, Sebastian; Xiong, Jieyi; Dehghani Amirabad, Azim; Behjati Ardakani, Fatemeh; Hutter, Barbara; Zipprich, Gideon; Felder, Bärbel; Eils, Jürgen; Brors, Benedikt; Chen, Wei; Hengstler, Jan G.; Hamann, Alf; Lengauer, Thomas; Rosenstiel, Philip; Walter, Jörn; Schulz, Marcel H.

    2017-01-01

    The binding and contribution of transcription factors (TF) to cell specific gene expression is often deduced from open-chromatin measurements to avoid costly TF ChIP-seq assays. Thus, it is important to develop computational methods for accurate TF binding prediction in open-chromatin regions (OCRs). Here, we report a novel segmentation-based method, TEPIC, to predict TF binding by combining sets of OCRs with position weight matrices. TEPIC can be applied to various open-chromatin data, e.g. DNaseI-seq and NOMe-seq. Additionally, Histone-Marks (HMs) can be used to identify candidate TF binding sites. TEPIC computes TF affinities and uses open-chromatin/HM signal intensity as quantitative measures of TF binding strength. Using machine learning, we find low affinity binding sites to improve our ability to explain gene expression variability compared to the standard presence/absence classification of binding sites. Further, we show that both footprints and peaks capture essential TF binding events and lead to a good prediction performance. In our application, gene-based scores computed by TEPIC with one open-chromatin assay nearly reach the quality of several TF ChIP-seq data sets. Finally, these scores correctly predict known transcriptional regulators as illustrated by the application to novel DNaseI-seq and NOMe-seq data for primary human hepatocytes and CD4+ T-cells, respectively. PMID:27899623

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

    Science.gov (United States)

    Krainer, Georg; Keller, Sandro

    2015-04-01

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

  1. Binding Affinity of Glycoconjugates to BACILLUS Spores and Toxins

    Science.gov (United States)

    Rasol, Aveen; Eassa, Souzan; Tarasenko, Olga

    2010-04-01

    Early recognition of Bacillus cereus group species is important since they can cause food-borne illnesses and deadly diseases in humans. Glycoconjugates (GCs) are carbohydrates covalently linked to non-sugar moieties including lipids, proteins or other entities. GCs are involved in recognition and signaling processes intrinsic to biochemical functions in cells. They also stimulate cell-cell adhesion and subsequent recognition and activation of receptors. We have demonstrated that GCs are involved in Bacillus cereus spore recognition. In the present study, we have investigated whether GCs possess the ability to bind and recognize B. cereus spores and Bacillus anthracis recombinant single toxins (sTX) and complex toxins (cTX). The affinity of GCs to spores + sTX and spores + cTX toxins was studied in the binding essay. Our results demonstrated that GC9 and GC10 were able to selectively bind to B. cereus spores and B. anthracis toxins. Different binding affinities for GCs were found toward Bacillus cereus spores + sTX and spores + cTX. Dilution of GCs does not impede the recognition and binding. Developed method provides a tool for simultaneous recognition and targeting of spores, bacteria toxins, and/or other entities.

  2. Membrane Modulates Affinity for Calcium Ion to Create an Apparent Cooperative Binding Response by Annexin a5

    Science.gov (United States)

    Gauer, Jacob W.; Knutson, Kristofer J.; Jaworski, Samantha R.; Rice, Anne M.; Rannikko, Anika M.; Lentz, Barry R.; Hinderliter, Anne

    2013-01-01

    Isothermal titration calorimetry was used to characterize the binding of calcium ion (Ca2+) and phospholipid to the peripheral membrane-binding protein annexin a5. The phospholipid was a binary mixture of a neutral and an acidic phospholipid, specifically phosphatidylcholine and phosphatidylserine in the form of large unilamellar vesicles. To stringently define the mode of binding, a global fit of data collected in the presence and absence of membrane concentrations exceeding protein saturation was performed. A partition function defined the contribution of all heat-evolving or heat-absorbing binding states. We find that annexin a5 binds Ca2+ in solution according to a simple independent-site model (solution-state affinity). In the presence of phosphatidylserine-containing liposomes, binding of Ca2+ differentiates into two classes of sites, both of which have higher affinity compared with the solution-state affinity. As in the solution-state scenario, the sites within each class were described with an independent-site model. Transitioning from a solution state with lower Ca2+ affinity to a membrane-associated, higher Ca2+ affinity state, results in cooperative binding. We discuss how weak membrane association of annexin a5 prior to Ca2+ influx is the basis for the cooperative response of annexin a5 toward Ca2+, and the role of membrane organization in this response. PMID:23746516

  3. Measuring Equilibrium Binding Affinity of Biological Macromolecules in Solution by Thermophoresis

    Science.gov (United States)

    2015-05-18

    Approved for Public Release; Distribution Unlimited Final Report: Measuring Equilibrium Binding Affinity of Biological Macromolecules in Solution by...Equilibrium Binding Affinity of Biological Macromolecules in Solution by Thermophoresis Report Title The primary research focus of the San Diego State...equilibrium binding affinities of biological macromolecules in solution. This qauntitative information plays a vital role in supporting the static

  4. Differential ligand binding affinities of human estrogen receptor-α isoforms.

    Directory of Open Access Journals (Sweden)

    Amanda H Y Lin

    Full Text Available Rapid non-genomic effects of 17β-estradiol are elicited by the activation of different estrogen receptor-α isoforms. Presence of surface binding sites for estrogen have been identified in cells transfected with full-length estrogen receptor-α66 (ER66 and the truncated isoforms, estrogen receptor-α46 (ER46 and estrogen receptor-α36 (ER36. However, the binding affinities of the membrane estrogen receptors (mERs remain unknown due to the difficulty of developing of stable mER-transfected cell lines with sufficient mER density, which has largely hampered biochemical binding studies. The present study utilized cell-free expression systems to determine the binding affinities of 17β-estradiol to mERs, and the relationship among palmitoylation, membrane insertion and binding affinities. Saturation binding assays of human mERs revealed that [³H]-17β-estradiol bound ER66 and ER46 with Kd values of 68.81 and 60.72 pM, respectively, whereas ER36 displayed no specific binding within the tested concentration range. Inhibition of palmitoylation or removal of the nanolipoprotein particles, used as membrane substitute, reduced the binding affinities of ER66 and ER46 to 17β-estradiol. Moreover, ER66 and ER46 bound differentially with some estrogen receptor agonists and antagonists, and phytoestrogens. In particular, the classical estrogen receptor antagonist, ICI 182,780, had a higher affinity for ER66 than ER46. In summary, the present study defines the binding affinities for human estrogen receptor-α isoforms, and demonstrates that ER66 and ER46 show characteristics of mERs. The present data also indicates that palmitoylation and membrane insertion of mERs are important for proper receptor conformation allowing 17β-estradiol binding. The differential binding of ER66 and ER46 with certain compounds substantiates the prospect of developing mER-selective drugs.

  5. Characterization of high affinity binding motifs for the discoidin domain receptor DDR2 in collagen.

    Science.gov (United States)

    Konitsiotis, Antonios D; Raynal, Nicolas; Bihan, Dominique; Hohenester, Erhard; Farndale, Richard W; Leitinger, Birgit

    2008-03-14

    The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that are activated by native triple-helical collagen. Here we have located three specific DDR2 binding sites by screening the entire triple-helical domain of collagen II, using the Collagen II Toolkit, a set of overlapping triple-helical peptides. The peptide sequence that bound DDR2 with highest affinity interestingly contained the sequence for the high affinity binding site for von Willebrand factor in collagen III. Focusing on this sequence, we used a set of truncated and alanine-substituted peptides to characterize the sequence GVMGFO (O is hydroxyproline) as the minimal collagen sequence required for DDR2 binding. Based on a recent NMR analysis of the DDR2 collagen binding domain, we generated a model of the DDR2-collagen interaction that explains why a triple-helical conformation is required for binding. Triple-helical peptides comprising the DDR2 binding motif not only inhibited DDR2 binding to collagen II but also activated DDR2 transmembrane signaling. Thus, DDR2 activation may be effected by single triple-helices rather than fibrillar collagen.

  6. Determinants of benzodiazepine brain uptake: lipophilicity versus binding affinity.

    Science.gov (United States)

    Arendt, R M; Greenblatt, D J; Liebisch, D C; Luu, M D; Paul, S M

    1987-01-01

    Factors influencing brain uptake of benzodiazepine derivatives were evaluated in adult Sprague Dawley rats (n = 8-10 per drug). Animals received single intraperitoneal doses of alprazolam, triazolam, lorazepam, flunitrazepam, diazepam, midazolam, desmethyldiazepam, or clobazam. Concentrations of each drug (and metabolites) in whole brain and serum 1 h after dosage were determined by gas chromatography. Serum free fraction was measured by equilibrium dialysis. In vitro binding affinity (apparent Ki) of each compound was estimated based on displacement of tritiated flunitrazepam in washed membrane preparations from rat cerebral cortex. Lipid solubility of each benzodiazepine was estimated using the reverse-phase liquid chromatographic (HPLC) retention index at physiologic pH. There was no significant relation between brain:total serum concentration ratio and either HPLC retention (r = 0.18) or binding Ki (r = -0.34). Correction of uptake ratios for free as opposed to total serum concentration yielded a highly significant correlation with HPLC retention (r = 0.78, P less than 0.005). However, even the corrected ratio was not correlated with binding Ki (r = -0.22). Thus a benzodiazepine's capacity to diffuse from systemic blood into brain tissue is much more closely associated with the physicochemical property of lipid solubility than with specific affinity. Unbound rather than total serum or plasma concentration most accurately reflects the quantity of drug available for diffusion.

  7. The serotonin transporter: Examination of the changes in transporter affinity induced by ligand binding

    Energy Technology Data Exchange (ETDEWEB)

    Humphreys, C.J.

    1989-01-01

    The plasmalemmal serotonin transporter uses transmembrane gradients of Na{sup +}, Cl{sup {minus}} and K{sup +} to accumulate serotonin within blood platelets. Transport is competitively inhibited by the antidepressant imipramine. Like serotonin transport, imipramine binding requires Na{sup +}. Unlike serotonin, however, imipramine does not appear to be transported. To gain insight into the mechanism of serotonin transport the author have analyzed the influences of Na{sup +} and Cl{sup {minus}}, the two ions cotransported with serotonin, on both serotonin transport and the interaction of imipramine and other antidepressant drugs with the plasmalemmal serotonin transporter of human platelets. Additionally, the author have synthesized, purified and characterized the binding of 2-iodoimipramine to the serotonin transporter. Finally, the author have conducted a preliminary study of the inhibition of serotonin transport and imipramine binding produced by dicyclohexylcarbodiimide. My results reveal many instances of positive heterotropic cooperativity in ligand binding to the serotonin transporter. Na{sup +} binding enhances the transporters affinity for imipramine and several other antidepressant drugs, and also increases the affinity for Cl{sup {minus}}. Cl{sup {minus}} enhances the transporters affinity for imipramine, as well as for Na{sup +}. At concentrations in the range of its K{sub M} for transport serotonin is a competitive inhibitor of imipramine binding. At much higher concentrations, however, serotonin also inhibits imipramines dissociation rate constant. This latter effect which is Na{sup +}-independent and species specific, is apparently produced by serotonin binding at a second, low affinity site on, or near, the transporter complex. Iodoimipramine competitively inhibit both ({sup 3}H)imipramine binding and ({sup 3}H)serotonin transport.

  8. Modulating uranium binding affinity in engineered calmodulin EF-hand peptides: effect of phosphorylation.

    Directory of Open Access Journals (Sweden)

    Romain Pardoux

    Full Text Available To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T(9TKE(12 sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from K(d = 25±6 nM to K(d = 5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (K(d = 0.25±0.06 nM. FTIR analyses showed that the phosphothreonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the ν(as(P-O and ν(s(P-O IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in ν(as(UO(2(2+ vibration (from 923 cm(-1 to 908 cm(-1 was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH.

  9. Modulating Uranium Binding Affinity in Engineered Calmodulin EF-Hand Peptides: Effect of Phosphorylation

    Science.gov (United States)

    Pardoux, Romain; Sauge-Merle, Sandrine; Lemaire, David; Delangle, Pascale; Guilloreau, Luc; Adriano, Jean-Marc; Berthomieu, Catherine

    2012-01-01

    To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T9TKE12 sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from Kd = 25±6 nM to Kd = 5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (Kd = 0.25±0.06 nM). FTIR analyses showed that the phosphothreonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the νas(P-O) and νs(P-O) IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in νas(UO2)2+ vibration (from 923 cm−1 to 908 cm−1) was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH. PMID:22870263

  10. CpG dinucleotide positioning patterns determine the binding affinity of methyl-binding domain to nucleosomes.

    Science.gov (United States)

    Mendonca, Agnes; Sanchez, Oscar F; Liu, Wenjie; Li, Zhe; Yuan, Chongli

    2017-06-01

    The methyl-binding domain of MBD1 is a common methyl CpG binding motif and has been linked to transcriptional repression. Understanding the dynamics of MBD1 binding to nucleosomal DNA is crucial, but the molecular interactions between MBD1 and chromatin remain elusive. In this study, we found the binding of MBD1 to nucleosomes demonstrates sequence preferences depending on the position of the methyl groups on the nucleosome. Specifically, binding was favored at (me)CpG sites in the dyad proximal region and facing towards the histone octamers. At locations where the (me)CpG sites face away from the histone octamer, the binding affinity was significantly lower. Interestingly, the binding of ΔMBD1 at methylated CpG sites facing away from histone octamers induces conformational changes of nucleosomes, resulting in a more "open" conformation. The biological implication of DNA methylation is thus likely to be synergistically regulated via DNA sequences contents and their nucleosome-positioning patterns based on our in vitro findings. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. High-affinity binding of fungal beta-glucan fragments to soybean (Glycine max L.) microsomal fractions and protoplasts.

    Science.gov (United States)

    Cosio, E G; Pöpperl, H; Schmidt, W E; Ebel, J

    1988-08-01

    We have recently reported the existence of binding sites in soybean membranes for a beta-glucan fraction derived from the fungal pathogen Phytophthora megasperma f. sp. glycinea, which may play a role in the elicitor-mediated phytoalexin response of this plant [Schmidt, W. E. & Ebel, J. (1987) Proc. Natl Acad. Sci. USA 84, 4117-4121]. The specificity of beta-glucan binding to soybean membranes has now been investigated using a variety of competing polyglucans and oligoglucans of fungal origin. P. megasperma beta-glucan binding showed high apparent affinity for branched glucans with degrees of polymerization greater than 12. Binding affinity showed good correlation with elicitor activity as measured in a soybean cotyledon bioassay. Modification of the glucans at the reducing end with phenylalkylamine reagents had no effect on binding affinity. This characteristic was used to synthesize an oligoglucosyl tyramine derivative suitable for radioiodination. The 125I-glucan (15-30 Ci/mmol) provided higher sensitivity and lower detection limits for the binding assays while behaving in a manner identical to the [3H]glucan used previously. More accurate determinations of the Kd value for glucan binding indicated a higher affinity than previously shown (37 nM versus 200 nM). The 125I-glucan was used to provide the first reported evidence of specific binding of a fungal beta-glucan fraction in vivo to soybean protoplasts. The binding affinity to protoplasts proved identical to that found in microsomal fractions.

  12. Species-dependent binding of tocainide analogues to albumin: affinity chromatography and circular dichroism study.

    Science.gov (United States)

    Pistolozzi, Marco; Fortugno, Cecilia; Franchini, Carlo; Corbo, Filomena; Muraglia, Marilena; Roy, Myriam; Félix, Guy; Bertucci, Carlo

    2014-10-01

    A series of novel tocainide analogues were characterized for their HSA and RSA binding, by using high-performance liquid affinity chromatography (HPLAC) and circular dichroism (CD). In this HPLAC study, HSA and RSA were covalently immobilized to the silica matrix of HPLC columns, with a procedure that maintained unaltered the binding properties of the proteins. The tocainide analogues were ranked for their affinity to HSA and RSA on the basis of their bound fractions measured by the two albumin-based columns. This technique was also applied to characterize the high affinity binding sites of these tocainide analogues to the protein. For this purpose displacement experiments were carried out by means of increasing concentrations in the mobile phase of competitors known to bind selectively to the main binding sites of HSA. The results obtained with the immobilized proteins were confirmed by investigating the same drug-protein systems in solution by circular dichroism. The comparison of the data collected with both methodologies highlighted the dramatic effect of small differences in the amino acidic sequences of the two proteins. In fact, despite their similar primary and secondary structures, a small difference in the amino acidic sequence leads to significant differences in their three-dimensional structure reflecting their different binding capacity and their stereoselectivity. Therefore, this study confirms how it is crucial to consider the significant differences among the animal models when performing pharmacokinetic studies. It is also clear that the knowledge of serum carrier binding parameters at an early stage of drug discovery represents a great advantage that may help to save time and efforts. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2016-09-01

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

  14. Differential ligand binding affinities of human estrogen receptor-α isoforms

    OpenAIRE

    Amanda H.Y. Lin; Li, Rachel W. S.; Ho, Eva Y. W.; George P H Leung; Susan W S Leung; Paul M Vanhoutte; Man, Ricky Y K

    2013-01-01

    Rapid non-genomic effects of 17β-estradiol are elicited by the activation of different estrogen receptor-α isoforms. Presence of surface binding sites for estrogen have been identified in cells transfected with full-length estrogen receptor-α66 (ER66) and the truncated isoforms, estrogen receptor-α46 (ER46) and estrogen receptor-α36 (ER36). However, the binding affinities of the membrane estrogen receptors (mERs) remain unknown due to the difficulty of developing of stable mER-transfected cel...

  15. THE RECEPTOR BINDING AFFINITIES, ANTIPROGESTERONE AND ANTIGLUCOCORTICOID ACTIVITIES OF MIFEPRISTONE AND LILOPRISTONE

    Institute of Scientific and Technical Information of China (English)

    LIUYong-Qiang; WUXi-Rui

    1989-01-01

    With radioligand binding assays, the receptor binding affmities of mifepristone and lilopristone to the rabbit uterus cytosol progesterone receptor and the rat fiver cytosol glucocorticoid receptor have been measured. The relative binding affinities ( RBA ) of

  16. Cooperative binding of LysM domains determines the carbohydrate affinity of a bacterial endopeptidase protein.

    Science.gov (United States)

    Wong, Jaslyn E M M; Alsarraf, Husam M A B; Kaspersen, Jørn Døvling; Pedersen, Jan Skov; Stougaard, Jens; Thirup, Søren; Blaise, Mickaël

    2014-02-01

    Cellulose, chitin and peptidoglycan are major long-chain carbohydrates in living organisms, and constitute a substantial fraction of the biomass. Characterization of the biochemical basis of dynamic changes and degradation of these β,1-4-linked carbohydrates is therefore important for both functional studies of biological polymers and biotechnology. Here, we investigated the functional role of multiplicity of the carbohydrate-binding lysin motif (LysM) domain that is found in proteins involved in bacterial peptidoglycan synthesis and remodelling. The Bacillus subtilis peptidoglycan-hydrolysing NlpC/P60 D,L-endopeptidase, cell wall-lytic enzyme associated with cell separation, possesses four LysM domains. The contribution of each LysM domain was determined by direct carbohydrate-binding studies in aqueous solution with microscale thermophoresis. We found that bacterial LysM domains have affinity for N-acetylglucosamine (GlcNac) polymers in the lower-micromolar range. Moreover, we demonstrated that a single LysM domain is able to bind carbohydrate ligands, and that LysM domains act additively to increase the binding affinity. Our study reveals that affinity for GlcNAc polymers correlates with the chain length of the carbohydrate, and suggests that binding of long carbohydrates is mediated by LysM domain cooperativity. We also show that bacterial LysM domains, in contrast to plant LysM domains, do not discriminate between GlcNAc polymers, and recognize both peptidoglycan fragments and chitin polymers with similar affinity. Finally, an Ala replacement study suggested that the carbohydrate-binding site in LysM-containing proteins is conserved across phyla. © 2013 FEBS.

  17. Understanding enzymic binding affinity : thermodynamics of binding of benzamidinium chloride inhibitors to trypsin

    NARCIS (Netherlands)

    Talhout, Reinskje

    2003-01-01

    Understanding enzymic binding affinity is of fundamental scientific importance as well as a prerequisite for structure-based drug design. In this study, the interactions of the serine proteinase trypsin with several artificial, benzamidinium-based inhibitors have been studied in aqueous solutions. I

  18. 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.

  19. Preferential affinity of /sup 3/H-2-oxo-quazepam for type I benzodiazepine recognition sites in the human brain

    Energy Technology Data Exchange (ETDEWEB)

    Corda, M.G.; Giorgi, O.; Longoni, B.; Ongini, E.; Montaldo, S.; Biggio, G.

    1988-01-01

    The hypnotic drug quazepam and its active metabolite 2-oxo-quazepam (2-oxo-quaz) are two benzodiazepines (BZ) containing a trifluoroethyl moiety on the ring nitrogen at position 1, characterized by their preferential affinity for Type I BZ recognition sites. In the present study we characterized the binding of /sup 3/H-2-oxo-quaz in discrete areas of the human brain. Saturation analysis demonstrated specific and saturable binding of /sup 3/H-2-oxo-quaz to membrane preparations from human cerebellum. Hill plot analysis of displacement curves of /sup 3/H-flunitrazepam binding by 2-oxo-quaz yielded Hill coefficients of approximately 1 in the cerebellum and significantly less than 1 in the cerebral cortex, hippocampus, caudate nucleus, thalamus and pons. Self and cross displacement curves for /sup 3/H-FNT and /sup 3/H-2-oxo-quaz binding in these brain areas indicated that 2-oxo-quaz binds with different affinities to two populations of binding sites. High affinity binding sites were more abundant in the cerebellum, cerebral cortex, hippocampus and thalamus, whereas low affinity sites were predominant in the caudate nucleus and pons. Competition studies of /sup 3/H-2-oxo-quaz and /sup 3/H-FNT using unlabelled ligands indicated that compounds which preferentially bind to Type I sites are more potent at displacing /sup 3/H-2-oxo-quaz than /sup 3/H-FNT from cerebral cortex membrane preparations. 26 references, 2 figures, 3 tables.

  20. Autologous peptides constitutively occupy the antigen binding site on Ia

    DEFF Research Database (Denmark)

    Buus, S; Sette, A; Colon, S M;

    1988-01-01

    Low molecular weight material associated with affinity-purified class II major histocompatibility complex (MHC) molecules of mouse (Ia) had the expected properties of peptides bound to the antigen binding site of Ia. Thus, the low molecular weight material derived from the I-Ad isotype...

  1. Generation of high-performance binding proteins for peptide motifs by affinity clamping

    OpenAIRE

    Koide, Shohei; Huang, Jin

    2013-01-01

    We describe concepts and methodologies for generating “Affinity Clamps”, a new class of recombinant binding proteins that achieve high affinity and high specificity toward short peptide motifs of biological importance, which is a major challenge in protein engineering. The Affinity Clamping concept exploits the potential of nonhomologous recombination of protein domains in generating large changes in protein function and the inherent binding affinity and specificity of the so-called modular i...

  2. Synthesis and binding affinity of an iodinated juvenile hormone

    Energy Technology Data Exchange (ETDEWEB)

    Prestwich, G.D.; Eng, W.S.; Robles, S.; Vogt, R.G.; Wisniewski, J.R.; Wawrzenczyk, C.

    1988-01-25

    The synthesis of the first iodinated juvenile hormone (JH) in enantiomerically enriched form is reported. This chiral compound, 12-iodo-JH I, has an iodine atom replacing a methyl group of the natural insect juvenile hormone, JH I, which is important in regulating morphogenesis and reproduction in the Lepidoptera. The unlabeled compound shows approximately 10% of the relative binding affinity for the larval hemolymph JH binding protein (JHBP) of Manduca sexta, which specifically binds natural /sup 3/H-10R,11S-JH I (labeled at 58 Ci/mmol) with a KD of 8 X 10(-8) M. It is also approximately one-tenth as biologically active as JH I in the black Manduca and epidermal commitment assays. The 12-hydroxy and 12-oxo compounds are poor competitors and are also biologically inactive. The radioiodinated (/sup 125/I)12-iodo-JH I can be prepared in low yield at greater than 2500 Ci/mmol by nucleophilic displacement using no-carrier-added /sup 125/I-labeled sodium iodide in acetone; however, synthesis using sodium iodide carrier to give the approximately 50 Ci/mmol radioiodinated ligand proceeds in higher radiochemical yield with fewer by-products and provides a radioligand which is more readily handled in binding assays. The KD of (/sup 125/I)12-iodo-JH I was determined for hemolymph JHBP of three insects: M. sexta, 795 nM; Galleria mellonella, 47 nM; Locusta migratoria, 77 nM. The selectivity of 12-iodo-JH I for the 32-kDa JHBP of M. sexta was demonstrated by direct autoradiography of a native polyacrylamide gel electrophoresis gel of larval hemolymph incubated with the radioiodinated ligand. Thus, the in vitro and in vivo activity of 12-iodo-JH I indicate that it can serve as an important new gamma-emitting probe in the search for JH receptor proteins in target tissues.

  3. Mechanisms of in vivo binding site selection of the hematopoietic master transcription factor PU.1.

    Science.gov (United States)

    Pham, Thu-Hang; Minderjahn, Julia; Schmidl, Christian; Hoffmeister, Helen; Schmidhofer, Sandra; Chen, Wei; Längst, Gernot; Benner, Christopher; Rehli, Michael

    2013-07-01

    The transcription factor PU.1 is crucial for the development of many hematopoietic lineages and its binding patterns significantly change during differentiation processes. However, the 'rules' for binding or not-binding of potential binding sites are only partially understood. To unveil basic characteristics of PU.1 binding site selection in different cell types, we studied the binding properties of PU.1 during human macrophage differentiation. Using in vivo and in vitro binding assays, as well as computational prediction, we show that PU.1 selects its binding sites primarily based on sequence affinity, which results in the frequent autonomous binding of high affinity sites in DNase I inaccessible regions (25-45% of all occupied sites). Increasing PU.1 concentrations and the availability of cooperative transcription factor interactions during lineage differentiation both decrease affinity thresholds for in vivo binding and fine-tune cell type-specific PU.1 binding, which seems to be largely independent of DNA methylation. Occupied sites were predominantly detected in active chromatin domains, which are characterized by higher densities of PU.1 recognition sites and neighboring motifs for cooperative transcription factors. Our study supports a model of PU.1 binding control that involves motif-binding affinity, PU.1 concentration, cooperativeness with neighboring transcription factor sites and chromatin domain accessibility, which likely applies to all PU.1 expressing cells.

  4. Specific high-affinity binding of fatty acids to epidermal cytosolic proteins

    Energy Technology Data Exchange (ETDEWEB)

    Raza, H.; Chung, W.L.; Mukhtar, H. (Department of Dermatology, University Hospitals of Cleveland, Case Western Reserve University, OH (USA))

    1991-08-01

    Cytosol from rat, mouse, and human skin or rat epidermis was incubated with (3H)arachidonic acid, (14C)retinoic acid, (14C)oleic acid, (3H)leukotriene A4, (3H)prostaglandin E2 (PGE2) or (3H) 15-hydroxyeicosatetraenoic acid (15-HETE), and protein-bound ligands were separated using Lipidex-1000 at 4C to assess the binding specificity. The binding of oleic acid and arachidonic acid with rat epidermal cytosol was rapid, saturable, and reversible. Binding of oleic acid was competed out with the simultaneous addition of other ligands and found to be in the following order: arachidonic acid greater than oleic acid greater than linoleic acid greater than lauric acid greater than leukotriene A4 greater than 15-HETE = PGE1 greater than PGE2 = PGF2. Scatchard analysis of the binding with arachidonic acid, oleic acid, and retinoic acid revealed high-affinity binding sites with the dissociation constant in the nM range. SDS-PAGE analysis of the oleic acid-bound epidermal cytosolic protein(s) revealed maximum binding at the 14.5 kDa region. The presence of the fatty acid-binding protein in epidermal cytosol and its binding to fatty acids and retinoic acid may be of significance both in the trafficking and the metabolism of fatty acids and retinoids across the skin.

  5. Crystallographic analysis reveals the structural basis of the high-affinity binding of iophenoxic acid to human serum albumin.

    Science.gov (United States)

    Ryan, Ali J; Chung, Chun-Wa; Curry, Stephen

    2011-04-18

    Iophenoxic acid is an iodinated radiocontrast agent that was withdrawn from clinical use because of its exceptionally long half-life in the body, which was due in part to its high-affinity binding to human serum albumin (HSA). It was replaced by Iopanoic acid, which has an amino rather than a hydroxyl group at position 3 on the iodinated benzyl ring and, as a result, binds to albumin with lower affinity and is excreted more rapidly from the body. To understand how iophenoxic acid binds so tightly to albumin, we wanted to examine the structural basis of its interaction with HSA. We have determined the co-crystal structure of HSA in complex with iophenoxic acid at 2.75 Å resolution, revealing a total of four binding sites, two of which--in drugs sites 1 and 2 on the protein--are likely to be occupied at clinical doses. High-affinity binding of iophenoxic acid occurs at drug site 1. The structure reveals that polar and apolar groups on the compound are involved in its interactions with drug site 1. In particular, the 3-hydroxyl group makes three hydrogen bonds with the side-chains of Tyr 150 and Arg 257. The mode of binding to drug site 2 is similar except for the absence of a binding partner for the hydroxyl group on the benzyl ring of the compound. The HSA-iophenoxic acid structure indicates that high-affinity binding to drug site 1 is likely to be due to extensive desolvation of the compound, coupled with the ability of the binding pocket to provide a full set of salt-bridging or hydrogen bonding partners for its polar groups. Consistent with this interpretation, the structure also suggests that the lower-affinity binding of iopanoic acid arises because replacement of the 3-hydroxyl by an amino group eliminates hydrogen bonding to Arg 257. This finding underscores the importance of polar interactions in high-affinity binding to albumin.

  6. Crystallographic analysis reveals the structural basis of the high-affinity binding of iophenoxic acid to human serum albumin

    Directory of Open Access Journals (Sweden)

    Chung Chun-wa

    2011-04-01

    Full Text Available Abstract Background Iophenoxic acid is an iodinated radiocontrast agent that was withdrawn from clinical use because of its exceptionally long half-life in the body, which was due in part to its high-affinity binding to human serum albumin (HSA. It was replaced by Iopanoic acid, which has an amino rather than a hydroxyl group at position 3 on the iodinated benzyl ring and, as a result, binds to albumin with lower affinity and is excreted more rapidly from the body. To understand how iophenoxic acid binds so tightly to albumin, we wanted to examine the structural basis of its interaction with HSA. Results We have determined the co-crystal structure of HSA in complex with iophenoxic acid at 2.75 Å resolution, revealing a total of four binding sites, two of which - in drugs sites 1 and 2 on the protein - are likely to be occupied at clinical doses. High-affinity binding of iophenoxic acid occurs at drug site 1. The structure reveals that polar and apolar groups on the compound are involved in its interactions with drug site 1. In particular, the 3-hydroxyl group makes three hydrogen bonds with the side-chains of Tyr 150 and Arg 257. The mode of binding to drug site 2 is similar except for the absence of a binding partner for the hydroxyl group on the benzyl ring of the compound. Conclusions The HSA-iophenoxic acid structure indicates that high-affinity binding to drug site 1 is likely to be due to extensive desolvation of the compound, coupled with the ability of the binding pocket to provide a full set of salt-bridging or hydrogen bonding partners for its polar groups. Consistent with this interpretation, the structure also suggests that the lower-affinity binding of iopanoic acid arises because replacement of the 3-hydroxyl by an amino group eliminates hydrogen bonding to Arg 257. This finding underscores the importance of polar interactions in high-affinity binding to albumin.

  7. 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.

  8. In vivo affinity label of a protein expressed in Escherichia coli. Coenzyme A occupied the AT(D)P binding site of the mutant F1-ATPase beta subunit (Y307C) through a disulfide bond.

    Science.gov (United States)

    Odaka, M; Kiribuchi, K; Allison, W S; Yoshida, M

    1993-12-27

    When Tyr-307 of the beta subunit of F1-ATPase from a thermophilic Bacillus strain PS3 is replaced by cysteine and expressed in Escherichia coli cells, about a half population of the mutant beta subunit are labeled by Coenzyme A at Cys-307 through a disulfide bond which is cleavable by reducing treatment. The mutant beta subunit can be reconstituted into the alpha 3 beta 3 complex of which ATPase activity is stimulated two-fold by reducing treatment either prior or after reconstitution. Since Tyr-307 has been supposed to be located at one of subdomains which form the ATP binding site of the beta subunit, Coenzyme A binds to the mutant beta subunit as an AT(D)P analogue in E. coli cells and then covalently attaches to Cys-307.

  9. Exploring binding affinity of oxaliplatin and carboplatin, to nucleoprotein structure of chromatin: spectroscopic study and histone proteins as a target.

    Science.gov (United States)

    Soori, Hosna; Rabbani-Chadegani, Azra; Davoodi, Jamshid

    2015-01-07

    Platinum drugs are potent chemotherapeutic agents widely used in cancer therapy. They exert their biological activity by binding to DNA, producing DNA adducts; however, in the cell nucleus, DNA is complexed with histone proteins into a nucleoprotein structure known as chromatin. The aim of this study was to explore the binding affinity of oxaliplatin and carboplatin to chromatin using spectroscopic as well as thermal denaturation and equilibrium dialysis techniques. The results showed that the drugs quenched with chromophores of chromatin and the quenching effect for oxaliplatin (Ksv = 3.156) was higher than carboplatin (Ksv = 0.28). The binding of the drugs exhibited hypochromicity both in thermal denaturation profiles and UV absorbance at 210 nm. The binding was positive cooperation with spontaneous reaction and oxaliplatin (Ka = 5.3 × 10(3) M(-1), n = 1.7) exhibited higher binding constant and number of binding sites than carboplatin (Ka = 0.33 × 10(3) M(-1), n = 1.0) upon binding to chromatin. Also secondary structure of chromatin proteins was altered upon drugs binding. It is concluded that oxaliplatin represents higher binding affinity to chromatin compared to carboplatin. In chromatin where DNA is compacted into nucleosomes structure with histones, the affinity of the platinated drugs is reduced and histone proteins may play a fundamental role in this binding process. Copyright © 2014. Published by Elsevier Masson SAS.

  10. Quinine binding by the cocaine-binding aptamer. Thermodynamic and hydrodynamic analysis of high-affinity binding of an off-target ligand.

    Science.gov (United States)

    Reinstein, Oren; Yoo, Mina; Han, Chris; Palmo, Tsering; Beckham, Simone A; Wilce, Matthew C J; Johnson, Philip E

    2013-12-03

    The cocaine-binding aptamer is unusual in that it tightly binds molecules other than the ligand it was selected for. Here, we study the interaction of the cocaine-binding aptamer with one of these off-target ligands, quinine. Isothermal titration calorimetry was used to quantify the quinine-binding affinity and thermodynamics of a set of sequence variants of the cocaine-binding aptamer. We find that the affinity of the cocaine-binding aptamer for quinine is 30-40 times stronger than it is for cocaine. Competitive-binding studies demonstrate that both quinine and cocaine bind at the same site on the aptamer. The ligand-induced structural-switching binding mechanism of an aptamer variant that contains three base pairs in stem 1 is retained with quinine as a ligand. The short stem 1 aptamer is unfolded or loosely folded in the free form and becomes folded when bound to quinine. This folding is confirmed by NMR spectroscopy and by the short stem 1 construct having a more negative change in heat capacity of quinine binding than is seen when stem 1 has six base pairs. Small-angle X-ray scattering (SAXS) studies of the free aptamer and both the quinine- and the cocaine-bound forms show that, for the long stem 1 aptamers, the three forms display similar hydrodynamic properties, and the ab initio shape reconstruction structures are very similar. For the short stem 1 aptamer there is a greater variation among the SAXS-derived ab initio shape reconstruction structures, consistent with the changes expected with its structural-switching binding mechanism.

  11. Specific Internalisation of Gold Nanoparticles into Engineered Porous Protein Cages via Affinity Binding

    Science.gov (United States)

    Peng, Tao; Free, Paul; Fernig, David G.; Lim, Sierin; Tomczak, Nikodem

    2016-01-01

    Porous protein cages are supramolecular protein self-assemblies presenting pores that allow the access of surrounding molecules and ions into their core in order to store and transport them in biological environments. Protein cages’ pores are attractive channels for the internalisation of inorganic nanoparticles and an alternative for the preparation of hybrid bioinspired nanoparticles. However, strategies based on nanoparticle transport through the pores are largely unexplored, due to the difficulty of tailoring nanoparticles that have diameters commensurate with the pores size and simultaneously displaying specific affinity to the cages’ core and low non-specific binding to the cages’ outer surface. We evaluated the specific internalisation of single small gold nanoparticles, 3.9 nm in diameter, into porous protein cages via affinity binding. The E2 protein cage derived from the Geobacillus stearothermophilus presents 12 pores, 6 nm in diameter, and an empty core of 13 nm in diameter. We engineered the E2 protein by site-directed mutagenesis with oligohistidine sequences exposing them into the cage’s core. Dynamic light scattering and electron microscopy analysis show that the structures of E2 protein cages mutated with bis- or penta-histidine sequences are well conserved. The surface of the gold nanoparticles was passivated with a self-assembled monolayer made of a mixture of short peptidols and thiolated alkane ethylene glycol ligands. Such monolayers are found to provide thin coatings preventing non-specific binding to proteins. Further functionalisation of the peptide coated gold nanoparticles with Ni2+ nitrilotriacetic moieties enabled the specific binding to oligohistidine tagged cages. The internalisation via affinity binding was evaluated by electron microscopy analysis. From the various mutations tested, only the penta-histidine mutated E2 protein cage showed repeatable and stable internalisation. The present work overcomes the limitations of

  12. Asparagine deamidation reduces DNA-binding affinity of the Drosophila melanogaster Scr homeodomain.

    Science.gov (United States)

    O'Connell, Nichole E; Lelli, Katherine; Mann, Richard S; Palmer, Arthur G

    2015-10-24

    Spontaneous deamidation of asparagine is a non-enzymatic post-translational modification of proteins. Residue Asn 321 is the main site of deamidation of the Drosophila melanogaster Hox transcription factor Sex Combs Reduced (Scr). Formation of iso-aspartate, the major deamidation product, is detected by HNCACB triple-resonance NMR spectroscopy. The rate of deamidation is quantified by fitting the decay of Asn NH2 side-chain signals in a time-series of (15)N-(1)H HSQC NMR spectra. The deamidated form of Scr binds to specific DNA target sequences with reduced affinity as determined by an electrophoretic mobility shift assay.

  13. Neurotensin decreases high affinity [3H]-ouabain binding to cerebral cortex membranes.

    Science.gov (United States)

    Rosin, Carina; Ordieres, María Graciela López; Arnaiz, Georgina Rodríguez de Lores

    2011-12-10

    Previous work from this laboratory showed the ability of neurotensin to inhibit synaptosomal membrane Na(+), K(+)-ATPase activity, the effect being blocked by SR 48692, a non-peptidic antagonist for high affinity neurotensin receptor (NTS1) [López Ordieres and Rodríguez de Lores Arnaiz 2000; 2001]. To further study neurotensin interaction with Na(+), K(+)-ATPase, peptide effect on high affinity [(3)H]-ouabain binding was studied in cerebral cortex membranes. It was observed that neurotensin modified binding in a dose-dependent manner, leading to 80% decrease with 1 × 10(-4)M concentration. On the other hand, the single addition of 1 × 10(-6)M, 1 × 10(-5)M and 1 × 10(-4)M SR 48692 (Sanofi-Aventis, U.S., Inc.) decreased [(3)H]-ouabain binding (in %) to 87 ± 16; 74 ± 16 and 34 ± 17, respectively. Simultaneous addition of neurotensin and SR 48692 led to additive or synergic effects. Partial NTS2 agonist levocabastine inhibited [(3)H]-ouabain binding likewise. Saturation assays followed by Scatchard analyses showed that neurotensin increased K(d) value whereas failed to modify B(max) value, indicating a competitive type interaction of the peptide at Na(+), K(+)-ATPase ouabain site. At variance, SR 48692 decreased B(max) value whereas it did not modify K(d) value. [(3)H]-ouabain binding was also studied in cerebral cortex membranes obtained from rats injected i. p. 30 min earlier with 100 μg and 250 μg/kg SR 48692. It was observed that the 250 μg/kg SR 48692 dose led to 19% decrease in basal [(3)H]-ouabain binding. After SR 48692 treatments, addition of 1 × 10(-6)M led to additive or synergic effect. Results suggested that [(3)H]-ouabain binding inhibition by neurotensin hardly involves NTS1 receptor.

  14. Experimental conditions can obscure the second high-affinity site in LeuT.

    Science.gov (United States)

    Quick, Matthias; Shi, Lei; Zehnpfennig, Britta; Weinstein, Harel; Javitch, Jonathan A

    2012-01-15

    Neurotransmitter:Na(+) symporters (NSSs), the targets of antidepressants and psychostimulants, recapture neurotransmitters from the synapse in a Na(+)-dependent symport mechanism. The crystal structure of the NSS homolog LeuT from Aquifex aeolicus revealed one leucine substrate in an occluded, centrally located (S1) binding site next to two Na(+) ions. Computational studies combined with binding and flux experiments identified a second substrate (S2) site and a molecular mechanism of Na(+)-substrate symport that depends upon the allosteric interaction of substrate molecules in the two high-affinity sites. Here we show that the S2 site, which has not yet been identified by crystallographic approaches, can be blocked during preparation of detergent-solubilized LeuT, thereby obscuring its crucial role in Na(+)-coupled symport. This finding points to the need for caution in selecting experimental environments in which the properties and mechanistic features of membrane proteins can be delineated.

  15. There is communication between all four Ca(2+)-bindings sites of calcineurin B.

    Science.gov (United States)

    Gallagher, S C; Gao, Z H; Li, S; Dyer, R B; Trewhella, J; Klee, C B

    2001-10-09

    We have used site-directed mutagenesis, flow dialysis, and Fourier transform infrared (FTIR) spectroscopy to study Ca(2+)-binding to the regulatory component of calcineurin. Single Glu-Gln(E --> Q) mutations were used to inactivate each of the four Ca(2+)-binding sites of CnB in turn, generating mutants Q1, Q2, Q3, and Q4, with the number indicating which Ca(2+) site is inactivated. The binding data derived from flow dialysis reveal two pairs of sites in the wild-type protein, one pair with very high affinity and the other with lower affinity Ca(2+)-binding sites. Also, only three sites are titratable in the wild-type protein because one site cannot be decalcified. Mutation of site 2 leaves the protein with only two titratable sites, while mutation of sites 1, 3, or 4 leave three titratable sites that are mostly filled with 3 Ca(2+) equiv added. The binding data further show that each of the single-site mutations Q2, Q3, and Q4 affects the affinities of at least one of the remaining sites. Mutation in either of sites 3 or 4 results in a protein with no high-affinity sites, indicating communication between the two high-affinity sites, most likely sites 3 and 4. Mutation in site 2 decreases the affinity of all three remaining sites, though still leaving two relatively high-affinity sites. The FTIR data support the conclusions from the binding data with respect to the number of titratable sites as well as the impact of each mutation on the affinities of the remaining sites. We conclude therefore that there is communication between all four Ca(2+)-binding sites. In addition, the Ca(2+) induced changes in the FTIR spectra for the wild-type and Q4 mutant are most similar, suggesting that the same three Ca(2+)-binding sites are being titrated, i.e., site 4 is the very high-affinity site under the conditions of the FTIR experiments.

  16. Grafting of protein-protein binding sites

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A strategy for grafting protein-protein binding sites is described. Firstly, key interaction residues at the interface of ligand protein to be grafted are identified and suitable positions in scaffold protein for grafting these key residues are sought. Secondly, the scaffold proteins are superposed onto the ligand protein based on the corresponding Ca and Cb atoms. The complementarity between the scaffold protein and the receptor protein is evaluated and only matches with high score are accepted. The relative position between scaffold and receptor proteins is adjusted so that the interface has a reasonable packing density. Then the scaffold protein is mutated to corresponding residues in ligand protein at each candidate position. And the residues having bad steric contacts with the receptor proteins, or buried charged residues not involved in the formation of any salt bridge are mutated. Finally, the mutated scaffold protein in complex with receptor protein is co-minimized by Charmm. In addition, we deduce a scoring function to evaluate the affinity between mutated scaffold protein and receptor protein by statistical analysis of rigid binding data sets.

  17. A novel non-opioid binding site for endomorphin-1.

    Science.gov (United States)

    Lengyel, I; Toth, F; Biyashev, D; Szatmari, I; Monory, K; Tomboly, C; Toth, G; Benyhe, S; Borsodi, A

    2016-08-01

    Endomorphins are natural amidated opioid tetrapeptides with the following structure: Tyr-Pro-Trp-Phe-NH2 (endomorphin-1), and Tyr-Pro-Phe-Phe-NH2 (endomorphin-2). Endomorphins interact selectively with the μ-opioid or MOP receptors and exhibit nanomolar or sub-nanomolar receptor binding affinities, therefore they suggested to be endogenous agonists for the μ-opioid receptors. Endomorphins mediate a number of characteristic opioid effects, such as antinociception, however there are several physiological functions in which endomorphins appear to act in a fashion that does not involve binding to and activation of the μ-opioid receptor. Our recent data indicate that a radiolabelled [(3)H]endomorphin-1 with a specific radioactivity of 2.35 TBq/mmol - prepared by catalytic dehalogenation of the diiodinated peptide precursor in the presence of tritium gas - is able to bind to a second, naloxone insensitive recognition site in rat brain membranes. Binding heterogeneity, i.e., the presence of higher (Kd = 0.4 nM / Bmax = 120 fmol/mg protein) and lower (Kd = 8.2 nM / Bmax = 432 fmol/mg protein) affinity binding components is observed both in saturation binding experiments followed by Schatchard analysis, and in equilibrium competition binding studies. The signs of receptor multiplicity, e.g., curvilinear Schatchard plots or biphasic displacement curves are seen only if the non-specific binding is measured in the presence of excess unlabeled endomorphin-1 and not in the presence of excess unlabeled naloxone. The second, lower affinity non-opioid binding site is not recognized by heterocyclic opioid alkaloid ligands, neither agonists such as morphine, nor antagonists such as naloxone. On the contrary, endomorphin-1 is displaced from its lower affinity, higher capacity binding site by several natural neuropeptides, including methionine-enkephalin-Arg-Phe, nociceptin-orphanin FQ, angiotensin and FMRF-amide. This naloxone-insensitive, consequently non-opioid binding site seems

  18. HDAC Inhibitors without an Active Site Zn2+-Binding Group

    DEFF Research Database (Denmark)

    Vickers, Chris J.; Olsen, Christian Adam; Leman, Luke J.

    2012-01-01

    Natural and synthetic histone deacetylase (HDAC) inhibitors generally derive their strong binding affinity and high potency from a key functional group that binds to the Zn2+ ion within the enzyme active site. However, this feature is also thought to carry the potential liability of undesirable off......-target interactions with other metalloenzymes. As a step toward mitigating this issue, here, we describe the design, synthesis, and structure−activity characterizations of cyclic α3β-tetrapeptide HDAC inhibitors that lack the presumed indispensable Zn2+-binding group. The lead compounds (e.g., 15 and 26) display good...

  19. Bifunctional avidin with covalently modifiable ligand binding site.

    Directory of Open Access Journals (Sweden)

    Jenni Leppiniemi

    Full Text Available The extensive use of avidin and streptavidin in life sciences originates from the extraordinary tight biotin-binding affinity of these tetrameric proteins. Numerous studies have been performed to modify the biotin-binding affinity of (streptavidin to improve the existing applications. Even so, (streptavidin greatly favours its natural ligand, biotin. Here we engineered the biotin-binding pocket of avidin with a single point mutation S16C and thus introduced a chemically active thiol group, which could be covalently coupled with thiol-reactive molecules. This approach was applied to the previously reported bivalent dual chain avidin by modifying one binding site while preserving the other one intact. Maleimide was then coupled to the modified binding site resulting in a decrease in biotin affinity. Furthermore, we showed that this thiol could be covalently coupled to other maleimide derivatives, for instance fluorescent labels, allowing intratetrameric FRET. The bifunctional avidins described here provide improved and novel tools for applications such as the biofunctionalization of surfaces.

  20. SiteOut: An Online Tool to Design Binding Site-Free DNA Sequences.

    Directory of Open Access Journals (Sweden)

    Javier Estrada

    Full Text Available DNA-binding proteins control many fundamental biological processes such as transcription, recombination and replication. A major goal is to decipher the role that DNA sequence plays in orchestrating the binding and activity of such regulatory proteins. To address this goal, it is useful to rationally design DNA sequences with desired numbers, affinities and arrangements of protein binding sites. However, removing binding sites from DNA is computationally non-trivial since one risks creating new sites in the process of deleting or moving others. Here we present an online binding site removal tool, SiteOut, that enables users to design arbitrary DNA sequences that entirely lack binding sites for factors of interest. SiteOut can also be used to delete sites from a specific sequence, or to introduce site-free spacers between functional sequences without creating new sites at the junctions. In combination with commercial DNA synthesis services, SiteOut provides a powerful and flexible platform for synthetic projects that interrogate regulatory DNA. Here we describe the algorithm and illustrate the ways in which SiteOut can be used; it is publicly available at https://depace.med.harvard.edu/siteout/.

  1. SiteOut: An Online Tool to Design Binding Site-Free DNA Sequences.

    Science.gov (United States)

    Estrada, Javier; Ruiz-Herrero, Teresa; Scholes, Clarissa; Wunderlich, Zeba; DePace, Angela H

    2016-01-01

    DNA-binding proteins control many fundamental biological processes such as transcription, recombination and replication. A major goal is to decipher the role that DNA sequence plays in orchestrating the binding and activity of such regulatory proteins. To address this goal, it is useful to rationally design DNA sequences with desired numbers, affinities and arrangements of protein binding sites. However, removing binding sites from DNA is computationally non-trivial since one risks creating new sites in the process of deleting or moving others. Here we present an online binding site removal tool, SiteOut, that enables users to design arbitrary DNA sequences that entirely lack binding sites for factors of interest. SiteOut can also be used to delete sites from a specific sequence, or to introduce site-free spacers between functional sequences without creating new sites at the junctions. In combination with commercial DNA synthesis services, SiteOut provides a powerful and flexible platform for synthetic projects that interrogate regulatory DNA. Here we describe the algorithm and illustrate the ways in which SiteOut can be used; it is publicly available at https://depace.med.harvard.edu/siteout/.

  2. The N-terminal domain determines the affinity and specificity of H1 binding to chromatin

    Energy Technology Data Exchange (ETDEWEB)

    Oeberg, Christine, E-mail: christine.oberg@ki.se [Karolinska Institute, Department of Cell and Molecular Biology, P.O. Box 285, SE-17177 Stockholm (Sweden); Belikov, Sergey, E-mail: sergey.belikov@ki.se [Karolinska Institute, Department of Cell and Molecular Biology, P.O. Box 285, SE-17177 Stockholm (Sweden)

    2012-04-06

    Highlights: Black-Right-Pointing-Pointer wt Human histone H1.4 and hH1.4 devoid of N-terminal domain, {Delta}N-hH1.4, were compared. Black-Right-Pointing-Pointer Both histones bind to chromatin, however, {Delta}N-hH1.4 displays lower binding affinity. Black-Right-Pointing-Pointer Interaction of {Delta}N-hH1.4 with chromatin includes a significant unspecific component. Black-Right-Pointing-Pointer N-terminal domain is a determinant of specificity of histone H1 binding to chromatin. -- Abstract: Linker histone H1, one of the most abundant nuclear proteins in multicellular eukaryotes, is a key component of the chromatin structure mainly due to its role in the formation and maintenance of the 30 nm chromatin fiber. It has a three-domain structure; a central globular domain flanked by a short N-terminal domain and a long, highly basic C-terminal domain. Previous studies have shown that the binding abilities of H1 are at large determined by the properties of the C-terminal domain; much less attention has been paid to role of the N-terminal domain. We have previously shown that H1 can be reconstituted via cytoplasmic mRNA injection in Xenopus oocytes, cells that lack somatic H1. The heterologously expressed H1 proteins are incorporated into in vivo assembled chromatin at specific sites and the binding event is monitored as an increase in nucleosomal repeat length (NRL). Using this setup we have here compared the binding properties of wt-H1.4 and hH1.4 devoid of its N-terminal domain ({Delta}N-hH1.4). The {Delta}N-hH1.4 displays a drastically lower affinity for chromatin binding as compared to the wild type hH1.4. Our data also indicates that {Delta}N-hH1.4 is more prone to unspecific chromatin binding than the wild type. We conclude that the N-terminal domain of H1 is an important determinant of affinity and specificity of H1-chromatin interactions.

  3. Evidence for a non-opioid sigma binding site din the guinea-pig myenteric plexus

    Energy Technology Data Exchange (ETDEWEB)

    Roman, F.; Pascaud, X.; Vauche, D.; Junien, J.

    1988-01-01

    The presence of a binding site to (+)-(/sup 3/H)SKF 10,047 was demonstrated in a guinea-pig myenteric plexus (MYP) membrane preparation. Specific binding to this receptor was saturable, reversible, linear with protein concentration and consisted of two components, a high affinity site and a low affinity site. Morphine and naloxone 10/sup -4/M were unable to displace (+)-(/sup 3/H)SKF 10,047 binding. Haloperidol, imipramine, ethylketocyclazocine and propranolol were among the most potent compounds to inhibit this specific binding. These results suggest the presence of a non-opioid haloperidol sensitive sigma receptor in the MYP of the guinea-pig.

  4. Affinity Probe Capillary Electrophoresis Evaluation of Aptamer Binding to Campylobacter jejuni Bacteria

    Science.gov (United States)

    2009-11-01

    Affinity Probe Capillary Electrophoresis Evaluation of Aptamer Binding to Campylobacter jejuni Bacteria by Dimitra N. Stratis-Cullum, Sun...Aptamer Binding to Campylobacter jejuni Bacteria Dimitra N. Stratis-Cullum, Sun McMasters, and Paul M. Pellegrino Sensors and Electron Devices...To) 2007–2008 4. TITLE AND SUBTITLE Affinity Probe Capillary Electrophoresis Evaluation of Aptamer Binding to Campylobacter jejuni Bacteria 5a

  5. Adaptive evolution of transcription factor binding sites

    Directory of Open Access Journals (Sweden)

    Berg Johannes

    2004-10-01

    Full Text Available Abstract Background The regulation of a gene depends on the binding of transcription factors to specific sites located in the regulatory region of the gene. The generation of these binding sites and of cooperativity between them are essential building blocks in the evolution of complex regulatory networks. We study a theoretical model for the sequence evolution of binding sites by point mutations. The approach is based on biophysical models for the binding of transcription factors to DNA. Hence we derive empirically grounded fitness landscapes, which enter a population genetics model including mutations, genetic drift, and selection. Results We show that the selection for factor binding generically leads to specific correlations between nucleotide frequencies at different positions of a binding site. We demonstrate the possibility of rapid adaptive evolution generating a new binding site for a given transcription factor by point mutations. The evolutionary time required is estimated in terms of the neutral (background mutation rate, the selection coefficient, and the effective population size. Conclusions The efficiency of binding site formation is seen to depend on two joint conditions: the binding site motif must be short enough and the promoter region must be long enough. These constraints on promoter architecture are indeed seen in eukaryotic systems. Furthermore, we analyse the adaptive evolution of genetic switches and of signal integration through binding cooperativity between different sites. Experimental tests of this picture involving the statistics of polymorphisms and phylogenies of sites are discussed.

  6. Guanine Nucleotides Modulate Cell Surface cAMP-Binding Sites in Membranes from Dictyostelium discoideum

    NARCIS (Netherlands)

    Haastert, Peter J.M. van

    1984-01-01

    D. discoideum contains kinetically distinguishable cell surface cAMP binding sites. One class, S, is slowly dissociating and has high affinity for cAMP (Kd = 15 nM, t½ = 15 s). A second class is fast dissociating (t½ about 1 s) and is composed of high affinity binding sites H (Kd ≈ 60 nM), and low a

  7. Synthesis and receptor binding affinity of new selective GluR5 ligands

    DEFF Research Database (Denmark)

    Bunch, L; Johansen, T H; Bräuner-Osborne, Hans;

    2001-01-01

    Two hybrid analogues of the kainic acid receptor agonists, 2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA) and (2S,4R)-4-methylglutamic acid ((2S,4R)-4-Me-Glu), were designed, synthesized, and characterized in radioligand binding assays using cloned ionotropic and metabotropi.......0 and 2.0 microM. respectively. Their affinities in the [3H]AMPA binding assay on native cortical receptors were shown to correlate with their GluR2 affinity rather than their GluR5 affinity. No affinity for GluR6 was detected (IC50 > 100 microM)....

  8. Statistics for Transcription Factor Binding Sites

    OpenAIRE

    2008-01-01

    Transcription factors (TFs) play a key role in gene regulation. They interact with specific binding sites or motifs on the DNA sequence and regulate expression of genes downstream of these binding sites. In silico prediction of potential binding of a TF to a binding site is an important task in computational biology. From a statistical point of view, the DNA sequence is a long text consisting of four different letters ('A','C','G', and 'T'). The binding of a TF to the sequence corresponds to ...

  9. Production and Characterization of Desmalonichrome Relative Binding Affinity for Uranyl Ions in Relation to Other Siderophores

    Energy Technology Data Exchange (ETDEWEB)

    Mo, Kai-For; Dai, Ziyu; Wunschel, David S.

    2016-06-24

    Siderophores are Fe binding secondary metabolites that have been investigated for their uranium binding properties. Much of the previous work has focused on characterizing hydroxamate types of siderophores, such as desferrioxamine B, for their uranyl binding affinity. Carboxylate forms of these metabolites hold potential to be more efficient chelators of uranyl, yet they have not been widely studied and are more difficult to obtain. Desmalonichrome is a carboxylate siderophore which is not commercially available and so was obtained from the ascomycete fungus Fusarium oxysporum cultivated under Fe depleted conditions. The relative affinity for uranyl binding of desmalonichrome was investigated using a competitive analysis of binding affinities between uranyl acetate and different concentrations of iron(III) chloride using electrospray ionization mass spectrometry (ESI-MS). In addition to desmalonichrome, three other siderophores, including two hydroxamates (desferrioxamine B and desferrichrome) and one carboxylate (desferrichrome A) were studied to understand their relative affinities for the uranyl ion at two pH values. The binding affinities of hydroxymate siderophores to uranyl ion were found to decrease to a greater degree at lower pH as the concentration of Fe (III) ion increases. On the other hand, lowering pH has little impact on the binding affinities between carboxylate siderophores and uranyl ion. Desmalonichrome was shown to have the greatest relative affinity for uranyl at any pH and Fe(III) concentration. These results suggest that acidic functional groups in the ligands are critical for strong chelation with uranium at lower pH.

  10. A protein engineered to bind uranyl selectively and with femtomolar affinity

    Science.gov (United States)

    Zhou, Lu; Bosscher, Mike; Zhang, Changsheng; Özçubukçu, Salih; Zhang, Liang; Zhang, Wen; Li, Charles J.; Liu, Jianzhao; Jensen, Mark P.; Lai, Luhua; He, Chuan

    2014-03-01

    Uranyl (UO22+), the predominant aerobic form of uranium, is present in the ocean at a concentration of ~3.2 parts per 109 (13.7 nM) however, the successful enrichment of uranyl from this vast resource has been limited by the high concentrations of metal ions of similar size and charge, which makes it difficult to design a binding motif that is selective for uranyl. Here we report the design and rational development of a uranyl-binding protein using a computational screening process in the initial search for potential uranyl-binding sites. The engineered protein is thermally stable and offers very high affinity and selectivity for uranyl with a Kd of 7.4 femtomolar (fM) and >10,000-fold selectivity over other metal ions. We also demonstrated that the uranyl-binding protein can repeatedly sequester 30-60% of the uranyl in synthetic sea water. The chemical strategy employed here may be applied to engineer other selective metal-binding proteins for biotechnology and remediation applications.

  11. In vitro characterization of cocaine binding sites in human hair.

    Science.gov (United States)

    Joseph, R E; Tsai, W J; Tsao, L I; Su, T P; Cone, E J

    1997-09-01

    In vitro studies were performed to characterize [3H]cocaine binding to dark and light ethnic hair types. In vitro binding to hair was selective, was reversible and increased linearly with increasing hair concentration. Scatchard analyses revealed high-affinity (6-112 nM) and low-affinity (906-4433 nM) binding in hair. Competition studies demonstrated that the potencies of 3beta-(4-bromophenyl)tropane-2beta-carboxylic acid methyl ester, and 5-(4-chlorophenyl)-2,5-dihydro-3H-imidazol[2,1-alpha]isoindole-5-ol and 2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane were similar to or less than that of (-)-cocaine. The potency of (-)-cocaine was 10-fold greater than that of (+)-cocaine at inhibiting radioligand specific binding to hair. Multivariate analysis indicated that significantly greater nonspecific and specific radioligand binding occurred in dark hair than in light hair. Multivariate analysis also demonstrated a significant ethnicity x sex effect on specific and nonspecific binding to hair. Greater radioligand binding occurred in male Africoid hair than in female Africoid hair and in all Caucasoid hair types. Melanin was considered the most likely binding site for cocaine in hair. Typically, the concentration of melanin is much greater in dark than in light hair. Scatchard analysis indicated that dark hair had a 5- to 43-fold greater binding capacity than light hair. Differences in radioligand binding between hair types appeared to be due to differences in the density of binding sites formed by melanin in hair.

  12. Characterization of the binding sites for dicarboxylic acids on bovine serum albumin.

    Science.gov (United States)

    Tonsgard, J H; Meredith, S C

    1991-06-15

    Dicarboxylic acids are prominent features of several diseases, including Reye's syndrome and inborn errors of mitochondrial and peroxisomal fatty acid oxidation. Moreover, dicarboxylic acids are potentially toxic to cellular processes. Previous studies [Tonsgard, Mendelson & Meredith (1988) J. Clin. Invest. 82, 1567-1573] demonstrated that long-chain dicarboxylic acids have a single high-affinity binding site and between one and three lower-affinity sites on albumin. Medium-chain-length dicarboxylic acids have a single low-affinity site. We further characterized dicarboxylic acid binding to albumin in order to understand the potential effects of drugs and other ligands on dicarboxylic acid binding and toxicity. Progesterone and oleate competitively inhibit octadecanedioic acid binding to the single high-affinity site. Octanoate inhibits binding to the low-affinity sites. Dansylated probes for subdomain 2AB inhibit dodecanedioic acid binding whereas probes for subdomain 3AB do not. In contrast, low concentrations of octadecanedioic acid inhibit the binding of dansylated probes to subdomain 3AB and 2AB. L-Tryptophan, which binds in subdomain 3AB, inhibits hexadecanedioic acid binding but has no effect on dodecanedioic acid. Bilirubin and acetylsalicylic acid, which bind in subdomain 2AB, inhibit the binding of medium-chain and long-chain dicarboxylic acids. Our results suggest that long-chain dicarboxylic acids bind in subdomains 2C, 3AB and 2AB. The single low-affinity binding site for medium-chain dicarboxylic acids is in subdomain 2AB. These studies suggest that dicarboxylic acids are likely to be unbound in disease states and may be potentially toxic.

  13. Molecular determinants of affinity for aminoglycoside binding to the aminoglycoside nucleotidyltransferase(2'')-Ia.

    Science.gov (United States)

    Wright, Edward; Serpersu, Engin H

    2006-08-29

    One of the most commonly occurring aminoglycoside resistance enzymes is aminoglycoside 2''-O-nucleotidyltransferase [ANT(2'')]. In the present study molecular determinants of affinity and specificity for aminoglycoside binding to this enzyme are investigated using isothermal titration calorimetry (ITC). Binding of aminoglycosides is enthalpically driven accompanied by negative entropy changes. The presence of metal-nucleotide increases the affinity for all but one of the aminoglycosides studied but has no effect on specificity. The substituents at positions 1, 2', and 6' are important determinants of substrate specificity. An amino group at these positions leads to greater affinity. No correlation is observed between the change in affinity and enthalpy. At the 2' position greater affinity results from a more negative enthalpy for an aminoglycoside containing an amino rather than a hydroxyl at that position. At the 6' position the greater affinity for an aminoglycoside containing an amino substituent results from a less disfavorable entropic contribution. The thermodynamic basis for the change in affinity at position 1 could not be determined because of the weak binding of one of the aminoglycoside substrates, amikacin. The effect of increasing osmotic stress on affinity was used to determine that a net release of approximately four water molecules occurs when tobramycin binds to ANT(2''). No measurable net change in the number of bound water molecules is observed when neomycin binds the enzyme. Data acquired in this work provide the rationale for the ability of ANT(2'') to confer resistance against kanamycins but not neomycins.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1988-01-01

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

  15. Cation binding site of cytochrome c oxidase: progress report.

    Science.gov (United States)

    Vygodina, Tatiana V; Kirichenko, Anna; Konstantinov, Alexander A

    2014-07-01

    Cytochrome c oxidase from bovine heart binds Ca(2+) reversibly at a specific Cation Binding Site located near the outer face of the mitochondrial membrane. Ca(2+) shifts the absorption spectrum of heme a, which allowed earlier the determination of the kinetic and equilibrium characteristics of the binding, and, as shown recently, the binding of calcium to the site inhibits cytochrome oxidase activity at low turnover rates of the enzyme [Vygodina, Т., Kirichenko, A., Konstantinov, A.A (2013). Direct Regulation of Cytochrome c Oxidase by Calcium Ions. PloS ONE 8, e74436]. This paper summarizes further progress in the studies of the Cation Binding Site in this group presenting the results to be reported at 18th EBEC Meeting in Lisbon, 2014. The paper revises specificity of the bovine oxidase Cation Binding Site for different cations, describes dependence of the Ca(2+)-induced inhibition on turnover rate of the enzyme and reports very high affinity binding of calcium with the "slow" form of cytochrome oxidase. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. Guest Editors: Manuela Pereira and Miguel Teixeira.

  16. Change in allosteric network affects binding affinities of PDZ domains: analysis through perturbation response scanning.

    Directory of Open Access Journals (Sweden)

    Z Nevin Gerek

    2011-10-01

    Full Text Available The allosteric mechanism plays a key role in cellular functions of several PDZ domain proteins (PDZs and is directly linked to pharmaceutical applications; however, it is a challenge to elaborate the nature and extent of these allosteric interactions. One solution to this problem is to explore the dynamics of PDZs, which may provide insights about how intramolecular communication occurs within a single domain. Here, we develop an advancement of perturbation response scanning (PRS that couples elastic network models with linear response theory (LRT to predict key residues in allosteric transitions of the two most studied PDZs (PSD-95 PDZ3 domain and hPTP1E PDZ2 domain. With PRS, we first identify the residues that give the highest mean square fluctuation response upon perturbing the binding sites. Strikingly, we observe that the residues with the highest mean square fluctuation response agree with experimentally determined residues involved in allosteric transitions. Second, we construct the allosteric pathways by linking the residues giving the same directional response upon perturbation of the binding sites. The predicted intramolecular communication pathways reveal that PSD-95 and hPTP1E have different pathways through the dynamic coupling of different residue pairs. Moreover, our analysis provides a molecular understanding of experimentally observed hidden allostery of PSD-95. We show that removing the distal third alpha helix from the binding site alters the allosteric pathway and decreases the binding affinity. Overall, these results indicate that (i dynamics plays a key role in allosteric regulations of PDZs, (ii the local changes in the residue interactions can lead to significant changes in the dynamics of allosteric regulations, and (iii this might be the mechanism that each PDZ uses to tailor their binding specificities regulation.

  17. Influence of sulfhydryl sites on metal binding by bacteria

    Science.gov (United States)

    Nell, Ryan M.; Fein, Jeremy B.

    2017-02-01

    The role of sulfhydryl sites within bacterial cell envelopes is still unknown, but the sites may control the fate and bioavailability of metals. Organic sulfhydryl compounds are important complexing ligands in aqueous systems and they can influence metal speciation in natural waters. Though representing only approximately 5-10% of the total available binding sites on bacterial surfaces, sulfhydryl sites exhibit high binding affinities for some metals. Due to the potential importance of bacterial sulfhydryl sites in natural systems, metal-bacterial sulfhydryl site binding constants must be determined in order to construct accurate models of the fate and distribution of metals in these systems. To date, only Cd-sulfhydryl binding has been quantified. In this study, the thermodynamic stabilities of Mn-, Co-, Ni-, Zn-, Sr- and Pb-sulfhydryl bacterial cell envelope complexes were determined for the bacterial species Shewanella oneidensis MR-1. Metal adsorption experiments were conducted as a function of both pH, ranging from 5.0 to 7.0, and metal loading, from 0.5 to 40.0 μmol/g (wet weight) bacteria, in batch experiments in order to determine if metal-sulfhydryl binding occurs. Initially, the data were used to calculate the value of the stability constants for the important metal-sulfhydryl bacterial complexes for each metal-loading condition studied, assuming a single binding reaction for the dominant metal-binding site type under the pH conditions of the experiments. For most of the metals that we studied, these calculated stability constant values increased significantly with decreasing metal loading, strongly suggesting that our initial assumption was not valid and that more than one type of binding occurs at the assumed binding site. We then modeled each dataset with two distinct site types with identical acidity constants: one site with a high metal-site stability constant value, which we take to represent metal-sulfhydryl binding and which dominates under low

  18. Cytisine binds with similar affinity to nicotinic alpha4beta2 receptors on the cell surface and in homogenates.

    Science.gov (United States)

    Zhang, Jessie; Steinbach, Joe Henry

    2003-01-03

    Cytisine and nicotine bound to specific sites in homogenates prepared from HEK 293 cells which stably express human neuronal nicotinic alpha4 and beta2 subunits. The number of sites was the same for both ligands and nicotine was a full competitive inhibitor of cytisine binding. However, when binding was done to intact cells the number of binding sites per cell for nicotine was approximately 4-fold the number of sites for cytisine. Nicotine fully blocked cytisine binding, but cytisine only partially blocked nicotine binding to intact cells. When cells were permeabilized with saponin, the number of sites for nicotine was unchanged, while the number of sites for cytisine was increased, and cytisine was able to fully block nicotine binding. These data indicate that cytisine binds only to surface receptors on intact cells. The apparent affinity of cytisine for surface receptors (K(d)=0.8 nM) was not significantly different from that for receptors in the cell homogenate (0.3 nM).

  19. Characterisation of the zebrafish serotonin transporter functionally links TM10 to the ligand binding site

    DEFF Research Database (Denmark)

    Severinsen, Kasper; Müller, Heidi Kaastrup; Wiborg, Ove

    2008-01-01

    and [(3)H]-escitalopram binding in transiently transfected human embryonic kidney cells; HEK-293-MSR. Residues responsible for altered affinities inhibitors were pinpointed by generating cross-species chimeras and subsequent point mutations by site directed mutagenesis. drSERT has a higher affinity...

  20. Affinity of Doripenem and Comparators to Penicillin-Binding Proteins in Escherichia coli and Pseudomonas aeruginosa▿

    OpenAIRE

    2008-01-01

    Doripenem, a parenteral carbapenem, exhibited high affinity for penicillin-binding protein 2 (PBP2) and PBP3 in Pseudomonas aeruginosa and PBP2 in Escherichia coli, the primary PBPs whose inhibition leads to cell death. This PBP affinity profile correlates with the broad-spectrum gram-negative activity observed with doripenem.

  1. The Binding of Biotin to Sepharose-Avidin Column: Demonstration of the Affinity Chromatography Technique

    Science.gov (United States)

    Landman, A. D.; Landman, N. N.

    1976-01-01

    Describes a biochemistry experiment that illustrates the methodology of affinity chromatography by attaching avidin, a glycoprotein in egg white, to a Sepharose matrix in order to bind biotin-containing proteins. (MLH)

  2. The Binding of Biotin to Sepharose-Avidin Column: Demonstration of the Affinity Chromatography Technique

    Science.gov (United States)

    Landman, A. D.; Landman, N. N.

    1976-01-01

    Describes a biochemistry experiment that illustrates the methodology of affinity chromatography by attaching avidin, a glycoprotein in egg white, to a Sepharose matrix in order to bind biotin-containing proteins. (MLH)

  3. Negative Cooperativity and High Affinity in Chitooligosaccharide Binding by a Mycobacterium smegmatis Protein Containing LysM and Lectin Domains.

    Science.gov (United States)

    Patra, Dhabaleswar; Mishra, Padmanabh; Vijayan, Mamannamana; Surolia, Avadhesha

    2016-01-12

    LysM domains have been recognized in bacteria and eukaryotes as carbohydrate-binding protein modules, but the mechanism of their binding to chitooligosaccharides has been underexplored. Binding of a Mycobacterium smegmatis protein containing a lectin (MSL) and one LysM domain to chitooligosaccharides has been studied using isothermal titration calorimetry and fluorescence titration that demonstrate the presence of two binding sites of nonidentical affinities per dimeric MSL-LysM molecule. The affinity of the molecule for chitooligosaccharides correlates with the length of the carbohydrate chain. Its binding to chitooligosaccharides is characterized by negative cooperativity in the interactions of the two domains. Apparently, the flexibility of the long linker that connects the LysM and MSL domains plays a facilitating role in this recognition. The LysM domain in the MSL-LysM molecule, like other bacterial domains but unlike plant LysM domains, recognizes equally well peptidoglycan fragments as well as chitin polymers. Interestingly, in the case presented here, two LysM domains are enough for binding to peptidoglycan in contrast to the three reportedly required by the LysM domains of Bacillus subtilis and Lactococcus lactis. Also, the affinity of the MSL-LysM molecule for chitooligosaccharides is higher than that of LysM-chitooligosaccharide interactions reported so far.

  4. Oestradiol and testosterone binding sites in mice tibiae and their relationship with bone growth.

    Science.gov (United States)

    Lopez, A; Ventanas, J; Burgos, J

    1986-11-01

    High affinity oestradiol and testosterone binding sites were found in tibiae cytosol from entire male and female of different ages. Scatchard assay allowed to estimate a Kd of 2.7 X 10(-9) M for oestradiol binding sites indicating that the 3H-oestradiol binding was of high affinity. Oestradiol and testosterone binding sites abundance in mice tibiae are subject to change with age. It is not easy to establish a direct correlation between these changes and the values reported here on bone growth in weight and length, however seems possible to point a negative relationship between bone lengthening and oestradiol binding site levels in female, as well a positive relationship with testosterone in both sexes. The presence of oestradiol and testosterone binding sites in epiphyses and not in the diaphyses reinforces the hypothesis that both are playing some role in bone growth.

  5. Involvement of nitrogen functional groups in high-affinity copper binding in tomato and wheat root apoplasts: spectroscopic and thermodynamic evidence.

    Science.gov (United States)

    Guigues, Stéphanie; Bravin, Matthieu N; Garnier, Cédric; Masion, Armand; Chevassus-Rosset, Claire; Cazevieille, Patrick; Doelsch, Emmanuel

    2016-03-01

    Carboxylic groups located in plant cell walls (CW) are generally considered to be the main copper binding sites in plant roots, despite the presence of other functional groups. The aim of this study was to investigate sites responsible for copper binding in root apoplasts, i.e. CW and outer surface of the plasma membrane (PM) continuum. Binding sites in root apoplasts were investigated by comparing isolated CW of a monocotyledon (Triticum aestivum L.) and dicotyledon (Solanum lycopersicum L.) crop with their respective whole roots. Copper speciation was examined by X-ray absorption (XAS) and (13)C-nuclear magnetic resonance spectroscopies while the affinity of ligands involved in copper binding was investigated by modeling copper sorption isotherms. Homogeneous speciation and binding of copper was found in wheat and tomato root apoplasts. Only Cu-N and Cu-O bonds were detected in wheat and tomato root apoplasts. Nitrogen/oxygen ligands were identified in slightly higher proportions (40-70%) than single oxygen ligands. Furthermore, low- and high-affinity binding sites contributed in an almost equivalent proportion to copper binding in root apoplasts. The high-affinity N functional groups embedded in root apoplasts participated in copper binding in the same magnitude than the low-affinity carboxylic groups.

  6. Probing binding hot spots at protein-RNA recognition sites.

    Science.gov (United States)

    Barik, Amita; Nithin, Chandran; Karampudi, Naga Bhushana Rao; Mukherjee, Sunandan; Bahadur, Ranjit Prasad

    2016-01-29

    We use evolutionary conservation derived from structure alignment of polypeptide sequences along with structural and physicochemical attributes of protein-RNA interfaces to probe the binding hot spots at protein-RNA recognition sites. We find that the degree of conservation varies across the RNA binding proteins; some evolve rapidly compared to others. Additionally, irrespective of the structural class of the complexes, residues at the RNA binding sites are evolutionary better conserved than those at the solvent exposed surfaces. For recognitions involving duplex RNA, residues interacting with the major groove are better conserved than those interacting with the minor groove. We identify multi-interface residues participating simultaneously in protein-protein and protein-RNA interfaces in complexes where more than one polypeptide is involved in RNA recognition, and show that they are better conserved compared to any other RNA binding residues. We find that the residues at water preservation site are better conserved than those at hydrated or at dehydrated sites. Finally, we develop a Random Forests model using structural and physicochemical attributes for predicting binding hot spots. The model accurately predicts 80% of the instances of experimental ΔΔG values in a particular class, and provides a stepping-stone towards the engineering of protein-RNA recognition sites with desired affinity.

  7. Probing binding hot spots at protein–RNA recognition sites

    Science.gov (United States)

    Barik, Amita; Nithin, Chandran; Karampudi, Naga Bhushana Rao; Mukherjee, Sunandan; Bahadur, Ranjit Prasad

    2016-01-01

    We use evolutionary conservation derived from structure alignment of polypeptide sequences along with structural and physicochemical attributes of protein–RNA interfaces to probe the binding hot spots at protein–RNA recognition sites. We find that the degree of conservation varies across the RNA binding proteins; some evolve rapidly compared to others. Additionally, irrespective of the structural class of the complexes, residues at the RNA binding sites are evolutionary better conserved than those at the solvent exposed surfaces. For recognitions involving duplex RNA, residues interacting with the major groove are better conserved than those interacting with the minor groove. We identify multi-interface residues participating simultaneously in protein–protein and protein–RNA interfaces in complexes where more than one polypeptide is involved in RNA recognition, and show that they are better conserved compared to any other RNA binding residues. We find that the residues at water preservation site are better conserved than those at hydrated or at dehydrated sites. Finally, we develop a Random Forests model using structural and physicochemical attributes for predicting binding hot spots. The model accurately predicts 80% of the instances of experimental ΔΔG values in a particular class, and provides a stepping-stone towards the engineering of protein–RNA recognition sites with desired affinity. PMID:26365245

  8. Dicyanovinylnaphthalenes for neuroimaging of amyloids and relationships of electronic structures and geometries to binding affinities.

    Science.gov (United States)

    Petric, Andrej; Johnson, Scott A; Pham, Hung V; Li, Ying; Ceh, Simon; Golobic, Amalija; Agdeppa, Eric D; Timbol, Gerald; Liu, Jie; Keum, Gyochang; Satyamurthy, Nagichettiar; Kepe, Vladimir; Houk, Kendall N; Barrio, Jorge R

    2012-10-09

    The positron-emission tomography (PET) probe 2-(1-[6-[(2-fluoroethyl)(methyl)amino]-2-naphthyl]ethylidene) (FDDNP) is used for the noninvasive brain imaging of amyloid-β (Aβ) and other amyloid aggregates present in Alzheimer's disease and other neurodegenerative diseases. A series of FDDNP analogs has been synthesized and characterized using spectroscopic and computational methods. The binding affinities of these molecules have been measured experimentally and explained through the use of a computational model. The analogs were created by systematically modifying the donor and the acceptor sides of FDDNP to learn the structural requirements for optimal binding to Aβ aggregates. FDDNP and its analogs are neutral, environmentally sensitive, fluorescent molecules with high dipole moments, as evidenced by their spectroscopic properties and dipole moment calculations. The preferred solution-state conformation of these compounds is directly related to the binding affinities. The extreme cases were a nonplanar analog t-butyl-FDDNP, which shows low binding affinity for Aβ aggregates (520 nM K(i)) in vitro and a nearly planar tricyclic analog cDDNP, which displayed the highest binding affinity (10 pM K(i)). Using a previously published X-ray crystallographic model of 1,1-dicyano-2-[6-(dimethylamino)naphthalen-2-yl]propene (DDNP) bound to an amyloidogenic Aβ peptide model, we show that the binding affinity is inversely related to the distortion energy necessary to avoid steric clashes along the internal surface of the binding channel.

  9. Opioid binding sites in the guinea pig and rat kidney: Radioligand homogenate binding and autoradiography

    Energy Technology Data Exchange (ETDEWEB)

    Dissanayake, V.U.; Hughes, J.; Hunter, J.C. (Parke-Davis Research Unit, Addenbrookes Hospital Site, Cambridge (England))

    1991-07-01

    The specific binding of the selective {mu}-, {delta}-, and {kappa}-opioid ligands (3H)(D-Ala2,MePhe4,Gly-ol5)enkephalin ((3H) DAGOL), (3H)(D-Pen2,D-Pen5)enkephalin ((3H)DPDPE), and (3H)U69593, respectively, to crude membranes of the guinea pig and rat whole kidney, kidney cortex, and kidney medulla was investigated. In addition, the distribution of specific 3H-opioid binding sites in the guinea pig and rat kidney was visualized by autoradiography. Homogenate binding and autoradiography demonstrated the absence of {mu}- and {kappa}-opioid binding sites in the guinea pig kidney. No opioid binding sites were demonstrable in the rat kidney. In the guinea pig whole kidney, cortex, and medulla, saturation studies demonstrated that (3H)DPDPE bound with high affinity (KD = 2.6-3.5 nM) to an apparently homogeneous population of binding sites (Bmax = 8.4-30 fmol/mg of protein). Competition studies using several opioid compounds confirmed the nature of the {delta}-opioid binding site. Autoradiography experiments demonstrated that specific (3H)DPDPE binding sites were distributed radially in regions of the inner and outer medulla and at the corticomedullary junction of the guinea pig kidney. Computer-assisted image analysis of saturation data yielded KD values (4.5-5.0 nM) that were in good agreement with those obtained from the homogenate binding studies. Further investigation of the {delta}-opioid binding site in medulla homogenates, using agonist ((3H)DPDPE) and antagonist ((3H)diprenorphine) binding in the presence of Na+, Mg2+, and nucleotides, suggested that the {delta}-opioid site is linked to a second messenger system via a GTP-binding protein. Further studies are required to establish the precise localization of the {delta} binding site in the guinea pig kidney and to determine the nature of the second messenger linked to the GTP-binding protein in the medulla.

  10. SYNTHESIS OF POLY(3,4-AZOPYRIDYLENE) AND OXYGEN-BINDING AFFINITY OF ITS COMPLEX WITH COBALTPORPHYRIN

    Institute of Scientific and Technical Information of China (English)

    Bao-qing Shentu; Zhi-xue Weng; Hiroyuki Nishide

    2005-01-01

    Synthesis and characteristics of poly(3,4-azopyridylene) (PAP), conductivity and oxygen-binding affinity of its complex with meso-α,α,α,α-tetrakis(o-pivalamidophenyl) porphyrinatocobalt(Ⅱ) (CoP) were studied. PAP was prepared by oxidative polymerization of 3,4-diaminopyridine (DAP) in DMF solution using CuCl/pyridine as the catalyst. IR and NMR results showed that the peak of amido group in DAP was converted to the azo group in PAP and a π conjugated polymer was synthesized. The average molecular weight of PAP was determined to be 5.0 × 103. The PAP-CoP complex was prepared by complexing the pyridyl group of PAP with the fifth coordination site of CoP in DMF solution. In comparison with the CoP complex with a non-π conjugated polymer, the PAP-CoP complex shows good electroconductivity of 5.8 × 10-6 Scm-1. The PAP-CoP complex displays a reversible change in the UV-Visible absorption spectrum from the deoxy form to the oxy or oxygen-binding one with an isosbestic point, in response to the partial oxygen pressure of the atmosphere. The oxygen-response behavior was monitored at the absorbance ascribed to the oxy form at 548 nm to give the oxygen-binding affinity.The oxygen-binding equilibrium curves of PAP-CoP complex obey a Langmuir isotherm. DMF has great effects on the oxygen-binding properties of the PAP-CoP complex. The oxygen-binding affinity of PAP-CoP complex in the solid state is higher than that in DMF solution. With decreasing temperature, the oxygen-binding affinity of the PAP-CoP complex increases.

  11. Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity.

    Science.gov (United States)

    Boder, E T; Midelfort, K S; Wittrup, K D

    2000-09-26

    Single-chain antibody mutants have been evolved in vitro with antigen-binding equilibrium dissociation constant K(d) = 48 fM and slower dissociation kinetics (half-time > 5 days) than those for the streptavidin-biotin complex. These mutants possess the highest monovalent ligand-binding affinity yet reported for an engineered protein by over two orders of magnitude. Optimal kinetic screening of randomly mutagenized libraries of 10(5)-10(7) yeast surface-displayed antibodies enabled a >1,000-fold decrease in the rate of dissociation after four cycles of affinity mutagenesis and screening. The consensus mutations are generally nonconservative by comparison with naturally occurring mouse Fv sequences and with residues that do not contact the fluorescein antigen in the wild-type complex. The existence of these mutants demonstrates that the antibody Fv architecture is not intrinsically responsible for an antigen-binding affinity ceiling during in vivo affinity maturation.

  12. Structure-affinity relationship of the cocaine-binding aptamer with quinine derivatives.

    Science.gov (United States)

    Slavkovic, Sladjana; Altunisik, Merve; Reinstein, Oren; Johnson, Philip E

    2015-05-15

    In addition to binding its target molecule, cocaine, the cocaine-binding aptamer tightly binds the alkaloid quinine. In order to understand better how the cocaine-binding aptamer interacts with quinine we have used isothermal titration calorimetry-based binding experiments to study the interaction of the cocaine-binding aptamer to a series of structural analogs of quinine. As a basis for comparison we also investigated the binding of the cocaine-binding aptamer to a set of cocaine metabolites. The bicyclic aromatic ring on quinine is essential for tight affinity by the cocaine-binding aptamer with 6-methoxyquinoline alone being sufficient for tight binding while the aliphatic portion of quinine, quinuclidine, does not show detectable binding. Compounds with three fused aromatic rings are not bound by the aptamer. Having a methoxy group at the 6-position of the bicyclic ring is important for binding as substituting it with a hydrogen, an alcohol or an amino group all result in lower binding affinity. For all ligands that bind, association is driven by a negative enthalpy compensated by unfavorable binding entropy.

  13. Receptor binding profiles and quantitative structure-affinity relationships of some 5-substituted-N,N-diallyltryptamines.

    Science.gov (United States)

    Cozzi, Nicholas V; Daley, Paul F

    2016-02-01

    N,N-Diallyltryptamine (DALT) and 5-methoxy-N,N-diallyltryptamine (5-MeO-DALT) are two tryptamines synthesized and tested by Alexander Shulgin. In self-experiments, 5-MeO-DALT was reported to be psychoactive in the 12-20mg range, while the unsubstituted compound DALT had few discernible effects in the 42-80 mg range. Recently, 5-MeO-DALT has been used in nonmedical settings for its psychoactive effects, but these effects have been poorly characterized and little is known of its pharmacological properties. We extended the work of Shulgin by synthesizing additional 5-substituted-DALTs. We then compared them to DALT and 5-MeO-DALT for their binding affinities at 45 cloned receptors and transporter proteins. Based on in vitro binding affinity, we identified 27 potential receptor targets for the 5-substituted-DALT compounds. Five of the DALT compounds had affinity in the 10-80 nM range for serotonin 5-HT1A and 5-HT2B receptors, while the affinity of DALT itself at 5-HT1A receptors was slightly lower at 100 nM. Among the 5-HT2 subtypes, the weakest affinity was at 5-HT2A receptors, spanning 250-730 nM. Five of the DALT compounds had affinity in the 50-400 nM range for serotonin 5-HT1D, 5-HT6, and 5-HT7 receptors; again, it was the unsubstituted DALT that had the weakest affinity at all three subtypes. The test drugs had even weaker affinity for 5-HT1B, 5-HT1E, and 5-HT5A subtypes and little or no affinity for the 5-HT3 subtype. These compounds also had generally nanomolar affinities for adrenergic α2A, α2B, and α2C receptors, sigma receptors σ1 and σ2, histamine H1 receptors, and norepinephrine and serotonin uptake transporters. They also bound to other targets in the nanomolar-to-low micromolar range. Based on these binding results, it is likely that multiple serotonin receptors, as well as several nonserotonergic sites are important for the psychoactive effects of DALT drugs. To learn whether any quantitative structure-affinity relationships existed, we evaluated

  14. Syntax compensates for poor binding sites to encode tissue specificity of developmental enhancers.

    Science.gov (United States)

    Farley, Emma K; Olson, Katrina M; Zhang, Wei; Rokhsar, Daniel S; Levine, Michael S

    2016-06-07

    Transcriptional enhancers are short segments of DNA that switch genes on and off in response to a variety of intrinsic and extrinsic signals. Despite the discovery of the first enhancer more than 30 y ago, the relationship between primary DNA sequence and enhancer activity remains obscure. In particular, the importance of "syntax" (the order, orientation, and spacing of binding sites) is unclear. A high-throughput screen identified synthetic notochord enhancers that are activated by the combination of ZicL and ETS transcription factors in Ciona embryos. Manipulation of these enhancers elucidated a "regulatory code" of sequence and syntax features for notochord-specific expression. This code enabled in silico discovery of bona fide notochord enhancers, including those containing low-affinity binding sites that would be excluded by standard motif identification methods. One of the newly identified enhancers maps upstream of the known enhancer that regulates Brachyury (Ci-Bra), a key determinant of notochord specification. This newly identified Ci-Bra shadow enhancer contains binding sites with very low affinity, but optimal syntax, and therefore mediates surprisingly strong expression in the notochord. Weak binding sites are compensated by optimal syntax, whereas enhancers containing high-affinity binding affinities possess suboptimal syntax. We suggest this balance has obscured the importance of regulatory syntax, as noncanonical binding motifs are typically disregarded by enhancer detection methods. As a result, enhancers with low binding affinities but optimal syntax may be a vastly underappreciated feature of the regulatory genome.

  15. Molecular modelling and competition binding study of Br-noscapine and colchicine provide insight into noscapinoid-tubulin binding site.

    Science.gov (United States)

    Naik, Pradeep K; Santoshi, Seneha; Rai, Ankit; Joshi, Harish C

    2011-06-01

    We have previously discovered the tubulin-binding anti-cancer properties of noscapine and its derivatives (noscapinoids). Here, we present three lines of evidence that noscapinoids bind at or near the well studied colchicine binding site of tubulin: (1) in silico molecular docking studies of Br-noscapine and noscapine yield highest docking score with the well characterised colchicine-binding site from the co-crystal structure; (2) the molecular mechanics-generalized Born/surface area (MM-GB/SA) scoring results ΔΔG(bind-cald) for both noscapine and Br-noscapine (3.915 and 3.025 kcal/mol) are in reasonably good agreement with our experimentally determined binding affinity (ΔΔG(bind-Expt) of 3.570 and 2.988 kcal/mol, derived from K(d) values); and (3) Br-noscapine competes with colchicine binding to tubulin. The simplest interpretation of these collective data is that Br-noscapine binds tubulin at a site overlapping with, or very close to colchicine-binding site of tubulin. Although we cannot rule out a formal possibility that Br-noscapine might bind to a site distinct and distant from the colchicine-binding site that might negatively influence the colchicine binding to tubulin.

  16. 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.

  17. Prediction of ligand binding affinity using a multiple-conformations-multiple-protonation scheme: application to estrogen receptor α.

    Science.gov (United States)

    Mizutani, Miho Y; Takamatsu, Yoshihiro; Ichinose, Tazuko; Itai, Akiko

    2012-01-01

    A fast method that can predict the binding affinities of chemicals to a target protein with a high degree of accuracy will be very useful in drug design and regulatory science. We have been developing a scoring function for affinity prediction, which can be applied to extensive protein systems, and also trying to generate a prediction scheme that specializes in each target protein, with as high a predictive power as possible. In this study, we have constructed a prediction scheme with target-specific scores for estimating ligand-binding affinities to human estrogen receptor α (ERα), considering the major conformational change between agonist- and antagonist-bound forms and the change in protonation states of histidine at the ligand-binding site. The generated scheme calibrated with fewer training compounds (23 for the agonist-bound form, 17 for the antagonist-bound form) demonstrated good predictive power (a predictive r(2) of 0.83 for 154 validation compounds); this was also true for compounds with frameworks that were quite different from those of the training compounds. Our prediction scheme will be useful in drug development targeting ERα and in primary screening of endocrine disruptors, and provides a successful method of affinity prediction considering the major conformational changes in a protein.

  18. Disease-associated mutation in SRSF2 misregulates splicing by altering RNA-binding affinities.

    Science.gov (United States)

    Zhang, Jian; Lieu, Yen K; Ali, Abdullah M; Penson, Alex; Reggio, Kathryn S; Rabadan, Raul; Raza, Azra; Mukherjee, Siddhartha; Manley, James L

    2015-08-25

    Serine/arginine-rich splicing factor 2 (SRSF2) is an RNA-binding protein that plays important roles in splicing of mRNA precursors. SRSF2 mutations are frequently found in patients with myelodysplastic syndromes and certain leukemias, but how these mutations affect SRSF2 function has only begun to be examined. We used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease to introduce the P95H mutation to SRSF2 in K562 leukemia cells, generating an isogenic model so that splicing alterations can be attributed solely to mutant SRSF2. We found that SRSF2 (P95H) misregulates 548 splicing events (RNA gel shift assays showed that a mutant SRSF2 derivative bound more tightly than its wild-type counterpart to RNA sites containing UCCAG but bound less tightly to UGGAG sites. Thus in most cases the pattern of exon inclusion or exclusion correlated with stronger or weaker RNA binding, respectively. We further show that the P95H mutation does not affect other functions of SRSF2, i.e., protein-protein interactions with key splicing factors. Our results thus demonstrate that the P95H mutation positively or negatively alters the binding affinity of SRSF2 for cognate RNA sites in target transcripts, leading to misregulation of exon inclusion. Our findings shed light on the mechanism of the disease-associated SRSF2 mutation in splicing regulation and also reveal a group of misspliced mRNA isoforms for potential therapeutic targeting.

  19. Binding-site assessment by virtual fragment screening.

    Directory of Open Access Journals (Sweden)

    Niu Huang

    Full Text Available The accurate prediction of protein druggability (propensity to bind high-affinity drug-like small molecules would greatly benefit the fields of chemical genomics and drug discovery. We have developed a novel approach to quantitatively assess protein druggability by computationally screening a fragment-like compound library. In analogy to NMR-based fragment screening, we dock approximately 11,000 fragments against a given binding site and compute a computational hit rate based on the fraction of molecules that exceed an empirically chosen score cutoff. We perform a large-scale evaluation of the approach on four datasets, totaling 152 binding sites. We demonstrate that computed hit rates correlate with hit rates measured experimentally in a previously published NMR-based screening method. Secondly, we show that the in silico fragment screening method can be used to distinguish known druggable and non-druggable targets, including both enzymes and protein-protein interaction sites. Finally, we explore the sensitivity of the results to different receptor conformations, including flexible protein-protein interaction sites. Besides its original aim to assess druggability of different protein targets, this method could be used to identifying druggable conformations of flexible binding site for lead discovery, and suggesting strategies for growing or joining initial fragment hits to obtain more potent inhibitors.

  20. Nonlinear scoring functions for similarity-based ligand docking and binding affinity prediction.

    Science.gov (United States)

    Brylinski, Michal

    2013-11-25

    A common strategy for virtual screening considers a systematic docking of a large library of organic compounds into the target sites in protein receptors with promising leads selected based on favorable intermolecular interactions. Despite a continuous progress in the modeling of protein-ligand interactions for pharmaceutical design, important challenges still remain, thus the development of novel techniques is required. In this communication, we describe eSimDock, a new approach to ligand docking and binding affinity prediction. eSimDock employs nonlinear machine learning-based scoring functions to improve the accuracy of ligand ranking and similarity-based binding pose prediction, and to increase the tolerance to structural imperfections in the target structures. In large-scale benchmarking using the Astex/CCDC data set, we show that 53.9% (67.9%) of the predicted ligand poses have RMSD of <2 Å (<3 Å). Moreover, using binding sites predicted by recently developed eFindSite, eSimDock models ligand binding poses with an RMSD of 4 Å for 50.0-39.7% of the complexes at the protein homology level limited to 80-40%. Simulations against non-native receptor structures, whose mean backbone rearrangements vary from 0.5 to 5.0 Å Cα-RMSD, show that the ratio of docking accuracy and the estimated upper bound is at a constant level of ∼0.65. Pearson correlation coefficient between experimental and predicted by eSimDock Ki values for a large data set of the crystal structures of protein-ligand complexes from BindingDB is 0.58, which decreases only to 0.46 when target structures distorted to 3.0 Å Cα-RMSD are used. Finally, two case studies demonstrate that eSimDock can be customized to specific applications as well. These encouraging results show that the performance of eSimDock is largely unaffected by the deformations of ligand binding regions, thus it represents a practical strategy for across-proteome virtual screening using protein models. eSimDock is freely

  1. High affinity binding of /sup 125/I-labeled mouse interferon to a specific cell surface receptor. II. Analysis of binding properties

    Energy Technology Data Exchange (ETDEWEB)

    Aguet, M.; Blanchard, B.

    1981-12-01

    Direct ligand-binding studies with highly purified /sup 125/I-labeled virus-induced mouse interferon on mouse lymphoma L 1210 cells revealed a direct correlation of specific high-affinity binding with the biologic response to interferon. Neutralization of the antiviral effect by anti-interferon gamma globulin occurred at the same antibody concentration as the inhibition of specific binding. These results suggest that specific high-affinity binding of /sup 125/I-interferon occurred at a biologically functional interferon receptor. Competitive inhibition experiments using /sup 125/I- and /sup 127/I-labeled interferon provided strong evidence that the fraction of /sup 125/I-interferon inactivated upon labeling did not bind specifically. Scatchard analysis of the binding data yielded linear plots and thus suggested that interferon binds to homogeneous noncooperative receptor sites. In contrast to a characteristic property of several peptide hormone systems, binding of /sup 125/I-interferon to its specific receptor did not induce subsequent ligand degradation. At 37/sup o/ bound interferon was rapidly released in a biologically active form without evidence for molecular degradation. The expression of interferon receptors was not modified by treatment with interferon. Trypsin treatment of target cells and inhibition of protein synthesis abolished the specific binding of /sup 125/I-interferon. Three major molecular weight species of Newcastle disease virus-induced mouse C 243 cell interferon were isolated, separated, and identified as mouse ..cap alpha.. and ..beta.. interferons. These interferons were shown to inhibit competitively the specific binding of the highly purified labeled starting material thus providing evidence for a common receptor site for mouse interferon.

  2. Binding affinity of five PBPs to Ostrinia sex pheromones

    Science.gov (United States)

    Pheromone binding proteins (PBPs) of Lepidoptera function in chemical communication, mate attraction and recognition, and may be involved in reinforcement of sexual isolation between recently diverged species. Directional selection was previously predicted between PBP3 orthologs of the corn borer si...

  3. Sequence2Vec: A novel embedding approach for modeling transcription factor binding affinity landscape

    KAUST Repository

    Dai, Hanjun

    2017-07-26

    Motivation: An accurate characterization of transcription factor (TF)-DNA affinity landscape is crucial to a quantitative understanding of the molecular mechanisms underpinning endogenous gene regulation. While recent advances in biotechnology have brought the opportunity for building binding affinity prediction methods, the accurate characterization of TF-DNA binding affinity landscape still remains a challenging problem. Results: Here we propose a novel sequence embedding approach for modeling the transcription factor binding affinity landscape. Our method represents DNA binding sequences as a hidden Markov model (HMM) which captures both position specific information and long-range dependency in the sequence. A cornerstone of our method is a novel message passing-like embedding algorithm, called Sequence2Vec, which maps these HMMs into a common nonlinear feature space and uses these embedded features to build a predictive model. Our method is a novel combination of the strength of probabilistic graphical models, feature space embedding and deep learning. We conducted comprehensive experiments on over 90 large-scale TF-DNA data sets which were measured by different high-throughput experimental technologies. Sequence2Vec outperforms alternative machine learning methods as well as the state-of-the-art binding affinity prediction methods.

  4. Interaction of benzimidazole anthelmintics with Haemonchus contortus tubulin: binding affinity and anthelmintic efficacy.

    Science.gov (United States)

    Lubega, G W; Prichard, R K

    1991-08-01

    The ability of various benzimidazoles (BZs) to bind tubulin under different conditions was assessed by determining their IC50 values (the concentration of unlabeled drug required to inhibit 50% of the labeled drug binding), Ka (the apparent equilibrium association constant) and Bmax (the maximum binding at infinite [BZ] = [drug-receptor]). The ability of unlabeled benzimidazoles--fenbendazole, mebendazole (MBZ), oxibendazole (OBZ), albendazole (ABZ), rycobendazole (albendazole sulfoxide, ABZSO), albendazole sulfone, oxfendazole (OFZ), and thiabendazole--to bind tubulin was determined from their ability to inhibit the binding of [3H]MBZ or [3H]OBZ to tubulin in supernatants derived from unembryonated eggs or adult worms of Haemonchus contortus. The binding constants (IC50, Ka, and Bmax) correlated with the known anthelmintic potency (recommended therapeutic doses) of the BZ compounds except for OFZ and ABZSO whose Ka values were lower than could be expected from anthelmintic potency. The binding of [3H]ABZ or [3H]OFZ to tubulin in supernatants derived from BZ-susceptible and BZ-resistant H. contortus was compared. [3H]ABZ demonstrated saturable high-affinity binding but [3H]OFZ bound with low affinity. The high-affinity binding of [3H]ABZ was reduced for the R strain. Tubulin bound BZ drugs at 4 degrees C with lower apparent Ka than at 37 degrees C.

  5. Multiple sup 3 H-oxytocin binding sites in rat myometrial plasma membranes

    Energy Technology Data Exchange (ETDEWEB)

    Crankshaw, D.; Gaspar, V.; Pliska, V. (McMaster Univ., Hamilton, Ontario, (Canada))

    1990-01-01

    The affinity spectrum method has been used to analyse binding isotherms for {sup 3}H-oxytocin to rat myometrial plasma membranes. Three populations of binding sites with dissociation constants (Kd) of 0.6-1.5 x 10(-9), 0.4-1.0 x 10(-7) and 7 x 10(-6) mol/l were identified and their existence verified by cluster analysis based on similarities between Kd, binding capacity and Hill coefficient. When experimental values were compared to theoretical curves constructed using the estimated binding parameters, good fits were obtained. Binding parameters obtained by this method were not influenced by the presence of GTP gamma S (guanosine-5'-O-3-thiotriphosphate) in the incubation medium. The binding parameters agree reasonably well with those found in uterine cells, they support the existence of a medium affinity site and may allow for an explanation of some of the discrepancies between binding and response in this system.

  6. Affinity of the heparin binding motif of Noggin1 to heparan sulfate and its visualization in the embryonic tissues.

    Science.gov (United States)

    Nesterenko, Alexey M; Orlov, Eugeny E; Ermakova, Galina V; Ivanov, Igor A; Semenyuk, Pavel I; Orlov, Victor N; Martynova, Natalia Y; Zaraisky, Andrey G

    Heparin binding motifs were found in many secreted proteins and it was suggested that they are responsible for retardation of the protein diffusion within the intercellular space due to the binding to heparan sulfate proteoglycanes (HSPG). Here we used synthetic FITC labeled heparin binding motif (HBM peptide) of the Xenopus laevis secreted BMP inhibitor Noggin1 to study its diffusion along the surface of the heparin beads by FRAP method. As a result, we have found out that diffusivity of HBM-labeled FITC was indeed much lesser than those predicted by theoretical calculations even for whole protein of the Noggin size. We also compared by isothermal titration calorimetry the binding affinity of HBM and the control oligolysine peptide to several natural polyanions including heparan sulfate (HS), heparin, the bacterial dextran sulfate and salmon sperm DNA, and demonstrated that HBM significantly exceeds oligolysine peptide in the affinity to HS, heparin and DNA. By contrast, oligolysine peptide bound with higher affinity to dextran sulfate. We speculate that such a difference may ensure specificity of the morphogen binding to HSPG and could be explained by steric constrains imposed by different distribution of the negative charges along a given polymeric molecule. Finally, by using EGFP-HBM recombinant protein we have visualized the natural pattern of the Noggin1 binding sites within the X. laevis gastrula and demonstrated that these sites forms a dorsal-ventral concentration gradient, with a maximum in the dorsal blastopore lip. In sum, our data provide a quantitative basis for modeling the process of Noggin1 diffusion in embryonic tissues, considering its interaction with HSPG.

  7. Quantitative affinity chromatographic studies of mitochondrial cytochrome c binding to bacterial photosynthetic reaction center, reconstituted in liposome membranes and immobilized by detergent dialysis and avidin--biotin binding.

    Science.gov (United States)

    Yang, Q; Liu, X Y; Hara, M; Lundahl, P; Miyake, J

    2000-04-10

    In order to study the affinity binding of c-type cytochromes to the photosynthetic reaction center (RC) by quantitative affinity chromatography (QAC), RC from Rhodobacter sphaeroides was reconstituted into liposomes composed of egg phosphatidylcholine (EPC) and 2 mol% of biotinyl phosphatidylethanolamine simultaneously as the liposomes were formed and immobilized in (strept)avidin-coupled gel beads by rotary detergent dialysis. The immobilized amount was up to 80 nmol of RC and 33 micromol of lipid/g of moist gel in streptavidin-coupled Sephacryl S-1000 gel. By QAC frontal runs, retardation of mitochondrial cyt c on immobilized RC liposome columns was demonstrated. The dissociation constant for the RC-cyt c interaction was determined to be 0.20-0.57 microM. QAC studies also allowed evaluation of the orientation of reconstituted RC in immobilized liposomes by comparison of the total amount of cyt c binding sites with the amount of available binding sites obtained by QAC. It seems that the RC proteoliposomes immobilized in Sephacryl S-1000 gel exposed the cyt c binding sites on the outer surface of the liposomes due to effects of the gel network pore size and the resulting liposomal size.

  8. Evaluation of ligand-binding affinity using polynomial empirical scoring functions.

    Science.gov (United States)

    de Azevedo, Walter Filgueira; Dias, Raquel

    2008-10-15

    Assessing protein-ligand interaction is of great importance for virtual screening initiatives in order to discover new drugs. The present work describes a set of empirical scoring functions to assess the binding affinity, involving terms for intermolecular hydrogen bonds and contact surface. The results show that our methodology works better to predict protein-ligand affinity when compared with XSCORE, a popular empirical scoring function.

  9. APPLICATION OF IMMUNOGLOBULIN-BINDING PROTEINS A, G, L IN THE AFFINITY CHROMATOGRAPHY

    Directory of Open Access Journals (Sweden)

    О. V. Sviatenko

    2014-04-01

    Full Text Available Proteins A, G and L are native or recombinant proteins of microbial origin that bind to mammalian immunoglobulins. Preferably recombinant variants of proteins A, G, L are used in biotechnology for affinity sorbents production. Сomparative characteristics of proteins A, G, L and affinity sorbents on the basis of them, advantages and disadvantages of these proteins application as ligands in the affinity chromatography are done. Analysis of proteins A, G, L properties is presented. Binding specificities and affinities of these proteins differ between species and antibody subclass. Protein А has high affinity to human IgG1, IgG2, IgG4, mouse IgG2a, IgG2b, IgG3, goat and sheep IgG2, dog, cat, guinea pig, rabbit IgG. Protein G binds strongly to human, mouse, cow, goat, sheep and rabbit IgG. Protein L has ability of strong binding to immunoglobulin kappa-chains of human, mouse, rat and pig. Expediency of application of affinity chromatography with usage of sorbents on the basis of immobilized proteins A, G, L are shown for isolation and purification of antibodies different classes. Previously mentioned method is used as an alternative to conventional methods of protein purification, such as ion-exchange, hydrophobic interactions, metal affinity chromatography, ethanol precipitation due to simplicity in usage, possibility of one-step purification process, obtaining of proteins high level purity, multiuse at maintenance of proper storage and usage conditions. Affinity sorbents on the basis of immobilized proteins A, G, L are used not only for antibodies purification, but also for extraction of different antibodies fractions from blood serum.

  10. Ganglioside embedded in reconstituted lipoprotein binds cholera toxin with elevated affinity.

    Science.gov (United States)

    Bricarello, Daniel A; Mills, Emily J; Petrlova, Jitka; Voss, John C; Parikh, Atul N

    2010-09-01

    The ability to exogenously present cell-surface receptors in high-affinity conformations in a synthetic system offers an opportunity to provide host cells with protection from pathogenic toxins. This strategy requires improvement of the synthetic receptor binding affinity against its native counterpart, particularly with polyvalent toxins where clustering of membrane receptors can hinder binding. Here we demonstrate that reconstituted lipoprotein, nanometer-sized discoidal lipid bilayers bounded by apolipoprotein and functionalized by incorporation of pathogen receptors, provides a means to enhance toxin-receptor binding through molecular-level control over the receptor microenvironment (specifically, its rigidity, composition, and heterogeneity). Using a Foerster Resonance Energy Transfer (FRET)-based assay, we found that reconstituted lipoprotein incorporating low concentrations of ganglioside monosialotetrahexosylganglioside (GM1) binds polymeric cholera toxin with significantly higher affinity than liposomes or supported lipid bilayers, most likely a result of the enhanced control over receptor clustering provided by the lipoprotein platform. Using wide-area epifluorescence, we found that this enhanced binding capacity can be effectively utilized to divert cholera toxin away from populations of healthy mammalian cells. In summary, we found that reconstitutions of high-density lipoprotein can be engineered to include specific pathogen receptors; that their pathogen binding affinity is altered, presumably due to attenuation of receptor aggregation; and that these assemblies are effective at protecting cells from biological toxins.

  11. Shark Attack: high affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation.

    Science.gov (United States)

    Zielonka, Stefan; Weber, Niklas; Becker, Stefan; Doerner, Achim; Christmann, Andreas; Christmann, Christine; Uth, Christina; Fritz, Janine; Schäfer, Elena; Steinmann, Björn; Empting, Martin; Ockelmann, Pia; Lierz, Michael; Kolmar, Harald

    2014-12-10

    A novel method for stepwise in vitro affinity maturation of antigen-specific shark vNAR domains is described that exclusively relies on semi-synthetic repertoires derived from non-immunized sharks. Target-specific molecules were selected from a CDR3-randomized bamboo shark (Chiloscyllium plagiosum) vNAR library using yeast surface display as platform technology. Various antigen-binding vNAR domains were easily isolated by screening against several therapeutically relevant antigens, including the epithelial cell adhesion molecule (EpCAM), the Ephrin type-A receptor 2 (EphA2), and the human serine protease HTRA1. Affinity maturation was demonstrated for EpCAM and HTRA1 by diversifying CDR1 of target-enriched populations which allowed for the rapid selection of nanomolar binders. EpCAM-specific vNAR molecules were produced as soluble proteins and more extensively characterized via thermal shift assays and biolayer interferometry. Essentially, we demonstrate that high-affinity binders can be generated in vitro without largely compromising the desirable high thermostability of the vNAR scaffold.

  12. Identification of an adeno-associated virus binding epitope for AVB sepharose affinity resin

    Directory of Open Access Journals (Sweden)

    Qiang Wang

    2015-01-01

    Full Text Available Recent successes of adeno-associated virus (AAV–based gene therapy have created a demand for large-scale AAV vector manufacturing and purification techniques for use in clinical trials and beyond. During the development of purification protocols for rh.10, hu.37, AAV8, rh.64R1, AAV3B, and AAV9 vectors, based on a widely used affinity resin, AVB sepharose (GE, we found that, under the same conditions, different serotypes have different affinities to the resin, with AAV3B binding the best and AAV9 the poorest. Further analysis revealed a surface-exposed residue (amino acid number 665 in AAV8 VP1 numbering differs between the high-affinity AAV serotypes (serine in AAV3B, rh.10, and hu.37 and the low-affinity ones (asparagine in AAV8, rh.64R1, and AAV9. The residue locates within a surface-exposed, variable epitope flanked by highly conserved residues. The substitution of the epitope in AAV8, rh.64R1, and AAV9 with the corresponding epitope of AAV3B (SPAKFA resulted in greatly increased affinity to AVB sepharose with no reduction in the vectors’ in vitro potency. The presence of the newly identified AVB-binding epitope will be useful for affinity resin selection for the purification of novel AAV serotypes. It also suggests the possibility of vector engineering to yield a universal affinity chromatography purification method for multiple AAV serotypes.

  13. A comparison of myocardial beta-adrenoreceptor density and ligand binding affinity among selected teleost fishes.

    Science.gov (United States)

    Olsson, H I; Yee, N; Shiels, H A; Brauner, C; Farrell, A P

    2000-11-01

    This study quantified the cell surface beta-adrenoreceptor density and ligand binding affinity in the ventricular tissue of seven teleost species; skipjack tuna (Katsowonus pelamis), yellowfin tuna (Thunnus albacares), Pacific mackerel (Scomber japonicus), mahimahi (dolphin fish; Coryphaena hippurus), sockeye salmon (Oncorhynchus nerka), rainbow trout (Oncorhynchus mykiss) and an Antarctic nototheniid (Trematomus bernacchii). Beta-Adrenoreceptor density varied by almost fourfold among these species, being highest for the athletic fish: sockeye salmon among the salmonids and skipjack tuna among the scombrids. Beta-Adrenoreceptor density was lowest for the Antarctic icefish. Beta-Adrenoreceptor binding affinity varied by almost threefold. We conclude that there is a significant species-specific variability in myocardial beta-adrenoreceptor density and binding affinity and these interspecific differences cannot be attributed to temperature even though intraspecifically cold temperature can stimulate an increase in myocardial beta-adrenoreceptor density. Instead, we suggest that interspecifically myocardial beta-adrenoreceptor density is highest in fish that inhabit tropical water.

  14. Influence of the galloyl moiety in tea catechins on binding affinity for human serum albumin.

    Science.gov (United States)

    Minoda, Kanako; Ichikawa, Tatsuya; Katsumata, Tomoharu; Onobori, Ken-ichi; Mori, Taiki; Suzuki, Yukiko; Ishii, Takeshi; Nakayama, Tsutomu

    2010-01-01

    The major catechins of green tea extract are (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg). Recent research has indicated that catechins form complexes with human serum albumin (HSA) in blood, and differences in their binding affinity toward HSA are believed to modulate their bioavailability. In this study, we kinetically investigated the interaction between the catechins and HSA immobilized on a quartz-crystal microbalance (QCM). The association constants obtained from the frequency changes of QCM revealed interactions of ECg and EGCg with HSA that are 100 times stronger than those of EC and EGC. Furthermore, comparisons of these catechins by native-gel electrophoresis/blotting with redox-cycling staining revealed that, in a phosphate buffer, ECg and EGCg have a higher binding affinity toward HSA than EC and EGC. These observations indicate that catechins with a galloyl moiety have higher binding affinities toward HSA than catechins lacking a galloyl moiety.

  15. Identification of consensus binding sites clarifies FMRP binding determinants.

    Science.gov (United States)

    Anderson, Bart R; Chopra, Pankaj; Suhl, Joshua A; Warren, Stephen T; Bassell, Gary J

    2016-08-19

    Fragile X mental retardation protein (FMRP) is a multifunctional RNA-binding protein with crucial roles in neuronal development and function. Efforts aimed at elucidating how FMRP target mRNAs are selected have produced divergent sets of target mRNA and putative FMRP-bound motifs, and a clear understanding of FMRP's binding determinants has been lacking. To clarify FMRP's binding to its target mRNAs, we produced a shared dataset of FMRP consensus binding sequences (FCBS), which were reproducibly identified in two published FMRP CLIP sequencing datasets. This comparative dataset revealed that of the various sequence and structural motifs that have been proposed to specify FMRP binding, the short sequence motifs TGGA and GAC were corroborated, and a novel TAY motif was identified. In addition, the distribution of the FCBS set demonstrates that FMRP preferentially binds to the coding region of its targets but also revealed binding along 3' UTRs in a subset of target mRNAs. Beyond probing these putative motifs, the FCBS dataset of reproducibly identified FMRP binding sites is a valuable tool for investigating FMRP targets and function. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  16. Predicted metal binding sites for phytoremediation.

    Science.gov (United States)

    Sharma, Ashok; Roy, Sudeep; Tripathi, Kumar Parijat; Roy, Pratibha; Mishra, Manoj; Khan, Feroz; Meena, Abha

    2009-09-05

    Metal ion binding domains are found in proteins that mediate transport, buffering or detoxification of metal ions. The objective of the study is to design and analyze metal binding motifs against the genes involved in phytoremediation. This is being done on the basis of certain pre-requisite amino-acid residues known to bind metal ions/metal complexes in medicinal and aromatic plants (MAP's). Earlier work on MAP's have shown that heavy metals accumulated by aromatic and medicinal plants do not appear in the essential oil and that some of these species are able to grow in metal contaminated sites. A pattern search against the UniProtKB/Swiss-Prot and UniProtKB/TrEMBL databases yielded true positives in each case showing the high specificity of the motifs designed for the ions of nickel, lead, molybdenum, manganese, cadmium, zinc, iron, cobalt and xenobiotic compounds. Motifs were also studied against PDB structures. Results of the study suggested the presence of binding sites on the surface of protein molecules involved. PDB structures of proteins were finally predicted for the binding sites functionality in their respective phytoremediation usage. This was further validated through CASTp server to study its physico-chemical properties. Bioinformatics implications would help in designing strategy for developing transgenic plants with increased metal binding capacity. These metal binding factors can be used to restrict metal update by plants. This helps in reducing the possibility of metal movement into the food chain.

  17. The Binding Affinity and Molecular Basis of the Structure-Binding Relationship between Urinary Tamm-Horsfall Glycoprotein and Tumor Necrosis Factor-α

    Directory of Open Access Journals (Sweden)

    Chia-Li Yu

    2012-10-01

    Full Text Available In a previous study we noted significant THP binding to TNF-α, but did not explore the molecular basis of the structure-binding relationship. In this study, we used lectin-binding ELISA to assess the carbohydrate compositions of THP, BSA, IgG, TNF-α, and IFN-g. We identified β(1,4-N-acetylglucosamine oligomers (GlcNAc and GlcNAc/branched mannose in BSA, IgG, TNF-α, and THP, but not in IFN-g. These carbohydrate moieties mediated binding with THP. Small amounts of Siaα(2,3Gal/ GalNAc, Sia(2,6Gal/GalNAc, and mannose residues were also present in THP and TNF-α. Binding affinity (Kd between THP and TNF-α by Scatchard plot analysis was 1.4–1.7 × 10−6 M, lower than antigen-antibody or ligand-receptor binding affinities. To elucidate the structure-binding relationship of THP-TNF-α, THP was digested with neuraminidase, β-galactosidase, O-sialoglycoprotein endopeptidase, carboxypeptidase Y, or proteinase K. β-galactosidase increased binding capacity of THP for TNF-α. Monosaccharide inhibition suggested that α-methyl-D-mannoside, GlcNAc, and GalNAc, but not sialic acid, suppress THP-TNF-α binding as detected by ELISA. We conclude that sugar-lectin and sugar-protein interactions between cognate sites in THP and TNF-α mediate their binding.

  18. A set of robust fluorescent peptide probes for quantification of Cu(ii) binding affinities in the micromolar to femtomolar range.

    Science.gov (United States)

    Young, Tessa R; Wijekoon, Chathuri J K; Spyrou, Benjamin; Donnelly, Paul S; Wedd, Anthony G; Xiao, Zhiguang

    2015-03-01

    Reliable quantification of copper binding affinities and identification of the binding sites provide a molecular basis for an understanding of the nutritional roles and toxic effects of copper ions. Sets of chromophoric probes are now available that can quantify Cu(i) binding affinities from nanomolar to attomolar concentrations on a unified scale under in vitro conditions. Equivalent probes for Cu(ii) are lacking. This work reports development of a set of four fluorescent dansyl peptide probes (DP1-4) that can quantify Cu(ii) binding affinities from micromolar to femtomolar concentrations, also on a unified scale. The probes were constructed by conjugation of a dansyl group to four short peptides of specific design. Each was characterised by its dissociation constant KD, its pH dependence and the nature of its binding site. One equivalent of Cu(ii) is bound by the individual probes that display different and well-separated affinities at pH 7.4 (log KD = -8.1, -10.1, -12.3 and -14.1, respectively). Intense fluorescence is emitted at λmax ∼ 550 nm upon excitation at ∼330 nm. Binding of Cu(ii) quenches the fluorescence intensity linearly until one equivalent of Cu(ii) is bound. Multiple approaches and multiple affinity standards were employed to ensure reliability. Selected examples of application to well-characterised Cu(ii) binding peptides and proteins are presented. These include Aβ16 peptides, two naturally occurring Cu(ii)-chelating motifs in human serum and cerebrospinal fluid with sequences GHK and DAHK and two copper binding proteins, CopC from Pseudomonas syringae and PcoC from Escherichia coli. Previously reported affinities are reproduced, demonstrating that peptides DP1-4 form a set of robust and reliable probes for Cu(ii) binding to peptides and protein targets.

  19. Characterization of a small acyl-CoA-binding protein (ACBP) from Helianthus annuus L. and its binding affinities.

    Science.gov (United States)

    Aznar-Moreno, Jose A; Venegas-Calerón, Mónica; Du, Zhi-Yan; Garcés, Rafael; Tanner, Julian A; Chye, Mee-Len; Martínez-Force, Enrique; Salas, Joaquín J

    2016-05-01

    Acyl-CoA-binding proteins (ACBPs) bind to acyl-CoA esters and promote their interaction with other proteins, lipids and cell structures. Small class I ACBPs have been identified in different plants, such as Arabidopsis thaliana (AtACBP6), Brassica napus (BnACBP) and Oryza sativa (OsACBP1, OsACBP2, OsACBP3), and they are capable of binding to different acyl-CoA esters and phospholipids. Here we characterize HaACBP6, a class I ACBP expressed in sunflower (Helianthus annuus) tissues, studying the specificity of its corresponding recombinant HaACBP6 protein towards various acyl-CoA esters and phospholipids in vitro, particularly using isothermal titration calorimetry and protein phospholipid binding assays. This protein binds with high affinity to de novo synthetized derivatives palmitoly-CoA, stearoyl-CoA and oleoyl-CoA (Kd 0.29, 0.14 and 0.15 μM respectively). On the contrary, it showed lower affinity towards linoleoyl-CoA (Kd 5.6 μM). Moreover, rHaACBP6 binds to different phosphatidylcholine species (dipalmitoyl-PC, dioleoyl-PC and dilinoleoyl-PC), yet it displays no affinity towards other phospholipids like lyso-PC, phosphatidic acid and lysophosphatidic acid derivatives. In the light of these results, the possible involvement of this protein in sunflower oil synthesis is considered.

  20. Further characterization of the low and high affinity binding components of the thyrotropin receptor.

    Science.gov (United States)

    McQuade, R; Thomas, C G; Nayfeh, S N

    1986-05-29

    Following cross-linking with disuccinimidyl suberate and analysis by SDS-PAGE and autoradiography, both the high- and low-affinity TSH binding components exhibited two similar 125I-TSH-labeled bands, with Mr values of 80,000 and 68,000. IgG fractions from patients with Graves' disease inhibited 125I-TSH binding to both components, while normal IgG had no effect. Although not entirely conclusive, these results suggest that the high- and low-affinity components share similar subunit composition and antigenic determinants.

  1. Further characterization of the low and high affinity binding components of the thyrotropin receptor

    Energy Technology Data Exchange (ETDEWEB)

    McQuade, R.; Thomas, C.G. Jr.; Nayfeh, S.N.

    1986-05-29

    Following cross-linking with disuccinimdiyl suberate and analysis by SDS-PAGE and autoradiography, both the high- and low-affinity TSH binding components exhibited two similar /sup 125/I-TSH-labeled bands, with Mr values of 80,000 and 68,000. IgG fractions from patients with Graves' disease inhibited /sup 125/I-TSH binding to both components, while normal IgG had no effect. Although not entirely conclusive, these results suggest that the high- and low-affinity components share similar subunit composition and antigenic determinants.

  2. Synthesis of Glutamic Acid-based Cluster Galactosides and Their Binding Affinities with Liver Cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG,Xiao-Ru(张晓茹); Ll,Ying-Xia(李英霞); CHU,Shi-Dong(褚世栋); DING,Ning(丁宁); Ll,Chun-Xia(李春霞); GUAN,Hua-Shi(管华诗)

    2004-01-01

    Structurally well defined di-,tri-and tetra-valent cluster galactosides were synthesized in a convenient way.Oligo-glutamic acids were assembled as scaffolds.The presence of amine groups in these three ligands is expected to couple with drugs or genes for delivery.The binding affinities of these cluster galactoses to liver cells were determined by in vitro binding studies.Among them,the tetravalent cluster galactose (19) showed the highest affinity to liver cell.It is therefore a promising targeting device for the specific delivery of drugs or genes to parenchymal liver cells.

  3. CORAL: prediction of binding affinity and efficacy of thyroid hormone receptor ligands.

    Science.gov (United States)

    Toropova, A P; Toropov, A A; Benfenati, E

    2015-08-28

    Quantitative structure - activity relationships (QSARs) for binding affinity of thyroid hormone receptors based on attributes of molecular structure extracted from simplified molecular input-line entry systems (SMILES) are established using the CORAL software (http://www.insilico.eu/coral). The half maximal inhibitory concentration (IC50) is used as the measure of the binding affinity of thyroid hormone receptors. Molecular features which are statistically reliable promoters of increase and decrease for IC50 are suggested. The examples of modifications of molecular structure which lead to the increase or to the decrease of the endpoint are represented. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  4. Fatty acid and drug binding to a low-affinity component of human serum albumin, purified by affinity chromatography

    DEFF Research Database (Denmark)

    Vorum, H; Pedersen, A O; Honoré, B

    1992-01-01

    of two albumin components about 40% of the albumin having high affinity and about 60% having low affinity. By affinity chromatography we succeeded in purifying the low-affinity component from the mixture. The high-affinity component, however, could not be isolated. We further analyzed the fatty acid...

  5. A novel method for protein-ligand binding affinity prediction and the related descriptors exploration.

    Science.gov (United States)

    Li, Shuyan; Xi, Lili; Wang, Chengqi; Li, Jiazhong; Lei, Beilei; Liu, Huanxiang; Yao, Xiaojun

    2009-04-30

    In this study, a novel method was developed to predict the binding affinity of protein-ligand based on a comprehensive set of structurally diverse protein-ligand complexes (PLCs). The 1300 PLCs with binding affinity (493 complexes with K(d) and 807 complexes with K(i)) from the refined dataset of PDBbind Database (release 2007) were studied in the predictive model development. In this method, each complex was described using calculated descriptors from three blocks: protein sequence, ligand structure, and binding pocket. Thereafter, the PLCs data were rationally split into representative training and test sets by full consideration of the validation of the models. The molecular descriptors relevant to the binding affinity were selected using the ReliefF method combined with least squares support vector machines (LS-SVMs) modeling method based on the training data set. Two final optimized LS-SVMs models were developed using the selected descriptors to predict the binding affinities of K(d) and K(i). The correlation coefficients (R) of training set and test set for K(d) model were 0.890 and 0.833. The corresponding correlation coefficients for the K(i) model were 0.922 and 0.742, respectively. The prediction method proposed in this work can give better generalization ability than other recently published methods and can be used as an alternative fast filter in the virtual screening of large chemical database. (c) 2008 Wiley Periodicals, Inc.

  6. Binding affinities of anti-acetylcholine receptor autoantibodies in myasthenia gravis

    Energy Technology Data Exchange (ETDEWEB)

    Bray, J.J.; Drachman, D.B.

    1982-01-01

    Antibodies directed against acetylcholine (ACh) receptors are present in the sera of nearly 90% of patients with myasthenia gravis (MG), and are involved in the pathogenesis of this autoimmune disease. However, the antibody titers measured by the standard radioimmunoassay correspond poorly with the clinical severity of the disease. To determine whether this disparity could be accounted for by differences in the binding affinities of anti-ACh receptor antibodies in different patients, we have measured the binding affinities of these autoantibodies in 15 sera from MG patients. The affinity constants (K/sub o/), as determined by Scatchard analysis, were all in the range of 10/sup 10/ M/sup -1/, comparable to the highest values reported in immunized animals. The affinity constants were truly representative of the population of autoantibodies detected by the radioimmunoassay, as shown by the remarkable linearity of the Scatchard plots (r/sup 2/>0.90) and the close correlation between the antibody titers determined by extrapolation of the Scatchard plots and by saturation analysis (r = 0.99; p < 0.001). There was only a 6-fold variation in affinity constants measured in this series of patients despite widely differing antibody titers and severity of the disease. Factors other than the titer and affinity of anti-ACh receptor antibodies may correlate better with the clinical manifestations of MG.

  7. The MM/PBSA and MM/GBSA methods to estimate ligand-binding affinities

    Science.gov (United States)

    Genheden, Samuel; Ryde, Ulf

    2015-01-01

    Introduction: The molecular mechanics energies combined with the Poisson–Boltzmann or generalized Born and surface area continuum solvation (MM/PBSA and MM/GBSA) methods are popular approaches to estimate the free energy of the binding of small ligands to biological macromolecules. They are typically based on molecular dynamics simulations of the receptor–ligand complex and are therefore intermediate in both accuracy and computational effort between empirical scoring and strict alchemical perturbation methods. They have been applied to a large number of systems with varying success. Areas covered: The authors review the use of MM/PBSA and MM/GBSA methods to calculate ligand-binding affinities, with an emphasis on calibration, testing and validation, as well as attempts to improve the methods, rather than on specific applications. Expert opinion: MM/PBSA and MM/GBSA are attractive approaches owing to their modular nature and that they do not require calculations on a training set. They have been used successfully to reproduce and rationalize experimental findings and to improve the results of virtual screening and docking. However, they contain several crude and questionable approximations, for example, the lack of conformational entropy and information about the number and free energy of water molecules in the binding site. Moreover, there are many variants of the method and their performance varies strongly with the tested system. Likewise, most attempts to ameliorate the methods with more accurate approaches, for example, quantum-mechanical calculations, polarizable force fields or improved solvation have deteriorated the results. PMID:25835573

  8. Development of predictive models for predicting binding affinity of endocrine disrupting chemicals to fish sex hormone-binding globulin.

    Science.gov (United States)

    Liu, Huihui; Yang, Xianhai; Yin, Cen; Wei, Mengbi; He, Xiao

    2017-02-01

    Disturbing the transport process is a crucial pathway for endocrine disrupting chemicals (EDCs) exerting disrupting endocrine function. However, this mechanism has not received enough attention compared with that of hormones receptors and synthetase. Recently, we have explored the interaction between EDCs and sex hormone-binding globulin of human (hSHBG). In this study, interactions between EDCs and sex hormone-binding globulin of eight fish species (fSHBG) were investigated by employing classification methods and quantitative structure-activity relationships (QSAR). In the modeling, the relative binding affinity (RBA) of a chemical with 17β-estradiol binding to fSHBG was selected as the endpoint. Classification models were developed for two fish species, while QSAR models were established for the other six fish species. Statistical results indicated that the models had satisfactory goodness of fit, robustness and predictive ability, and that application domain covered a large number of endogenous and exogenous steroidal and non-steroidal chemicals. Additionally, by comparing the log RBA values, it was found that the same chemical may have different affinities for fSHBG from different fish species, thus species diversity should be taken into account. However, the affinity of fSHBG showed a high correlation for fishes within the same Order (i.e., Salmoniformes, Cypriniformes, Perciformes and Siluriformes), thus the fSHBG binding data for one fish species could be used to extrapolate other fish species in the same Order.

  9. Interplay between binding affinity and kinetics in protein-protein interactions.

    Science.gov (United States)

    Cao, Huaiqing; Huang, Yongqi; Liu, Zhirong

    2016-07-01

    To clarify the interplay between the binding affinity and kinetics of protein-protein interactions, and the possible role of intrinsically disordered proteins in such interactions, molecular simulations were carried out on 20 protein complexes. With bias potential and reweighting techniques, the free energy profiles were obtained under physiological affinities, which showed that the bound-state valley is deep with a barrier height of 12 - 33 RT. From the dependence of the affinity on interface interactions, the entropic contribution to the binding affinity is approximated to be proportional to the interface area. The extracted dissociation rates based on the Arrhenius law correlate reasonably well with the experimental values (Pearson correlation coefficient R = 0.79). For each protein complex, a linear free energy relationship between binding affinity and the dissociation rate was confirmed, but the distribution of the slopes for intrinsically disordered proteins showed no essential difference with that observed for ordered proteins. A comparison with protein folding was also performed. Proteins 2016; 84:920-933. © 2016 Wiley Periodicals, Inc.

  10. Calculating protein-ligand binding affinities with MMPBSA: Method and error analysis.

    Science.gov (United States)

    Wang, Changhao; Nguyen, Peter H; Pham, Kevin; Huynh, Danielle; Le, Thanh-Binh Nancy; Wang, Hongli; Ren, Pengyu; Luo, Ray

    2016-10-15

    Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) methods have become widely adopted in estimating protein-ligand binding affinities due to their efficiency and high correlation with experiment. Here different computational alternatives were investigated to assess their impact to the agreement of MMPBSA calculations with experiment. Seven receptor families with both high-quality crystal structures and binding affinities were selected. First the performance of nonpolar solvation models was studied and it was found that the modern approach that separately models hydrophobic and dispersion interactions dramatically reduces RMSD's of computed relative binding affinities. The numerical setup of the Poisson-Boltzmann methods was analyzed next. The data shows that the impact of grid spacing to the quality of MMPBSA calculations is small: the numerical error at the grid spacing of 0.5 Å is already small enough to be negligible. The impact of different atomic radius sets and different molecular surface definitions was further analyzed and weak influences were found on the agreement with experiment. The influence of solute dielectric constant was also analyzed: a higher dielectric constant generally improves the overall agreement with experiment, especially for highly charged binding pockets. The data also showed that the converged simulations caused slight reduction in the agreement with experiment. Finally the direction of estimating absolute binding free energies was briefly explored. Upon correction of the binding-induced rearrangement free energy and the binding entropy lost, the errors in absolute binding affinities were also reduced dramatically when the modern nonpolar solvent model was used, although further developments were apparently necessary to further improve the MMPBSA methods. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  11. Mannosylerythritol lipid, a yeast extracellular glycolipid, shows high binding affinity towards human immunoglobulin G

    Directory of Open Access Journals (Sweden)

    Ikegami Toru

    2001-09-01

    Full Text Available Abstract Background There have been many attempts to develop new materials with stability and high affinity towards immunoglobulins. Some of glycolipids such as gangliosides exhibit a high affinity toward immunoglobulins. However, it is considerably difficult to develop these glycolipids into the practical separation ligand due to their limited amounts. We thus focused our attention on the feasible use of "mannosylerythritol lipid A", a yeast glycolipid biosurfactant, as an alternative ligand for immunoglobulins, and undertook the investigation on the binding between mannosylerythritol lipid A (MEL-A and human immunoglobulin G (HIgG. Results In ELISA assay, MEL-A showed nearly the same binding affinity towards HIgG as that of bovine ganglioside GM1. Fab of human IgG was considered to play a more important role than Fc in the binding of HIgG by MEL-A. The bound amount of HIgG increased depending on the attached amount of MEL-A onto poly (2-hydroxyethyl methacrylate (polyHEMA beads, whereas the amount of human serum albumin slightly decreased. Binding-amount and -selectivity of HIgG towards MEL-A were influenced by salt species, salt concentration and pH in the buffer solution. The composite of MEL-A and polyHEMA, exhibited a significant binding constant of 1.43 × 106 (M-1 for HIgG, which is approximately 4-fold greater than that of protein A reported. Conclusions MEL-A shows high binding-affinity towards HIgG, and this is considered to be due to "multivalent effect" based on the binding molar ratio. This is the first report on the binding of a natural human antibody towards a yeast glycolipid.

  12. Peptide Nucleic Acids Having Enhanced Binding Affinity, Sequence Specificity and Solubility

    DEFF Research Database (Denmark)

    1998-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary DNA and RNA strands more strongly than a corresponding DNA strand, and exhibit increased sequence specificity and solubility. The peptide nucleic acids comprise ligands selected from a group consisting of naturally......-occurring nucleobases and non-naturally-occurring nucleobases attached to a polyamide backbone, and contain C1-C8 alkylamine side chains. Methods of enhancing the solubility, binding affinity and sequence specificity of PNAs are provided....

  13. Phylogenetic distribution of [3H]kainic acid receptor binding sites in neuronal tissue.

    Science.gov (United States)

    London, E D; Klemm, N; Coyle, J T

    1980-06-23

    The phylogenetic distribution of specific binding sites for kainic acid was determined in 14 species including invertebrates and vertebrates. The highest level of binding was observed in brains of the frog (Xenopus laevis), followed by the spiny dogfish (Heterodontus francisci), the goldfish (Carasius auratus) and the chick (Gallus domesticus). Although significant specific binding was noted in some of the lowest forms tested (e.g. Hydra littoralis), this was not a consistent observation in the invertebrates. In most cases, specific binding to both high and low affinity sites was detected; notable exceptions were the cockroach brain (Periplaneta americana), which had negligible high affinity binding, and the crayfish brain (Procambarus) which had negligible low affinity binding. In the spiny dogfish, the smooth dogfish and the chick, the highest level of binding occurred in cerebellum with less in the forebrain and the least in the medulla; in the mammalian species, the highest level of binding occurred in the forebrain structures with less in the cerebellum and least in the medulla. Eadie plots of the saturation isotherms for [3H]kainic acid revealed similar kinetics of binding for frog whole brain, rat forebrain and human parietal cortex with two apparent populations of binding sites: KD1 = 25--50 nM and KD2 = 3--14 nM. While binding in the spiny dogfish forebrain and human caudate nucleus occurred exclusively at a high affinity component, the cerebella of chick, rat and man exhibited only a low affinity binding site. In the 3 species studied most extensively, frog, rat and man, unlabeled kainic acid was the most potent inhibitor of the specific binding of [3H]-kainic acid. L-Glutamic acid was 20--20-fold less potent than kainic acid, and D-glutamic acid was 4--2500-fold less potent than its L-isomer. Reduction of the isopropylene side chain of kainic acid to form dihydrokainic acid decreased the affinity of the derivative 115--30,000-fold. Hill coefficients

  14. Relationships of the molecular structure of aldosterone derivatives with their binding affinity for mineralocorticoid receptor.

    Science.gov (United States)

    Yamakawa, M; Ezumi, K; Shiro, M; Nakai, H; Kamata, S; Matsui, T; Haga, N

    1986-12-01

    The molecular structures of 19-nor-11-deoxycorticosterone (III) and 21-hydroxypregna-4,11-diene-3,20-dione (IV) were determined by X-ray crystallographic analysis and the factors affecting the binding affinities for the mineralocorticoid receptor were examined with six aldosterone derivatives (I-VI) containing these two compounds. The most important factor was found to be the steric one; affinity increased with increasing flatness of the structure. The electronic factor may be a minor influence although a good relationship was found between the affinity and the 13C-NMR chemical shift of the C(5) atom. The factor playing no role in the binding is the hydrophobic one.

  15. Routes to improve binding capacities of affinity resins demonstrated for Protein A chromatography.

    Science.gov (United States)

    Müller, Egbert; Vajda, Judith

    2016-05-15

    Protein A chromatography is a well-established platform in downstream purification of monoclonal antibodies. Dynamic binding capacities are continuously increasing with almost every newly launched Protein A resin. Nevertheless, binding capacities of affinity chromatography resins cannot compete with binding capacities obtained with modern ion exchange media. Capacities of affinity resins are roughly 50% lower. High binding capacities of ion exchange media are supported by spacer technologies. In this article, we review existing spacer technologies of affinity chromatography resins. A yet known effective approach to increase the dynamic binding capacity of Protein A resins is oligomerization of the particular Protein A motifs. This resembles the tentacle technology used in ion exchange chromatography. Dynamic binding capacities of a hexameric ligand are roughly twice as high compared to capacities obtained with a tetrameric ligand. Further capacity increases up to 130mg/ml can be realized with the hexamer ligand, if the sodium phosphate buffer concentration is increased from 20 to 100mM. Equilibrium isotherms revealed a BET shape for the hexamer ligand at monoclonal antibody liquid phase concentrations higher than 9mg/ml. The apparent multilayer formation may be due to hydrophobic forces. Other quality attributes such as recovery, aggregate content, and overall purity of the captured monoclonal antibody are not affected.

  16. Prediction of the binding affinities of peptides to class II MHC using a regularized thermodynamic model

    Directory of Open Access Journals (Sweden)

    Mittelmann Hans D

    2010-01-01

    Full Text Available Abstract Background The binding of peptide fragments of extracellular peptides to class II MHC is a crucial event in the adaptive immune response. Each MHC allotype generally binds a distinct subset of peptides and the enormous number of possible peptide epitopes prevents their complete experimental characterization. Computational methods can utilize the limited experimental data to predict the binding affinities of peptides to class II MHC. Results We have developed the Regularized Thermodynamic Average, or RTA, method for predicting the affinities of peptides binding to class II MHC. RTA accounts for all possible peptide binding conformations using a thermodynamic average and includes a parameter constraint for regularization to improve accuracy on novel data. RTA was shown to achieve higher accuracy, as measured by AUC, than SMM-align on the same data for all 17 MHC allotypes examined. RTA also gave the highest accuracy on all but three allotypes when compared with results from 9 different prediction methods applied to the same data. In addition, the method correctly predicted the peptide binding register of 17 out of 18 peptide-MHC complexes. Finally, we found that suboptimal peptide binding registers, which are often ignored in other prediction methods, made significant contributions of at least 50% of the total binding energy for approximately 20% of the peptides. Conclusions The RTA method accurately predicts peptide binding affinities to class II MHC and accounts for multiple peptide binding registers while reducing overfitting through regularization. The method has potential applications in vaccine design and in understanding autoimmune disorders. A web server implementing the RTA prediction method is available at http://bordnerlab.org/RTA/.

  17. Calculation of Absolute Protein-Ligand Binding Affinity Using Path and Endpoint Approaches

    Science.gov (United States)

    2006-02-01

    an explicit solvent layer width of 10 Å. The hybrid solvent model (35) involves encapsulating a biological solute by a layer of water molecules...of cyclodextrin binding affinities: energy, entropy, and implications for drug design. Biophys. J. 87:3035–3049. 42. Janezic, D., R. M. Venable, and

  18. High-affinity small molecule-phospholipid complex formation: binding of siramesine to phosphatidicacid

    DEFF Research Database (Denmark)

    Khandelia, Himanshu

    2008-01-01

    , comparable to the affinities for the binding of small molecule ligands to proteins, was measured for phosphatidic acid (PA, mole fraction of XPA ) 0.2 in phosphatidylcholine vesicles), yielding a molecular partition coefficient of 240 ( 80 × 106. An MD simulation on the siramesine:PA interaction...

  19. Identifying Affinity Classes of Inorganic Materials Binding Sequences via a Graph-Based Model.

    Science.gov (United States)

    Du, Nan; Knecht, Marc R; Swihart, Mark T; Tang, Zhenghua; Walsh, Tiffany R; Zhang, Aidong

    2015-01-01

    Rapid advances in bionanotechnology have recently generated growing interest in identifying peptides that bind to inorganic materials and classifying them based on their inorganic material affinities. However, there are some distinct characteristics of inorganic materials binding sequence data that limit the performance of many widely-used classification methods when applied to this problem. In this paper, we propose a novel framework to predict the affinity classes of peptide sequences with respect to an associated inorganic material. We first generate a large set of simulated peptide sequences based on an amino acid transition matrix tailored for the specific inorganic material. Then the probability of test sequences belonging to a specific affinity class is calculated by minimizing an objective function. In addition, the objective function is minimized through iterative propagation of probability estimates among sequences and sequence clusters. Results of computational experiments on two real inorganic material binding sequence data sets show that the proposed framework is highly effective for identifying the affinity classes of inorganic material binding sequences. Moreover, the experiments on the structural classification of proteins (SCOP) data set shows that the proposed framework is general and can be applied to traditional protein sequences.

  20. Affinity maturation generates greatly improved xyloglucan-specific carbohydrate binding modules

    Directory of Open Access Journals (Sweden)

    Cicortas Gunnarsson Lavinia

    2009-10-01

    Full Text Available Abstract Background Molecular evolution of carbohydrate binding modules (CBM is a new approach for the generation of glycan-specific molecular probes. To date, the possibility of performing affinity maturation on CBM has not been investigated. In this study we show that binding characteristics such as affinity can be improved for CBM generated from the CBM4-2 scaffold by using random mutagenesis in combination with phage display technology. Results Two modified proteins with greatly improved affinity for xyloglucan, a key polysaccharide abundant in the plant kingdom crucial for providing plant support, were generated. Both improved modules differ from other existing xyloglucan probes by binding to galactose-decorated subunits of xyloglucan. The usefulness of the evolved binders was verified by staining of plant sections, where they performed better than the xyloglucan-binding module from which they had been derived. They discriminated non-fucosylated from fucosylated xyloglucan as shown by their ability to stain only the endosperm, rich in non-fucosylated xyloglucan, but not the integument rich in fucosylated xyloglucan, on tamarind seed sections. Conclusion We conclude that affinity maturation of CBM selected from molecular libraries based on the CBM4-2 scaffold is possible and has the potential to generate new analytical tools for detection of plant carbohydrates.

  1. Central melatonin binding sites in rainbow trout (Onchorhynchus mykiss).

    Science.gov (United States)

    Davies, B; Hannah, L T; Randall, C F; Bromage, N; Williams, L M

    1994-10-01

    A combination of in vitro autoradiography and membrane homogenate receptor assays has been used to localize and characterized 2-[125I]iodomelatonin binding sites in the brain of the rainbow trout (Onchorhynchus mykiss). Specific 2-[125I]iodomelatonin binding, defined as that displaced by 1 microM melatonin, increased linearly with increasing protein concentration in membrane homogenates of whole trout brain. Specific binding was both time and temperature dependent and reversible in the presence of 1 microM melatonin. Binding was saturable at between 100-150 pM 2-[125I]iodomelatonin and Scatchard analysis of saturation isotherms revealed a dissociation constant (Kd) of 15.00 +/- 0.95 pM and a maximum receptor number (Bmax) of 42.35 +/- 2.70 fm/mg protein (n = 16). Addition of 10(-4) M GTP gamma S (an analogue of guanosine triphosphate) to saturation isotherms apparently reduced the Bmax by 75% on average with no apparent change in the affinity of the binding. Scatchard analysis of saturation isotherms generated from whole brain membrane homogenates of trout kept on long days (15 hr light:9 hr dark) and killed either during the midlight or middark phase showed no significant differences in either the Kd or the Bmax of 2-[125I]iodomelatonin binding, although a robust rhythm in melatonin concentration was confirmed in these fish. Displacement of 2-[125I]iodomelatonin binding with increasing concentrations of competing ligands gave an order of potency of 2-iodomelatonin > melatonin > 5-HT. Localization of specific central 2-[125I]iodomelatonin binding in the rainbow trout showed high levels of binding associated with neuronal areas involved in the processing of visual signals, particularly the optic tectum and nucleus rotundus.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. A strategy to enhance the binding affinity of fluorophore-aptamer pairs for RNA tagging with neomycin conjugation.

    Science.gov (United States)

    Jeon, Jongho; Lee, Kyung Hyun; Rao, Jianghong

    2012-10-14

    Fluorogenic sulforhodamine-neomycin conjugates have been designed and synthesized for RNA tagging. Conjugates were fluorescently activated by binding to RNA aptamers and exhibited greater than 250-400 fold enhancement in binding affinity relative to corresponding unconjugated fluorophores.

  3. Structure-Based Understanding of Binding Affinity and Mode of Estrogen Receptor α Agonists and Antagonists

    Science.gov (United States)

    Barron, Mace G.

    2017-01-01

    The flexible hydrophobic ligand binding pocket (LBP) of estrogen receptor α (ERα) allows the binding of a wide variety of endocrine disruptors. Upon ligand binding, the LBP reshapes around the contours of the ligand and stabilizes the complex by complementary hydrophobic interactions and specific hydrogen bonds with the ligand. Here we present a framework for quantitative analysis of the steric and electronic features of the human ERα-ligand complex using three dimensional (3D) protein-ligand interaction description combined with 3D-QSAR approach. An empirical hydrophobicity density field is applied to account for hydrophobic contacts of ligand within the LBP. The obtained 3D-QSAR model revealed that hydrophobic contacts primarily determine binding affinity and govern binding mode with hydrogen bonds. Several residues of the LBP appear to be quite flexible and adopt a spectrum of conformations in various ERα-ligand complexes, in particular His524. The 3D-QSAR was combined with molecular docking based on three receptor conformations to accommodate receptor flexibility. The model indicates that the dynamic character of the LBP allows accommodation and stable binding of structurally diverse ligands, and proper representation of the protein flexibility is critical for reasonable description of binding of the ligands. Our results provide a quantitative and mechanistic understanding of binding affinity and mode of ERα agonists and antagonists that may be applicable to other nuclear receptors. PMID:28061508

  4. Influence of length and flexibility of spacers on the binding affinity of divalent ligands

    Directory of Open Access Journals (Sweden)

    Susanne Liese

    2015-05-01

    Full Text Available We present a quantitative model for the binding of divalent ligand–receptor systems. We study the influence of length and flexibility of the spacers on the overall binding affinity and derive general rules for the optimal ligand design. To this end, we first compare different polymeric models and determine the probability to simultaneously bind to two neighboring receptor binding pockets. In a second step the binding affinity of divalent ligands in terms of the IC50 value is derived. We find that a divalent ligand has the potential to bind more efficiently than its monovalent counterpart only, if the monovalent dissociation constant is lower than a critical value. This critical monovalent dissociation constant depends on the ligand-spacer length and flexibility as well as on the size of the receptor. Regarding the optimal ligand-spacer length and flexibility, we find that the average spacer length should be equal or slightly smaller than the distance between the receptor binding pockets and that the end-to-end spacer length fluctuations should be in the same range as the size of a receptor binding pocket.

  5. Influence of length and flexibility of spacers on the binding affinity of divalent ligands.

    Science.gov (United States)

    Liese, Susanne; Netz, Roland R

    2015-01-01

    We present a quantitative model for the binding of divalent ligand-receptor systems. We study the influence of length and flexibility of the spacers on the overall binding affinity and derive general rules for the optimal ligand design. To this end, we first compare different polymeric models and determine the probability to simultaneously bind to two neighboring receptor binding pockets. In a second step the binding affinity of divalent ligands in terms of the IC50 value is derived. We find that a divalent ligand has the potential to bind more efficiently than its monovalent counterpart only, if the monovalent dissociation constant is lower than a critical value. This critical monovalent dissociation constant depends on the ligand-spacer length and flexibility as well as on the size of the receptor. Regarding the optimal ligand-spacer length and flexibility, we find that the average spacer length should be equal or slightly smaller than the distance between the receptor binding pockets and that the end-to-end spacer length fluctuations should be in the same range as the size of a receptor binding pocket.

  6. Predicting the relative binding affinity of mineralocorticoid receptor antagonists by density functional methods

    Science.gov (United States)

    Roos, Katarina; Hogner, Anders; Ogg, Derek; Packer, Martin J.; Hansson, Eva; Granberg, Kenneth L.; Evertsson, Emma; Nordqvist, Anneli

    2015-12-01

    In drug discovery, prediction of binding affinity ahead of synthesis to aid compound prioritization is still hampered by the low throughput of the more accurate methods and the lack of general pertinence of one method that fits all systems. Here we show the applicability of a method based on density functional theory using core fragments and a protein model with only the first shell residues surrounding the core, to predict relative binding affinity of a matched series of mineralocorticoid receptor (MR) antagonists. Antagonists of MR are used for treatment of chronic heart failure and hypertension. Marketed MR antagonists, spironolactone and eplerenone, are also believed to be highly efficacious in treatment of chronic kidney disease in diabetes patients, but is contra-indicated due to the increased risk for hyperkalemia. These findings and a significant unmet medical need among patients with chronic kidney disease continues to stimulate efforts in the discovery of new MR antagonist with maintained efficacy but low or no risk for hyperkalemia. Applied on a matched series of MR antagonists the quantum mechanical based method gave an R2 = 0.76 for the experimental lipophilic ligand efficiency versus relative predicted binding affinity calculated with the M06-2X functional in gas phase and an R2 = 0.64 for experimental binding affinity versus relative predicted binding affinity calculated with the M06-2X functional including an implicit solvation model. The quantum mechanical approach using core fragments was compared to free energy perturbation calculations using the full sized compound structures.

  7. Computational Prediction of RNA-Binding Proteins and Binding Sites

    Directory of Open Access Journals (Sweden)

    Jingna Si

    2015-11-01

    Full Text Available Proteins and RNA interaction have vital roles in many cellular processes such as protein synthesis, sequence encoding, RNA transfer, and gene regulation at the transcriptional and post-transcriptional levels. Approximately 6%–8% of all proteins are RNA-binding proteins (RBPs. Distinguishing these RBPs or their binding residues is a major aim of structural biology. Previously, a number of experimental methods were developed for the determination of protein–RNA interactions. However, these experimental methods are expensive, time-consuming, and labor-intensive. Alternatively, researchers have developed many computational approaches to predict RBPs and protein–RNA binding sites, by combining various machine learning methods and abundant sequence and/or structural features. There are three kinds of computational approaches, which are prediction from protein sequence, prediction from protein structure, and protein-RNA docking. In this paper, we review all existing studies of predictions of RNA-binding sites and RBPs and complexes, including data sets used in different approaches, sequence and structural features used in several predictors, prediction method classifications, performance comparisons, evaluation methods, and future directions.

  8. Computational Prediction of RNA-Binding Proteins and Binding Sites.

    Science.gov (United States)

    Si, Jingna; Cui, Jing; Cheng, Jin; Wu, Rongling

    2015-01-01

    Proteins and RNA interaction have vital roles in many cellular processes such as protein synthesis, sequence encoding, RNA transfer, and gene regulation at the transcriptional and post-transcriptional levels. Approximately 6%-8% of all proteins are RNA-binding proteins (RBPs). Distinguishing these RBPs or their binding residues is a major aim of structural biology. Previously, a number of experimental methods were developed for the determination of protein-RNA interactions. However, these experimental methods are expensive, time-consuming, and labor-intensive. Alternatively, researchers have developed many computational approaches to predict RBPs and protein-RNA binding sites, by combining various machine learning methods and abundant sequence and/or structural features. There are three kinds of computational approaches, which are prediction from protein sequence, prediction from protein structure, and protein-RNA docking. In this paper, we review all existing studies of predictions of RNA-binding sites and RBPs and complexes, including data sets used in different approaches, sequence and structural features used in several predictors, prediction method classifications, performance comparisons, evaluation methods, and future directions.

  9. Ceruloplasmin has two nearly identical sites that bind myeloperoxidase.

    Science.gov (United States)

    Bakhautdin, Bakytzhan; Goksoy Bakhautdin, Esen; Fox, Paul L

    2014-10-31

    Ceruloplasmin (Cp) is a copper-containing ferroxidase with potent antioxidant activity. Cp is expressed by hepatocytes and activated macrophages and has been known as physiologic inhibitor of myeloperoxidase (MPO). Enzymatic activity of MPO produces anti-microbial agents and strong prooxidants such as hypochlorous acid and has a potential to damage host tissue at the sites of inflammation and infection. Thus Cp-MPO interaction and inhibition of MPO has previously been suggested as an important control mechanism of excessive MPO activity. Our aim in this study was to identify minimal Cp domain or peptide that interacts with MPO. We first confirmed Cp-MPO interaction by ELISA and surface plasmon resonance (SPR). SPR analysis of the interaction yielded 30nM affinity between Cp and MPO. We then designed and synthesized 87 overlapping peptides spanning the entire amino acid sequence of Cp. Each of the peptides was tested whether it binds to MPO by direct binding ELISA. Two of the 87 peptides, P18 and P76 strongly interacted with MPO. Amino acid sequence analysis of identified peptides revealed high sequence and structural homology between them. Further structural analysis of Cp's crystal structure by PyMOL software unfolded that both peptides represent surface-exposed sites of Cp and face nearly the same direction. To confirm our finding we raised anti-P18 antisera in rabbit and demonstrated that this antisera disrupts Cp-MPO binding and rescues MPO activity. Collectively, our results confirm Cp-MPO interaction and identify two nearly identical sites on Cp that specifically bind MPO. We propose that inhibition of MPO by Cp requires two nearly identical sites on Cp to bind homodimeric MPO simultaneously and at an angle of at least 120degrees, which, in turn, exerts tension on MPO and results in conformational change.

  10. SKF 525-A and cytochrome P-450 ligands inhibit with high affinity the binding of ( sup 3 H)dextromethorphan and. sigma. ligands to guinea pig brain

    Energy Technology Data Exchange (ETDEWEB)

    Klein, M.; Canoll, P.D.; Musacchio, J.M. (New York Univ. Medical Center, New York, NY (USA))

    1991-01-01

    The DM{sub 1}/{sigma}{sub 1} site binds dextromethorphan (DM) and {sigma} receptor ligands. The broad binding specificity of this site and its peculiar subcellular distribution prompted us to explore the possibility that this site is a member of the cytochrome P-450 superfamily of enzymes. We tested the effects of the liver microsomal monooxygenase inhibitor SKF 525-A (Proadifen), and other P-450 substrates on the binding of ({sup 3}H)dextromethorphan, ({sup 3}H)3- (3-Hydroxyphenyl) -N- (1-propyl) piperidine and (+)-({sup 3}H)1,3-Di-o-tolyl-guanidine (({sup 3}H)DTG) to the guinea pig brain. SKF 525-A, l-lobeline and GBR-12909 inhibited the binding of the three labeled ligands with nM affinity. Each drug has identical nM K{sub i} values for the high-affinity site labeled by the three ligands. This indicated that they displaced the labeled ligands from the common DM{sub 1}{sigma}{sub 1} site. Debrisoquine and sparteine, prototypical substrates for liver debrisoquine 4-hydroxylase, displayed K{sub i} values of 9-13 and 3-4 {mu}M respectively against the three labeled ligands. These results, the broad specificity of the DM{sub 1}/{sigma}{sub 1} binding site, and its peculiar subcellular distribution, raises the possibility that this binding site is a member of the cytochrome P-450 superfamily of isozymes, rather than a neurotransmitter receptor.

  11. Binding of lipoic acid induces conformational change and appearance of a new binding site in methylglyoxal modified serum albumin.

    Science.gov (United States)

    Suji, George; Khedkar, Santosh A; Singh, Sreelekha K; Kishore, Nand; Coutinho, Evans C; Bhor, Vikrant M; Sivakami, S

    2008-06-01

    The binding of lipoic acid (LA), to methylglyoxal (MG) modified BSA was studied using isothermal titration calorimetry in combination with enzyme kinetics and molecular modelling. The binding of LA to BSA was sequential with two sites, one with higher binding constant and another comparatively lower. In contrast the modified protein showed three sequential binding sites with a reduction in affinity at the high affinity binding site by a factor of 10. CD results show appreciable changes in conformation of the modified protein as a result of binding to LA. The inhibition of esterase like activity of BSA by LA revealed that it binds to site II in domain III of BSA. The pH dependence of esterase activity of native BSA indicated a catalytic group with a pK(a) = 7.9 +/- 0.1, assigned to Tyr411 with the conjugate base stabilised by interaction with Arg410. Upon modification by MG, this pK(a) increased to 8.13. A complex obtained by docking of LA to BSA and BSA in which Arg410 is modified to hydroimidazolone showed that the long hydrocarbon chain of lipoic acid sits in a cavity different from the one observed for unmodified BSA. The molecular electrostatic potential showed that the modification of Arg410 reduced the positive electrostatic potential around the protein-binding site. Thus it can be concluded that the modification of BSA by MG resulted in altered ligand binding characteristics due to changes in the internal geometry and electrostatic potential at the binding site.

  12. Screening for oligonucleotide binding affinity by a convenient fluorescence competition assay.

    Science.gov (United States)

    Harrison, J G; Liu, X; Balasubramanian, S

    1999-09-01

    A competitive homogeneous quenched fluorescence assay system is described for the high throughput screening of DNA conjugates that bind to single-stranded DNA. Fluorescence signal is generated by competitive binding of the sample molecule to a target strand labelled with a quencher probe, which is otherwise hybridised to a complementary strand containing a fluorescent probe. Thus fluorescence generated is related to the affinity of the sample. Competitive analysis of a number of peptide-oligonucleotide conjugates gave data that correlated well with the corresponding UV melting data. The assay will be useful for screening of large numbers of potential single-stranded binding molecules.

  13. Osteopontin binding to the alpha 4 integrin requires highest affinity integrin conformation, but is independent of post-translational modifications of osteopontin

    DEFF Research Database (Denmark)

    Hui, Tommy; Sørensen, Esben Skipper; Rittling, Susan R.

    2015-01-01

    with α4 integrin to that of VCAM and fibronectin. Jurkat cells, whose α4 integrins are inherently activated, adhered to different preparations of OPN in the presence of Mn2+: the EC50 of adhesion was not affected by phosphorylation or glycosylation status. Thrombin cleavage of OPN at the C......-terminus of the α4 integrin binding site also did not affect binding affinity. THP-1 cells express a low affinity conformation of the integrin and adhered to OPN only in the presence of Mn2+ plus PMA or an activating antibody. This was in contrast to VCAM and fibronectin: THP-1 cells adhered to these ligands...

  14. Ligand binding affinities of arctigenin and its demethylated metabolites to estrogen receptor alpha.

    Science.gov (United States)

    Jin, Jong-Sik; Lee, Jong-Hyun; Hattori, Masao

    2013-01-16

    Phytoestrogens are defined as plant-derived compounds with estrogen-like activities according to their chemical structures and activities. Plant lignans are generally categorized as phytoestrogens. It was reported that (-)-arctigenin, the aglycone of arctiin, was demethylated to (-)-dihydroxyenterolactone (DHENL) by Eubacterium (E.) sp. ARC-2. Through stepwise demethylation, E. sp. ARC-2 produced six intermediates, three mono-desmethylarctigenins and three di-desmethylarctigenins. In the present study, ligand binding affinities of (-)-arctigenin and its seven metabolites, including DHENL, were investigated for an estrogen receptor alpha, and found that demethylated metabolites had stronger binding affinities than (-)-arctigenin using a ligand binding screen assay method. The IC(50) value of (2R,3R)-2-(4-hydroxy-3-methoxybenzyl)-3-(3,4-dihydroxybenzyl)-butyrolactone was 7.9 × 10⁻⁴ M.

  15. Ligand Binding Affinities of Arctigenin and Its Demethylated Metabolites to Estrogen Receptor Alpha

    Directory of Open Access Journals (Sweden)

    Masao Hattori

    2013-01-01

    Full Text Available Phytoestrogens are defined as plant-derived compounds with estrogen-like activities according to their chemical structures and activities. Plant lignans are generally categorized as phytoestrogens. It was reported that (−-arctigenin, the aglycone of arctiin, was demethylated to (−-dihydroxyenterolactone (DHENL by Eubacterium (E. sp. ARC-2. Through stepwise demethylation, E. sp. ARC-2 produced six intermediates, three mono-desmethylarctigenins and three di-desmethylarctigenins. In the present study, ligand binding affinities of (−-arctigenin and its seven metabolites, including DHENL, were investigated for an estrogen receptor alpha, and found that demethylated metabolites had stronger binding affinities than (−-arctigenin using a ligand binding screen assay method. The IC50 value of (2R,3R-2-(4-hydroxy-3-methoxybenzyl-3-(3,4-dihydroxybenzyl-butyrolactone was 7.9 × 10−4 M.

  16. Novel cyclen-based linear polymer as a high-affinity binding material for DNA condensation

    Institute of Scientific and Technical Information of China (English)

    XIANG YongZhe; WANG Na; ZHANG Ji; LI Kun; ZHANG ZhongWei; LIN HongHui; YU XiaoQi

    2009-01-01

    A novel cyclen-based linear polyamine (POGEC) was designed and synthesized from the reaction be-tween 1,3-propanediol diglycidyl ether and 1,7-bis(diethoxyphosphory)-1,4,7,10-tetraazacyclod- odecane.High-affinity binding between POGEC and DNA was demonstrated by agarose gel electrophoresis and scanning electron microscopy (SEM). Moreover, the formed POGEC/DNA complex (termed polyplex) could be disassociated to release the free DNA through addition of the physiological concentration of NaCl solution. Fluorescence spectrum was used to measure the high-affinity binding and DNA con-densation capability of POGEC. Circular dichroism (CD) spectrum indicates that the DNA conformation did not change after binding to POEGC.

  17. False positive RNA binding activities after Ni-affinity purification from Escherichia coli.

    Science.gov (United States)

    Milojevic, Tetyana; Sonnleitner, Elisabeth; Romeo, Alessandra; Djinović-Carugo, Kristina; Bläsi, Udo

    2013-06-01

    A His-tag is often added by means of recombinant DNA technology to a heterologous protein of interest, which is then over-produced in Escherchia coli and purified by one-step immobilized metal-affinity chromatography (IMAC). Owing to the presence of 24 histidines at the C-termini of the hexameric E. coli RNA chaperone Hfq, the protein co-purifies with His-tagged proteins of interest. As Hfq can bind to distinct RNA substrates with high affinity, its presence can obscure studies performed with (putative) RNA binding activities purified by IMAC. Here, we present results for a seemingly positive RNA-binding activity, exemplifying that false-positive results can be avoided if the protein of interest is either subjected to further purification step(s) or produced in an E. coli hfq- strain.

  18. Novel cyclen-based linear polymer as a high-affinity binding material for DNA condensation

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    A novel cyclen-based linear polyamine (POGEC) was designed and synthesized from the reaction between 1,3-propanediol diglycidyl ether and 1,7-bis(diethoxyphosphory)-1,4,7,10-tetraazacyclod-odecane. High-affinity binding between POGEC and DNA was demonstrated by agarose gel electrophoresis and scanning electron microscopy (SEM). Moreover,the formed POGEC/DNA complex (termed polyplex) could be disassociated to release the free DNA through addition of the physiological concentration of NaCl solution. Fluorescence spectrum was used to measure the high-affinity binding and DNA condensation capability of POGEC. Circular dichroism (CD) spectrum indicates that the DNA conformation did not change after binding to POEGC.

  19. Effect of cysteamine on cytosolic somatostatin binding sites in rabbit duodenal mucosa.

    Science.gov (United States)

    Gonzalez-Guijarro, L; Lopez-Ruiz, M P; Bodegas, G; Prieto, J C; Arilla, E

    1987-04-01

    Administration of cysteamine in rabbits elicited a rapid depletion of both duodenal mucosa and plasma somatostatin. A significant reduction was observed within 5 min, returning toward control values by 150 min. The depletion of somatostatin was associated with an increase in the binding capacity and a decrease in the affinity of both high- and low-affinity binding sites present in cytosol of duodenal mucosa. Incubation of cytosolic fraction from control rabbits with 1 mM cysteamine did not modify somatostatin binding. Furthermore, addition of cysteamine at the time of binding assay did not affect the integrity of 125I-Tyr11-somatostatin. It is concluded that in vivo administration of cysteamine to rabbits depletes both duodenal mucosa and plasma somatostatin and leads to up-regulation of duodenal somatostatin binding sites.

  20. Is There Consistency between the Binding Affinity and Inhibitory Potential of Natural Polyphenols as α-amylase Inhibitors?

    Science.gov (United States)

    Xu, Wei; Shao, Rong; Xiao, Jianbo

    2016-07-26

    The inhibitory potential of natural polyphenols for α-amylases has attracted great interests among researchers. The structure-affinity properties of natural polyphenols binding to α-amylase and the structure-activity relationship of dietary polyphenols inhibiting α-amylase were deeply investigated. There is a lack of consistency between the structure-affinity relationship and the structure-activity relationship of natural polyphenols as α-amylase inhibitors. Is it consistent between the binding affinity and inhibitory potential of natural polyphenols as with α-amylase inhibitors? It was found that the consistency between the binding affinity and inhibitory potential of natural polyphenols as with α-amylase inhibitors is not equivocal. For example, there is no consistency between the binding affinity and the inhibitory potential of quercetin and its glycosides as α-amylase inhibitors. However, catechins with higher α-amylase inhibitory potential exhibited higher affinity with α-amylase.

  1. Selectivity and affinity determinants for ligand binding to the aromatic amino acid hydroxylases.

    Science.gov (United States)

    Teigen, Knut; McKinney, Jeffrey Alan; Haavik, Jan; Martínez, Aurora

    2007-01-01

    Hydroxylation of the aromatic amino acids phenylalanine, tyrosine and tryptophan is carried out by a family of non-heme iron and tetrahydrobiopterin (BH4) dependent enzymes, i.e. the aromatic amino acid hydroxylases (AAHs). The reactions catalyzed by these enzymes are important for biomedicine and their mutant forms in humans are associated with phenylketonuria (phenylalanine hydroxylase), Parkinson's disease and DOPA-responsive dystonia (tyrosine hydroxylase), and possibly neuropsychiatric and gastrointestinal disorders (tryptophan hydroxylase 1 and 2). We attempt to rationalize current knowledge about substrate and inhibitor specificity based on the three-dimensional structures of the enzymes and their complexes with substrates, cofactors and inhibitors. In addition, further insights on the selectivity and affinity determinants for ligand binding in the AAHs were obtained from molecular interaction field (MIF) analysis. We applied this computational structural approach to a rational analysis of structural differences at the active sites of the enzymes, a strategy that can help in the design of novel selective ligands for each AAH.

  2. Quantifying high-affinity binding of hydrophobic ligands by isothermal titration calorimetry.

    Science.gov (United States)

    Krainer, Georg; Broecker, Jana; Vargas, Carolyn; Fanghänel, Jörg; Keller, Sandro

    2012-12-18

    A fast and reliable quantification of the binding thermodynamics of hydrophobic high-affinity ligands employing a new calorimetric competition experiment is described. Although isothermal titration calorimetry is the method of choice for a quantitative characterization of intermolecular interactions in solution, a reliable determination of a dissociation constant (K(D)) is typically limited to the range 100 μM > K(D) > 1 nM. Interactions displaying higher or lower K(D) values can be assessed indirectly, provided that a suitable competing ligand is available whose K(D) falls within the directly accessible affinity window. This established displacement assay, however, requires the high-affinity ligand to be soluble at high concentrations in aqueous buffer and, consequently, poses serious problems in the study of protein binding involving small-molecule ligands dissolved in organic solvents--a familiar case in many drug-discovery projects relying on compound libraries. The calorimetric competition assay introduced here overcomes this limitation, thus allowing for a detailed thermodynamic description of high-affinity receptor-ligand interactions involving poorly water-soluble compounds. Based on a single titration of receptor into a dilute mixture of the two competing ligands, this competition assay provides accurate and precise values for the dissociation constants and binding enthalpies of both high- and moderate-affinity ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation and high-affinity protein-inhibitor interactions, and explore its potential and limitations with the aid of simulations and statistical analyses.

  3. Design of cyclic peptides that bind protein surfaces with antibody-like affinity.

    Science.gov (United States)

    Millward, Steven W; Fiacco, Stephen; Austin, Ryan J; Roberts, Richard W

    2007-09-21

    There is a pressing need for new molecular tools to target protein surfaces with high affinity and specificity. Here, we describe cyclic messenger RNA display with a trillion-member covalent peptide macrocycle library. Using this library, we have designed a number of high-affinity, redox-insensitive, cyclic peptides that target the signaling protein G alpha i1. In addition to cyclization, our library construction took advantage of an expanded genetic code, utilizing nonsense suppression to insert N-methylphenylalanine as a 21st amino acid. The designed macrocycles exhibit several intriguing features. First, the core motif seen in all of the selected variants is the same and shares an identical context with respect to the macrocyclic scaffold, consistent with the idea that selection simultaneously optimizes both the cyclization chemistry and the structural placement of the binding epitope. Second, detailed characterization of one molecule, cyclic G alpha i binding peptide (cycGiBP), demonstrates substantially enhanced proteolytic stability relative to that of the parent linear molecule. Third and perhaps most important, the cycGiBP peptide binds the target with very high affinity ( K i approximately 2.1 nM), similar to those of many of the best monoclonal antibodies and higher than that of the betagamma heterodimer, an endogenous G alpha i1 ligand. Overall the work provides a general route to design novel, low-molecular-weight, high-affinity ligands that target protein surfaces.

  4. Evaluation of Caesalpinia pulcherrima endospermic gum as affinity matrices for galactose-binding lectins interaction

    Directory of Open Access Journals (Sweden)

    Renata Chastinet Braga

    2011-04-01

    Full Text Available Lectins are proteins or glycoproteins able to bind, specifically and reversibly carbohydrates and glycoconjugates. Considering this ability, the utilization of Caesalpinia pulcherrima seeds polysaccharides as an affinity matrix was tested. The endospermic gum were solubilized in distinct concentrations of NaOH and treated with different amounts of epichlorohydrin (ECH forming affinity gels with variable capacity for interaction with galactose- binding lectins. The gel with an ECH/gum ration of 6.0mmol/g was selected as the best affinity matrix. The matrix presented different efficiencies in terms of isolating galactose-binding lectins. C. pulcherrima endospermic galactomannans were purified by ethanol precipitation and the purified galactomannan was crosslinked with the best formulation of gel. The Artocarpus incisa, Ricinus communis and Abrus precatorius lectins showed interactions of 11.5, 17.7 and 47.2mg of retained protein in 1g of gel, respectively; the Artocarpus integrifolia lectin showed the highest affinities (79.7mg/g. The heamaglutination assays confirmed the activity and SDS-PAGE electrophoresis confirmed the isolation of the lectins in a single-step procedure.

  5. Partial enterectomy decreases somatostatin-binding sites in residual intestine of rabbits.

    Science.gov (United States)

    Colas, B; Bodegas, G; Sanz, M; Prieto, J C; Arilla, E

    1988-05-01

    1. Three weeks after partial enterectomy in the rabbit there was an increased somatostatin concentration and a decreased number of somatostatin-binding sites (without changes in the corresponding affinity values) in the cytosol of the residual intestinal tissue, except in the terminal ileum and the colon. 2. Five weeks after surgery both the somatostatin concentration and the number of somatostatin-binding sites returned towards control values. 3. These results suggest that an increase in bowel somatostatin content could lead to down-regulation of somatostatin-binding sites in the intestinal mucosa.

  6. Low density and high affinity of platelet [3H]paroxetine binding in women with bulimia nervosa.

    Science.gov (United States)

    Ekman, Agneta; Sundblad-Elverfors, Charlotta; Landén, Mikael; Eriksson, Tomas; Eriksson, Elias

    2006-06-15

    Impaired serotonin transmission has been suggested to be implicated in the pathophysiology of bulimia nervosa. As an indirect measure of brain serotonergic activity, the binding of tritiated ligands to platelet serotonin transporters has been studied in bulimia nervosa as well as in other putatively serotonin-related psychiatric disorders. In this study, the density and affinity of platelet serotonin transporters were assessed in 20 women meeting the DSM-IV criteria for bulimia nervosa and in 14 controls without previous or ongoing eating disorder using [(3)H]paroxetine as a ligand. In comparison to controls, women with bulimia nervosa had a significantly reduced number of platelet binding sites (B(max) = 721 +/- 313 vs. 1145 +/- 293 fmol/mg protein) and an increase in the affinity for the ligand demonstrated by a lower dissociaton constant (K(d) = 33 +/- 10 vs. 44 +/- 10 pM). A significant correlation between B(max) and K(d) values was found in patients but not in controls. Our results support the notion that bulimia nervosa is associated with a reduction in platelet serotonin transporter density. In addition, our study is the first to report that this reduced transporter density in women with bulimia nervosa is accompanied by an increase in the affinity of the transporter for the ligand.

  7. Histidine-rich glycoprotein binds fibrin(ogen) with high affinity and competes with thrombin for binding to the gamma'-chain.

    Science.gov (United States)

    Vu, Trang T; Stafford, Alan R; Leslie, Beverly A; Kim, Paul Y; Fredenburgh, James C; Weitz, Jeffrey I

    2011-09-01

    Histidine-rich glycoprotein (HRG) is an abundant protein that binds fibrinogen and other plasma proteins in a Zn(2+)-dependent fashion but whose function is unclear. HRG has antimicrobial activity, and its incorporation into fibrin clots facilitates bacterial entrapment and killing and promotes inflammation. Although these findings suggest that HRG contributes to innate immunity and inflammation, little is known about the HRG-fibrin(ogen) interaction. By immunoassay, HRG-fibrinogen complexes were detected in Zn(2+)-supplemented human plasma, a finding consistent with a high affinity interaction. Surface plasmon resonance determinations support this concept and show that in the presence of Zn(2+), HRG binds the predominant γ(A)/γ(A)-fibrinogen and the γ-chain elongated isoform, γ(A)/γ'-fibrinogen, with K(d) values of 9 nm. Likewise, (125)I-labeled HRG binds γ(A)/γ(A)- or γ(A)/γ'-fibrin clots with similar K(d) values when Zn(2+) is present. There are multiple HRG binding sites on fibrin(ogen) because HRG binds immobilized fibrinogen fragment D or E and γ'-peptide, an analog of the COOH terminus of the γ'-chain that mediates the high affinity interaction of thrombin with γ(A)/γ'-fibrin. Thrombin competes with HRG for γ'-peptide binding and displaces (125)I-HRG from γ(A)/γ'-fibrin clots and vice versa. Taken together, these data suggest that (a) HRG circulates in complex with fibrinogen and that the complex persists upon fibrin formation, and (b) by competing with thrombin for γ(A)/γ'-fibrin binding, HRG may modulate coagulation. Therefore, the HRG-fibrin interaction may provide a novel link between coagulation, innate immunity, and inflammation.

  8. Defining the sequence specificity of DNA-binding proteins by selecting binding sites from random-sequence oligonucleotides: analysis of yeast GCN4 protein.

    OpenAIRE

    Oliphant, A R; Brandl, C J; Struhl, K

    1989-01-01

    We describe a new method for accurately defining the sequence recognition properties of DNA-binding proteins by selecting high-affinity binding sites from random-sequence DNA. The yeast transcriptional activator protein GCN4 was coupled to a Sepharose column, and binding sites were isolated by passing short, random-sequence oligonucleotides over the column and eluting them with increasing salt concentrations. Of 43 specifically bound oligonucleotides, 40 contained the symmetric sequence TGA(C...

  9. The high-affinity peptidoglycan binding domain of Pseudomonas phage endolysin KZ144

    Energy Technology Data Exchange (ETDEWEB)

    Briers, Yves [Division of Gene Technology, Department of Biosystems, Katholieke Universiteit Leuven, Kasteelpark Arenberg 21, B-3001 Leuven (Belgium); Schmelcher, Mathias; Loessner, Martin J. [Institute of Food Science and Nutrition, ETH Zuerich, Schmelzbergstrasse 7, CH-8092 Zuerich (Switzerland); Hendrix, Jelle; Engelborghs, Yves [Laboratory of Biomolecular Dynamics, Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200G, B-3001 Leuven (Belgium); Volckaert, Guido [Division of Gene Technology, Department of Biosystems, Katholieke Universiteit Leuven, Kasteelpark Arenberg 21, B-3001 Leuven (Belgium); Lavigne, Rob, E-mail: rob.lavigne@biw.kuleuven.be [Division of Gene Technology, Department of Biosystems, Katholieke Universiteit Leuven, Kasteelpark Arenberg 21, B-3001 Leuven (Belgium)

    2009-05-29

    The binding affinity of the N-terminal peptidoglycan binding domain of endolysin KZ144 (PBD{sub KZ}), originating from Pseudomonas aeruginosa bacteriophage {phi}KZ, has been examined using a fusion protein of PBD{sub KZ} and green fluorescent protein (PBD{sub KZ}-GFP). A fluorescence recovery after photobleaching analysis of bound PBD{sub KZ}-GFP molecules showed less than 10% fluorescence recovery in the bleached area within 15 min. Surface plasmon resonance analysis confirmed this apparent high binding affinity revealing an equilibrium affinity constant of 2.95 x 10{sup 7} M{sup -1} for the PBD{sub KZ}-peptidoglycan interaction. This unique domain, which binds to the peptidoglycan of all tested Gram-negative species, was harnessed to improve the specific activity of the peptidoglycan hydrolase domain KMV36C. The chimeric peptidoglycan hydrolase (PBD{sub KZ}-KMV36C) exhibits a threefold higher specific activity than the native catalytic domain (KMV36C). These results demonstrate that the modular assembly of functional domains is a rational approach to improve the specific activity of endolysins from phages infecting Gram-negatives.

  10. A Combinatorial Approach to Biophysically Characterise Chemokine-Glycan Binding Affinities for Drug Development

    Directory of Open Access Journals (Sweden)

    Tanja Gerlza

    2014-07-01

    Full Text Available Chemokine binding to glycosaminoglycans (GAGs is recognised to be an important step in inflammation and other pathological disorders like tumor growth and metastasis. Although different ways and strategies to interfere with these interactions are being pursued, no major breakthrough in the development of glycan-targeting drugs has been reported so far. We have engineered CXCL8 towards a dominant-negative form of this chemokine (dnCXCL8 which was shown to be highly active in various inflammatory animal models due to its inability to bind/activate the cognate CXCL8 GPC receptors on neutrophils in combination with its significantly increased GAG-binding affinity [1]. For the development of GAG-targeting chemokine-based biopharmaceuticals, we have established a repertoire of methods which allow the quantification of protein-GAG interactions. Isothermal fluorescence titration (IFT, surface plasmon resonance (SPR, isothermal titration calorimetry (ITC, and a novel ELISA-like competition assay (ELICO have been used to determine Kd and IC50 values for CXCL8 and dnCXCL8 interacting with heparin and heparan sulfate (HS, the proto-typical members of the GAG family. Although the different methods gave different absolute affinities for the four protein-ligand pairs, the relative increase in GAG-binding affinity of dnCXCL8 compared to the wild type chemokine was found by all methods. In combination, these biophysical methods allow to discriminate between unspecific and specific protein-GAG interactions.

  11. Protein-binding affinity of leucaena condensed tannins of differing molecular weights.

    Science.gov (United States)

    Huang, Xiao Dan; Liang, Juan Boo; Tan, Hui Yin; Yahya, Rosiyah; Long, Ruijun; Ho, Yin Wan

    2011-10-12

    Depending on their source, concentration, chemical structure, and molecular weight, condensed tannins (CTs) form insoluble complexes with protein, which could lead to ruminal bypass protein, benefiting animal production. In this study, CTs from Leuceana leucocephala hybrid were fractionated into five fractions by a size exclusion chromatography procedure. The molecular weights of the CT fractions were determined using Q-TOF LC-MS, and the protein-binding affinities of the respective CT fractions were determined using a protein precipitation assay with bovine serum albumin (BSA) as the standard protein. The calculated number-average molecular weights (M(n)) were 1348.6, 857.1, 730.1, 726.0, and 497.1, and b values (the b value represents the CT quantity that is needed to bind half of the maximum precipitable BSA) of the different molecular weight fractions were 0.381, 0.510, 0.580, 0.636, and 0.780 for fractions 1, 2, 3, 4, and 5, respectively. The results indicated that, in general, CTs of higher molecular weight fractions have stronger protein-binding affinity than those of lower molecular weights. However, the number of hydroxyl units within the structure of CT polymers also affects the protein-binding affinity.

  12. Affinity and activity of non-native quinones at the QB site of bacterial photosynthetic reaction centers

    Science.gov (United States)

    Zhang, Xinyu; Gunner, M. R.

    2014-01-01

    Purple, photosynthetic reaction centers (RCs) from Rb. sphaeroides bacteria use UQ10 as primary (QA) and secondary (QB) electron acceptors. Many quinones reconstitute QA function, while few will act as QB. Nine quinones were tested for their ability to bind and reconstitute QA and QB function. Only ubiquinone (UQ) reconstitutes both QA and QB function in the same protein. The affinities of the non-native quinones for the QB site were determined by a competitive inhibition assay. The affinities of benzoquinones (BQ), napthoquinone (NQ) and 2-methyl-NQ for the QB site are 7±3 times weaker than for the QA site. However, di-ortho substituted NQs and anthraquinone bind tightly to the QA site (Kd ≤200 nM) and ≥1000 times more weakly to the QB site, perhaps setting a limit on the size of the site. With a low potential electron donor (2-methyl, 3-dimethylamino-1,4-Napthoquinone (Me-diMeAm-NQ)) at QA, QB reduction is 260 meV more favorable than with UQ as QA. Electron transfer from Me-diMeAm-NQ at the QA site to NQ at the QB site can be detected. In the QB site the NQ semiquinone is estimated to be ≈ 60–100 meV higher in energy than the UQ semiquinone, while in the QA site the semiquinone energy level is similar or lower with NQ than with UQ. Thus, the NQ semiquinone is more stable in the QA than QB site. In contrast, the native UQ semiquinone is ≈ 60 meV lower in energy in the QB than the QA site, stabilizing forward electron transfer from QA to QB. PMID:23715773

  13. Site-specific fab fragment biotinylation at the conserved nucleotide binding site for enhanced Ebola detection.

    Science.gov (United States)

    Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

    2015-07-01

    The nucleotide binding site (NBS) is a highly conserved region between the variable light and heavy chains at the Fab domains of all antibodies, and a small molecule that we identified, indole-3-butyric acid (IBA), binds specifically to this site. Fab fragment, with its small size and simple production methods compared to intact antibody, is good candidate for use in miniaturized diagnostic devices and targeted therapeutic applications. However, commonly used modification techniques are not well suited for Fab fragments as they are often more delicate than intact antibodies. Fab fragments are of particular interest for sensor surface functionalization but immobilization results in damage to the antigen binding site and greatly reduced activity due to their truncated size that allows only a small area that can bind to surfaces without impeding antigen binding. In this study, we describe an NBS-UV photocrosslinking functionalization method (UV-NBS(Biotin) in which a Fab fragment is site-specifically biotinylated with an IBA-EG11-Biotin linker via UV energy exposure (1 J/cm(2)) without affecting its antigen binding activity. This study demonstrates successful immobilization of biotinylated Ebola detecting Fab fragment (KZ52 Fab fragment) via the UV-NBS(Biotin) method yielding 1031-fold and 2-fold better antigen detection sensitivity compared to commonly used immobilization methods: direct physical adsorption and NHS-Biotin functionalization, respectively. Utilization of the UV-NBS(Biotin) method for site-specific conjugation to Fab fragment represents a proof of concept use of Fab fragment for various diagnostic and therapeutic applications with numerous fluorescent probes, affinity molecules and peptides.

  14. Specificity residues determine binding affinity for two-component signal transduction systems.

    Science.gov (United States)

    Willett, Jonathan W; Tiwari, Nitija; Müller, Susanne; Hummels, Katherine R; Houtman, Jon C D; Fuentes, Ernesto J; Kirby, John R

    2013-11-05

    Two-component systems (TCS) comprise histidine kinases and their cognate response regulators and allow bacteria to sense and respond to a wide variety of signals. Histidine kinases (HKs) phosphorylate and dephosphorylate their cognate response regulators (RRs) in response to stimuli. In general, these reactions appear to be highly specific and require an appropriate association between the HK and RR proteins. The Myxococcus xanthus genome encodes one of the largest repertoires of signaling proteins in bacteria (685 open reading frames [ORFs]), including at least 127 HKs and at least 143 RRs. Of these, 27 are bona fide NtrC-family response regulators, 21 of which are encoded adjacent to their predicted cognate kinases. Using system-wide profiling methods, we determined that the HK-NtrC RR pairs display a kinetic preference during both phosphotransfer and phosphatase functions, thereby defining cognate signaling systems in M. xanthus. Isothermal titration calorimetry measurements indicated that cognate HK-RR pairs interact with dissociation constants (Kd) of approximately 1 µM, while noncognate pairs had no measurable binding. Lastly, a chimera generated between the histidine kinase, CrdS, and HK1190 revealed that residues conferring phosphotransfer and phosphatase specificity dictate binding affinity, thereby establishing discrete protein-protein interactions which prevent cross talk. The data indicate that binding affinity is a critical parameter governing system-wide signaling fidelity for bacterial signal transduction proteins. Using in vitro phosphotransfer and phosphatase profiling assays and isothermal titration calorimetry, we have taken a system-wide approach to demonstrate specificity for a family of two-component signaling proteins in Myxococcus xanthus. Our results demonstrate that previously identified specificity residues dictate binding affinity and that phosphatase specificity follows phosphotransfer specificity for cognate HK-RR pairs. The data

  15. Computational identification of uncharacterized cruzain binding sites.

    Directory of Open Access Journals (Sweden)

    Jacob D Durrant

    Full Text Available Chagas disease, caused by the unicellular parasite Trypanosoma cruzi, claims 50,000 lives annually and is the leading cause of infectious myocarditis in the world. As current antichagastic therapies like nifurtimox and benznidazole are highly toxic, ineffective at parasite eradication, and subject to increasing resistance, novel therapeutics are urgently needed. Cruzain, the major cysteine protease of Trypanosoma cruzi, is one attractive drug target. In the current work, molecular dynamics simulations and a sequence alignment of a non-redundant, unbiased set of peptidase C1 family members are used to identify uncharacterized cruzain binding sites. The two sites identified may serve as targets for future pharmacological intervention.

  16. Cyr61/CCN1 displays high-affinity binding to the somatomedin B(1-44 domain of vitronectin.

    Directory of Open Access Journals (Sweden)

    Ivo M B Francischetti

    Full Text Available BACKGROUND: Cyr61 is a member of the CCN (Cyr61, connective tissue growth, NOV family of extracellular-associated (matricellular proteins that present four distinct functional modules, namely insulin-like growth factor binding protein (IGFBP, von Willebrand factor type C (vWF, thrombospondin type 1 (TSP, and C-terminal growth factor cysteine knot (CT domain. While heparin sulphate proteoglycans reportedly mediate the interaction of Cyr61 with the matrix and cell surface, the role of other extracellular associated proteins has not been revealed. METHODS AND FINDINGS: In this report, surface plasmon resonance (SPR experiments and solid-phase binding assays demonstrate that recombinant Cyr61 interacts with immobilized monomeric or multimeric vitronectin (VTNC with K(D in the nanomolar range. Notably, the binding site for Cyr61 was identified as the somatomedin B domain (SMTB(1-44 of VTNC, which mediates its interaction with PAI-1, uPAR, and integrin alphav beta3. Accordingly, PAI-1 outcompetes Cyr61 for binding to immobilized SMTB(1-44, and Cyr61 attenuates uPAR-mediated U937 adhesion to VTNC. In contrast, isothermal titration calorimetry shows that Cyr61 does not display high-affinity binding for SMTB(1-44 in solution. Nevertheless, competitive ELISA revealed that multimeric VTNC, heat-modified monomeric VTNC, or SMTB(1-44 at high concentrations attenuate Cyr61 binding to immobilized VTNC, while monomeric VTNC was ineffective. Therefore, immobilization of VTNC exposes cryptic epitopes that recognize Cyr61 with high affinity, as reported for a number of antibodies, beta-endorphin, and other molecules. CONCLUSIONS: The finding that Cyr61 interacts with the SMTB(1-44 domain suggests that VTNC represent a point of anchorage for CCN family members to the matrix. Results are discussed in the context of the role of CCN and VTNC in matrix biology and angiogenesis.

  17. Quick and Simple Detection Technique to Assess the Binding of Antimicrotubule Agents to the Colchicine-Binding Site

    Directory of Open Access Journals (Sweden)

    Fortin Sébastien

    2010-04-01

    Full Text Available Abstract Development of antimitotic binding to the colchicine-binding site for the treatment of cancer is rapidly expanding. Numerous antimicrotubule agents are prepared every year, and the determination of their binding affinity to tubulin requires the use of purified tubulins and radiolabeled ligands. Such a procedure is costly and time-consuming and therefore is limited to the most promising candidates. Here, we report a quick and inexpensive method that requires only usual laboratory resources to assess the binding of antimicrotubules to colchicine-binding site. The method is based on the ability of N,N'-ethylene-bis(iodoacetamide (EBI to crosslink in living cells the cysteine residues at position 239 and 354 of β-tubulin, residues which are involved in the colchicine-binding site. The β-tubulin adduct formed by EBI is easily detectable by Western blot as a second immunoreacting band of β-tubulin that migrates faster than β-tubulin. The occupancy of colchicine-binding site by pertinent antimitotics inhibits the formation of the EBI: β-tubulin adduct, resulting in an assay that allows the screening of new molecules targeting this binding site.

  18. Quick and Simple Detection Technique to Assess the Binding of Antimicrotubule Agents to the Colchicine-Binding Site

    Directory of Open Access Journals (Sweden)

    Moreau Emmanuel

    2010-01-01

    Full Text Available Abstract Development of antimitotic binding to the colchicine-binding site for the treatment of cancer is rapidly expanding. Numerous antimicrotubule agents are prepared every year, and the determination of their binding affinity to tubulin requires the use of purified tubulins and radiolabeled ligands. Such a procedure is costly and time-consuming and therefore is limited to the most promising candidates. Here, we report a quick and inexpensive method that requires only usual laboratory resources to assess the binding of antimicrotubules to colchicine-binding site. The method is based on the ability of N,N'-ethylene-bis(iodoacetamide (EBI to crosslink in living cells the cysteine residues at position 239 and 354 of β-tubulin, residues which are involved in the colchicine-binding site. The β-tubulin adduct formed by EBI is easily detectable by Western blot as a second immunoreacting band of β-tubulin that migrates faster than β-tubulin. The occupancy of colchicine-binding site by pertinent antimitotics inhibits the formation of the EBI: β-tubulin adduct, resulting in an assay that allows the screening of new molecules targeting this binding site.

  19. Functional identification and characterization of sodium binding sites in Na symporters.

    Science.gov (United States)

    Loo, Donald D F; Jiang, Xuan; Gorraitz, Edurne; Hirayama, Bruce A; Wright, Ernest M

    2013-11-19

    Sodium cotransporters from several different gene families belong to the leucine transporter (LeuT) structural family. Although the identification of Na(+) in binding sites is beyond the resolution of the structures, two Na(+) binding sites (Na1 and Na2) have been proposed in LeuT. Na2 is conserved in the LeuT family but Na1 is not. A biophysical method has been used to measure sodium dissociation constants (Kd) of wild-type and mutant human sodium glucose cotransport (hSGLT1) proteins to identify the Na(+) binding sites in hSGLT1. The Na1 site is formed by residues in the sugar binding pocket, and their mutation influences sodium binding to Na1 but not to Na2. For the canonical Na2 site formed by two -OH side chains, S392 and S393, and three backbone carbonyls, mutation of S392 to cysteine increased the sodium Kd by sixfold. This was accompanied by a dramatic reduction in the apparent sugar and phlorizin affinities. We suggest that mutation of S392 in the Na2 site produces a structural rearrangement of the sugar binding pocket to disrupt both the binding of the second Na(+) and the binding of sugar. In contrast, the S393 mutations produce no significant changes in sodium, sugar, and phlorizin affinities. We conclude that the Na2 site is conserved in hSGLT1, the side chain of S392 and the backbone carbonyl of S393 are important in the first Na(+) binding, and that Na(+) binding to Na2 promotes binding to Na1 and also sugar binding.

  20. A Unitary Anesthetic Binding Site at High Resolution

    Energy Technology Data Exchange (ETDEWEB)

    Vedula, L. Sangeetha; Brannigan, Grace; Economou, Nicoleta J.; Xi, Jin; Hall, Michael A.; Liu, Renyu; Rossi, Matthew J.; Dailey, William P.; Grasty, Kimberly C.; Klein, Michael L.; Eckenhoff, Roderic G.; Loll, Patrick J.; (Drexel-MED); (UPENN)

    2009-10-21

    Propofol is the most widely used injectable general anesthetic. Its targets include ligand-gated ion channels such as the GABA{sub A} receptor, but such receptor-channel complexes remain challenging to study at atomic resolution. Until structural biology methods advance to the point of being able to deal with systems such as the GABA{sub A} receptor, it will be necessary to use more tractable surrogates to probe the molecular details of anesthetic recognition. We have previously shown that recognition of inhalational general anesthetics by the model protein apoferritin closely mirrors recognition by more complex and clinically relevant protein targets; here we show that apoferritin also binds propofol and related GABAergic anesthetics, and that the same binding site mediates recognition of both inhalational and injectable anesthetics. Apoferritin binding affinities for a series of propofol analogs were found to be strongly correlated with the ability to potentiate GABA responses at GABA{sub A} receptors, validating this model system for injectable anesthetics. High resolution x-ray crystal structures reveal that, despite the presence of hydrogen bond donors and acceptors, anesthetic recognition is mediated largely by van der Waals forces and the hydrophobic effect. Molecular dynamics simulations indicate that the ligands undergo considerable fluctuations about their equilibrium positions. Finally, apoferritin displays both structural and dynamic responses to anesthetic binding, which may mimic changes elicited by anesthetics in physiologic targets like ion channels.

  1. A Unitary Anesthetic Binding Site at High Resolution

    Energy Technology Data Exchange (ETDEWEB)

    L Vedula; G Brannigan; N Economou; J Xi; M Hall; R Liu; M Rossi; W Dailey; K Grasty; et. al.

    2011-12-31

    Propofol is the most widely used injectable general anesthetic. Its targets include ligand-gated ion channels such as the GABA{sub A} receptor, but such receptor-channel complexes remain challenging to study at atomic resolution. Until structural biology methods advance to the point of being able to deal with systems such as the GABA{sub A} receptor, it will be necessary to use more tractable surrogates to probe the molecular details of anesthetic recognition. We have previously shown that recognition of inhalational general anesthetics by the model protein apoferritin closely mirrors recognition by more complex and clinically relevant protein targets; here we show that apoferritin also binds propofol and related GABAergic anesthetics, and that the same binding site mediates recognition of both inhalational and injectable anesthetics. Apoferritin binding affinities for a series of propofol analogs were found to be strongly correlated with the ability to potentiate GABA responses at GABA{sub A} receptors, validating this model system for injectable anesthetics. High resolution x-ray crystal structures reveal that, despite the presence of hydrogen bond donors and acceptors, anesthetic recognition is mediated largely by van der Waals forces and the hydrophobic effect. Molecular dynamics simulations indicate that the ligands undergo considerable fluctuations about their equilibrium positions. Finally, apoferritin displays both structural and dynamic responses to anesthetic binding, which may mimic changes elicited by anesthetics in physiologic targets like ion channels.

  2. A Unitary Anesthetic-Binding Site at High Resolution

    Energy Technology Data Exchange (ETDEWEB)

    Vedula, L.; Brannigan, G; Economou, N; Xi, J; Hall, M; Liu, R; Rossi, M; Dailey, W; Grasty, K; et. al.

    2009-01-01

    Propofol is the most widely used injectable general anesthetic. Its targets include ligand-gated ion channels such as the GABAA receptor, but such receptor-channel complexes remain challenging to study at atomic resolution. Until structural biology methods advance to the point of being able to deal with systems such as the GABA{sub A} receptor, it will be necessary to use more tractable surrogates to probe the molecular details of anesthetic recognition. We have previously shown that recognition of inhalational general anesthetics by the model protein apoferritin closely mirrors recognition by more complex and clinically relevant protein targets; here we show that apoferritin also binds propofol and related GABAergic anesthetics, and that the same binding site mediates recognition of both inhalational and injectable anesthetics. Apoferritin binding affinities for a series of propofol analogs were found to be strongly correlated with the ability to potentiate GABA responses at GABA{sub A} receptors, validating this model system for injectable anesthetics. High resolution x-ray crystal structures reveal that, despite the presence of hydrogen bond donors and acceptors, anesthetic recognition is mediated largely by van der Waals forces and the hydrophobic effect. Molecular dynamics simulations indicate that the ligands undergo considerable fluctuations about their equilibrium positions. Finally, apoferritin displays both structural and dynamic responses to anesthetic binding, which may mimic changes elicited by anesthetics in physiologic targets like ion channels.

  3. Six independent fucose-binding sites in the crystal structure of Aspergillus oryzae lectin

    Energy Technology Data Exchange (ETDEWEB)

    Makyio, Hisayoshi [Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), 1-1 Oho, Tsukuba, Ibaraki, 305-0801 (Japan); Shimabukuro, Junpei; Suzuki, Tatsuya [Department of Applied Bioorganic Chemistry, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193 (Japan); Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501 (Japan); Imamura, Akihiro; Ishida, Hideharu [Department of Applied Bioorganic Chemistry, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193 (Japan); Kiso, Makoto [Department of Applied Bioorganic Chemistry, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193 (Japan); Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501 (Japan); Ando, Hiromune, E-mail: hando@gifu-u.ac.jp [Department of Applied Bioorganic Chemistry, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193 (Japan); Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501 (Japan); Kato, Ryuichi, E-mail: ryuichi.kato@kek.jp [Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), 1-1 Oho, Tsukuba, Ibaraki, 305-0801 (Japan)

    2016-08-26

    The crystal structure of AOL (a fucose-specific lectin of Aspergillus oryzae) has been solved by SAD (single-wavelength anomalous diffraction) and MAD (multi-wavelength anomalous diffraction) phasing of seleno-fucosides. The overall structure is a six-bladed β-propeller similar to that of other fucose-specific lectins. The fucose moieties of the seleno-fucosides are located in six fucose-binding sites. Although the Arg and Glu/Gln residues bound to the fucose moiety are common to all fucose-binding sites, the amino-acid residues involved in fucose binding at each site are not identical. The varying peak heights of the seleniums in the electron density map suggest that each fucose-binding site has a different carbohydrate binding affinity. - Highlights: • The six-bladed β-propeller structure of AOL was solved by seleno-sugar phasing. • The mode of fucose binding is essentially conserved at all six binding sites. • The seleno-fucosides exhibit slightly different interactions and electron densities. • These findings suggest that the affinity for fucose is not identical at each site.

  4. A computational study of ligand binding affinities in iron(III) porphine and protoporphyrin IX complexes.

    Science.gov (United States)

    Durrant, Marcus C

    2014-07-01

    The search for novel anti-malarial drugs that can disrupt biomineralization of ferriprotoporphyrin IX to haemozoin requires an understanding of the fundamental chemistry of the porphyrin's iron(iii) centre at the water-lipid interface. Towards this end, the binding affinities for a diverse set of 31 small ligands with iron(iii) porphine have been calculated using density functional theory, in the gas phase and also with implicit solvent corrections for both water and n-octanol. In addition, the binding of hydroxide, chloride, acetate, methylamine and water to ferriprotoporphyrin IX has been studied, and very similar trends are observed for the smaller and larger models. Anionic ligands generally give stronger binding than neutral ones; the strongest binding is observed for RO(-) and OH(-) ligands, whilst acetate binds relatively weakly among the anions studied. Electron-rich nitrogen donors tend to bind more strongly than electron-deficient ones, and the weakest binding is found for neutral O and S donors such as oxazole and thiophene. In all cases, ligand binding is stronger in n-octanol than in water, and the differences in binding energies for the two solvents are greater for ionic ligands than for neutrals. Finally, dimerization of ferriprotoporphyrin IX by means of iron(iii)-carboxylate bond formation has been modelled. The results are discussed in terms of haemozoin crystal growth and its disruption by known anti-malarial drugs.

  5. Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

    Science.gov (United States)

    Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.

    2000-01-01

    Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

  6. Single-nucleotide mutation matrix: a new model for predicting the NF-κB DNA binding sites.

    Directory of Open Access Journals (Sweden)

    Wenxin Du

    Full Text Available In this study, we established a single nucleotide mutation matrix (SNMM model based on the relative binding affinities of NF-κB p50 homodimer to a wild-type binding site (GGGACTTTCC and its all single-nucleotide mutants detected with the double-stranded DNA microarray. We evaluated this model by scoring different groups of 10-bp DNA sequences with this model and analyzing the correlations between the scores and the relative binding affinities detected with three wet experiments, including the electrophoresis mobility shift assay (EMSA, the protein-binding microarray (PBM and the systematic evolution of ligands by exponential enrichment-sequencing (SELEX-Seq. The results revealed that the SNMM scores were strongly correlated with the detected binding affinities. We also scored the DNA sequences with other three models, including the principal coordinate (PC model, the position weight matrix scoring algorithm (PWMSA model and the Match model, and analyzed the correlations between the scores and the detected binding affinities. In comparison with these models, the SNMM model achieved reliable results. We finally determined 0.747 as the optimal threshold for predicting the NF-κB DNA-binding sites with the SNMM model. The SNMM model thus provides a new alternative model for scoring the relative binding affinities of NF-κB to the 10-bp DNA sequences and predicting the NF-κB DNA-binding sites.

  7. Single-nucleotide mutation matrix: a new model for predicting the NF-κB DNA binding sites.

    Science.gov (United States)

    Du, Wenxin; Gao, Jing; Wang, Tingting; Wang, Jinke

    2014-01-01

    In this study, we established a single nucleotide mutation matrix (SNMM) model based on the relative binding affinities of NF-κB p50 homodimer to a wild-type binding site (GGGACTTTCC) and its all single-nucleotide mutants detected with the double-stranded DNA microarray. We evaluated this model by scoring different groups of 10-bp DNA sequences with this model and analyzing the correlations between the scores and the relative binding affinities detected with three wet experiments, including the electrophoresis mobility shift assay (EMSA), the protein-binding microarray (PBM) and the systematic evolution of ligands by exponential enrichment-sequencing (SELEX-Seq). The results revealed that the SNMM scores were strongly correlated with the detected binding affinities. We also scored the DNA sequences with other three models, including the principal coordinate (PC) model, the position weight matrix scoring algorithm (PWMSA) model and the Match model, and analyzed the correlations between the scores and the detected binding affinities. In comparison with these models, the SNMM model achieved reliable results. We finally determined 0.747 as the optimal threshold for predicting the NF-κB DNA-binding sites with the SNMM model. The SNMM model thus provides a new alternative model for scoring the relative binding affinities of NF-κB to the 10-bp DNA sequences and predicting the NF-κB DNA-binding sites.

  8. An in silico analysis of the binding modes and binding affinities of small molecule modulators of PDZ-peptide interactions.

    Directory of Open Access Journals (Sweden)

    Garima Tiwari

    Full Text Available Inhibitors of PDZ-peptide interactions have important implications in a variety of biological processes including treatment of cancer and Parkinson's disease. Even though experimental studies have reported characterization of peptidomimetic inhibitors of PDZ-peptide interactions, the binding modes for most of them have not been characterized by structural studies. In this study we have attempted to understand the structural basis of the small molecule-PDZ interactions by in silico analysis of the binding modes and binding affinities of a set of 38 small molecules with known K(i or K(d values for PDZ2 and PDZ3 domains of PSD-95 protein. These two PDZ domains show differential selectivity for these compounds despite having a high degree of sequence similarity and almost identical peptide binding pockets. Optimum binding modes for these ligands for PDZ2 and PDZ3 domains were identified by using a novel combination of semi-flexible docking and explicit solvent molecular dynamics (MD simulations. Analysis of the binding modes revealed most of the peptidomimectic ligands which had high K(i or K(d moved away from the peptide binding pocket, while ligands with high binding affinities remained in the peptide binding pocket. The differential specificities of the PDZ2 and PDZ3 domains primarily arise from differences in the conformation of the loop connecting βB and βC strands, because this loop interacts with the N-terminal chemical moieties of the ligands. We have also computed the MM/PBSA binding free energy values for these 38 compounds with both the PDZ domains from multiple 5 ns MD trajectories on each complex i.e. a total of 228 MD trajectories of 5 ns length each. Interestingly, computational binding free energies show good agreement with experimental binding free energies with a correlation coefficient of approximately 0.6. Thus our study demonstrates that combined use of docking and MD simulations can help in identification of potent inhibitors

  9. Antidepressant Binding Site in a Bacterial Homologue of Neurotransmitter Transporters

    Energy Technology Data Exchange (ETDEWEB)

    Singh,S.; Yamashita, A.; Gouaux, E.

    2007-01-01

    Sodium-coupled transporters are ubiquitous pumps that harness pre-existing sodium gradients to catalyse the thermodynamically unfavourable uptake of essential nutrients, neurotransmitters and inorganic ions across the lipid bilayer. Dysfunction of these integral membrane proteins has been implicated in glucose/galactose malabsorption, congenital hypothyroidism, Bartter's syndrome, epilepsy, depression, autism and obsessive-compulsive disorder. Sodium-coupled transporters are blocked by a number of therapeutically important compounds, including diuretics, anticonvulsants and antidepressants, many of which have also become indispensable tools in biochemical experiments designed to probe antagonist binding sites and to elucidate transport mechanisms. Steady-state kinetic data have revealed that both competitive and noncompetitive modes of inhibition exist. Antagonist dissociation experiments on the serotonin transporter (SERT) have also unveiled the existence of a low-affinity allosteric site that slows the dissociation of inhibitors from a separate high-affinity site. Despite these strides, atomic-level insights into inhibitor action have remained elusive. Here we screen a panel of molecules for their ability to inhibit LeuT, a prokaryotic homologue of mammalian neurotransmitter sodium symporters, and show that the tricyclic antidepressant (TCA) clomipramine noncompetitively inhibits substrate uptake. Cocrystal structures show that clomipramine, along with two other TCAs, binds in an extracellular-facing vestibule about 11 {angstrom} above the substrate and two sodium ions, apparently stabilizing the extracellular gate in a closed conformation. Off-rate assays establish that clomipramine reduces the rate at which leucine dissociates from LeuT and reinforce our contention that this TCA inhibits LeuT by slowing substrate release. Our results represent a molecular view into noncompetitive inhibition of a sodium-coupled transporter and define principles for the

  10. De novo analysis of receptor binding affinity data of xanthine adenosine receptor antagonists.

    Science.gov (United States)

    Dalpiaz, A; Gardenghi, A; Borea, P A

    1995-03-01

    The receptor binding affinity data to adenosine A1 and A2 receptors of a wide series of xanthine derivatives have been analyzed by means of the Free-Wilson model. The analysis of the individual group contribution shows, for both A1 and A2 receptors, the primary importance of the presence of bulky substituents at position 8 for an optimum receptor binding. Moreover, considering the different aij contributions of bulky substituents at position 8 for affinity to A1 with respect to A2 receptors, this position appears to be the most important for the synthesis of highly A1 selective xanthine derivatives. Moreover the analysis of group contributions for other substitution positions of the xanthine moiety allows to state that suitable substitutions at positions 3 and 7 could confer some degree of A2 selectivity.

  11. Microscale characterization of the binding specificity and affinity of a monoclonal antisulfotyrosyl IgG antibody

    DEFF Research Database (Denmark)

    Lassen, K.S.; Bradbury, A.R.; Heegaard, N.H.;

    2008-01-01

    peptides and proteins. The data show that the anti-Tyr(SO(3)H) antibody is completely specific for compounds containing sulfated tyrosyls. Affinity electrophoresis experiments allowed us to estimate dissociation constants for sulfated hirudin fragment (56-65), gastrin-17, and cholecystokinin octapeptide...... (CCK8) in the 1-3 microM range. The affinity of the antibody toward complement 4 protein that contains three sulfotyrosines was analyzed by surface plasmon resonance technology and modeled according to a bivalent-binding model which yielded a K(d1) of 20.1 microM for the monovalent complex. The same...... binding was studied by CE and found to be in the micromolar scale albeit with some uncertainty due to complex separation patterns. The work illustrates the amount of information on antibody-antigen interactions that may be obtained with microelectrophoretic methods consuming minute quantities of material...

  12. Binding and elution strategy for improved performance of arginine affinity chromatography in supercoiled plasmid DNA purification.

    Science.gov (United States)

    Sousa, F; Prazeres, D M F; Queiroz, J A

    2009-02-01

    New interesting strategies for plasmid DNA (pDNA) purification were designed, exploiting affinity interactions between amino acids and nucleic acids. The potential application of arginine-based chromatography to purify pDNA has been recently described in our work; however, to achieve higher efficiency and selectivity in arginine affinity chromatography, it is essential to characterize the behaviour of binding/elution of supercoiled (sc) isoforms. In this study, two different strategies based on increased sodium chloride (225-250 mm) or arginine (20-70 mm) stepwise gradients are described to purify sc isoforms. Thus, it was proved that well-defined binding/elution conditions are crucial to enhance the purification performance, resulting in an improvement of the final plasmids yields and transfection efficiency, as this could represent a significant impact on therapeutic applications of the purified sc isoform. Copyright (c) 2008 John Wiley & Sons, Ltd.

  13. Comparison of high affinity binding of {sup 3}H-proadifen and {sup 3}H-(-)-cocaine t rat liver membranes

    Energy Technology Data Exchange (ETDEWEB)

    Ross, S.B. [Astra Arcus AB, Dept. of Neuropharmacology, Soedertaelje (Sweden)

    1995-06-01

    The characteristics of the binding of {sup 3}H-proadifen to rat liver membranes were studied and compared to those of {sup 3}H-cocaine. It was found that {sup 3}H-proadifen was bound reversibly with high affinity (K{sub D}=1.8{+-}0.5 nM) and large capacity (B{sub max}=2010{+-}340 pmol/g wet tissue) to liver membranes. The corresponding values for the {sup 3}H-cocaine binding were 3.5 nM and 1000 pmol/g wet tissue. The binding of {sup 3}H-proadifen was mainly localised to the microsomal fraction. The number of binding sites was not increased by treatment of rats with phenobarbitone. With 1 {mu}M CdCl{sub 2} in the incubation buffer it was possible to differentiate between two {sup 3}H-cocaine binding sites with K{sub d} values of 1.6 and 7.7 nM and B{sub max} values of 280 and 940 pmol/g wet liver tissue. S-(-)-Alaproclate inhibited the binding of {sup 3}H-proadifen and {sup 3}H-cocaine inhibited the binding of {sup 3}H-proadifen (IC{sub 50}=10 nM) and proadifen that of {sup 3}H-cocaine (IC{sub 50}=1 nM). There was a high correlation coefficient (r{sub r}=0.972; P<0.01; n=12) in the Spearman rank test between the inhibitory potencies of compounds examined in both systems. Beside some potent alaproclate analogues a couple of compounds had moderately high affinity (IC{sub 50}=100-500 nM): chloroquine, phenoxybenzamine, amitriptyline, ajmaline, remoxipride, imipramine and (-)-alaprenolol. CdCl{sub 2}, ZnCl{sub 2} and CuCl{sub 2} inhibited the binding of both ligands with low Hill coefficients, indicating heterogeneous binding sites. The inhibition curve of Cd{sup 2+} on the cocaine binding was biphasic with a high affinity part around 50 nM and a low affinity part at 15{mu}M. The similarity of the characteristics of the binding of these ligands with that of {sup 3}H-alaproclate is discussed. It is suggested that all three compounds bind to the same sites, although additional binding sites seem to exist for proadifen. (au) (9 refs.).

  14. Kinetic properties of a single nucleotide binding site on chloroplast coupling factor 1 (CF1).

    Science.gov (United States)

    Günther, S; Huchzermeyer, B

    1998-12-01

    The kinetics of nucleotide binding to spinach chloroplast coupling factor CF1 in a fully inhibited state were investigated by stopped-flow experiments using the fluorescent trinitrophenyl analogue (NO2)3Ph-ADP. The CF1 was in a state in which two of the three binding sites on the beta subunits were irreversibly blocked with ADP, Mg2+ and fluoroaluminate, while the three binding sites on the alpha subunits were occupied by nucleotides [Garin, J., Vincon, M., Gagnon, J. & Vignais, P. V. (1994) Biochemistry 33, 3772-3777)]. Thus, it was possible to characterise a single nucleotide-binding site without superimposed nucleotide exchange or binding to an additional site. (NO2)3Ph-ADP binding to the remaining site on the third beta subunit was characterised by a high dissociation rate of 15 s(-1), leading to a very low affinity (dissociation constant higher than 150 microM). Subsequent to isolation, CF1 preparations contained two endogenously bound nucleotides. Pre-loading with ATP yielded CF1 with five tightly bound nucleotides and one free nucleotide-binding site on a beta subunit. Pre-loading with ADP, however, resulted in a CF1 preparation containing four tightly bound nucleotides and two free nucleotide binding sites. One of the two free binding sites was located on a beta subunit, while the other was probably located on an alpha subunit.

  15. Localization of the substrate binding site in the homodimeric mannitol transporter, EIImtl, of Escherichia coli

    NARCIS (Netherlands)

    Opacic, Milena; Vos, Erwin P. P.; Hesp, Ben H.; Broos, Jaap

    2010-01-01

    The mannitol transporter from Escherichia coli, EIImtl, belongs to a class of membrane proteins coupling the transport of substrates with their chemical modification. EIImtl is functional as a homodimer, and it harbors one high affinity mannitol-binding site in the membrane-embedded C domain (IICmtl

  16. Azaflavones compared to flavones as ligands to the benzodiazepine binding site of brain GABAA receptors

    DEFF Research Database (Denmark)

    Nilsson, Jakob; Nielsen, Elsebet Østergaard; Liljefors, Tommy

    2008-01-01

    A series of azaflavone derivatives and analogues were prepared and evaluated for their affinity to the benzodiazepine binding site of the GABA(A) receptor, and compared to their flavone counterparts. Three of the compounds, the azaflavones 9 and 12 as well as the new flavone 13, were also assayed...

  17. Six independent fucose-binding sites in the crystal structure of Aspergillus oryzae lectin.

    Science.gov (United States)

    Makyio, Hisayoshi; Shimabukuro, Junpei; Suzuki, Tatsuya; Imamura, Akihiro; Ishida, Hideharu; Kiso, Makoto; Ando, Hiromune; Kato, Ryuichi

    2016-08-26

    The crystal structure of AOL (a fucose-specific lectin of Aspergillus oryzae) has been solved by SAD (single-wavelength anomalous diffraction) and MAD (multi-wavelength anomalous diffraction) phasing of seleno-fucosides. The overall structure is a six-bladed β-propeller similar to that of other fucose-specific lectins. The fucose moieties of the seleno-fucosides are located in six fucose-binding sites. Although the Arg and Glu/Gln residues bound to the fucose moiety are common to all fucose-binding sites, the amino-acid residues involved in fucose binding at each site are not identical. The varying peak heights of the seleniums in the electron density map suggest that each fucose-binding site has a different carbohydrate binding affinity.

  18. Weak binding affinity of human 4EHP for mRNA cap analogs.

    Science.gov (United States)

    Zuberek, Joanna; Kubacka, Dorota; Jablonowska, Agnieszka; Jemielity, Jacek; Stepinski, Janusz; Sonenberg, Nahum; Darzynkiewicz, Edward

    2007-05-01

    Ribosome recruitment to the majority of eukaryotic mRNAs is facilitated by the interaction of the cap binding protein, eIF4E, with the mRNA 5' cap structure. eIF4E stimulates translation through its interaction with a scaffolding protein, eIF4G, which helps to recruit the ribosome. Metazoans also contain a homolog of eIF4E, termed 4EHP, which binds the cap structure, but not eIF4G, and thus cannot stimulate translation, but it instead inhibits the translation of only one known, and possibly subset mRNAs. To understand why 4EHP does not inhibit general translation, we studied the binding affinity of 4EHP for cap analogs using two methods: fluorescence titration and stopped-flow measurements. We show that 4EHP binds cap analogs m(7)GpppG and m(7)GTP with 30 and 100 lower affinity than eIF4E. Thus, 4EHP cannot compete with eIF4E for binding to the cap structure of most mRNAs.

  19. Synergistic binding of the phosphorylated S233- and S259-binding sites of C-RAF to one 14-3-3ζ dimer.

    Science.gov (United States)

    Molzan, Manuela; Ottmann, Christian

    2012-11-02

    C-RAF kinase is a central component of the Ras-RAF-MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase)-ERK (extracellular signal-regulated kinase) pathway, which has been shown to be activated in 30% of human tumors. 14-3-3 proteins inactivate C-RAF by binding to the two N-terminal phosphorylation-dependent binding sites surrounding S233 and S259. 14-3-3 proteins can bind two target sequences located on one polypeptide chain simultaneously, thereby increasing binding affinity compared to single-site binding and possibly allowing regulated 14-3-3 binding through gatekeeper phosphorylation. To date, it was unclear whether 14-3-3 proteins can bind the two N-terminal phosphorylation-dependent binding sites of C-RAF simultaneously. Fluorescence polarization using phosphorylated peptides demonstrated that S233 is the low-affinity and S259 is the high-affinity binding site, while simultaneous engagement of both sites by 14-3-3ζ enhances affinity compared to single-site binding. Determination of a 1:1 stoichiometry for the di-phosphorylated peptide binding to one 14-3-3ζ dimer with isothermal titration calorimetry was supported by the crystal structure of the 14-3-3ζ/C-RAFpS233,pS259 complex. Cellular localization studies validate the significance of these sites for cytoplasmic retention of C-RAF, suggesting an extended mechanism of RAF regulation by 14-3-3 proteins. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. A knowledge-guided strategy for improving the accuracy of scoring functions in binding affinity prediction

    Directory of Open Access Journals (Sweden)

    Wang Renxiao

    2010-04-01

    Full Text Available Abstract Background Current scoring functions are not very successful in protein-ligand binding affinity prediction albeit their popularity in structure-based drug designs. Here, we propose a general knowledge-guided scoring (KGS strategy to tackle this problem. Our KGS strategy computes the binding constant of a given protein-ligand complex based on the known binding constant of an appropriate reference complex. A good training set that includes a sufficient number of protein-ligand complexes with known binding data needs to be supplied for finding the reference complex. The reference complex is required to share a similar pattern of key protein-ligand interactions to that of the complex of interest. Thus, some uncertain factors in protein-ligand binding may cancel out, resulting in a more accurate prediction of absolute binding constants. Results In our study, an automatic algorithm was developed for summarizing key protein-ligand interactions as a pharmacophore model and identifying the reference complex with a maximal similarity to the query complex. Our KGS strategy was evaluated in combination with two scoring functions (X-Score and PLP on three test sets, containing 112 HIV protease complexes, 44 carbonic anhydrase complexes, and 73 trypsin complexes, respectively. Our results obtained on crystal structures as well as computer-generated docking poses indicated that application of the KGS strategy produced more accurate predictions especially when X-Score or PLP alone did not perform well. Conclusions Compared to other targeted scoring functions, our KGS strategy does not require any re-parameterization or modification on current scoring methods, and its application is not tied to certain systems. The effectiveness of our KGS strategy is in theory proportional to the ever-increasing knowledge of experimental protein-ligand binding data. Our KGS strategy may serve as a more practical remedy for current scoring functions to improve their

  1. Novel high-affinity and selective biaromatic 4-substituted gamma-hydroxybutyric acid (GHB) analogues as GHB ligands: design, synthesis, and binding studies.

    Science.gov (United States)

    Høg, Signe; Wellendorph, Petrine; Nielsen, Birgitte; Frydenvang, Karla; Dahl, Ivar F; Bräuner-Osborne, Hans; Brehm, Lotte; Frølund, Bente; Clausen, Rasmus P

    2008-12-25

    Gamma-hydroxybutyrate (GHB) is a metabolite of gamma-aminobutyric acid (GABA) and has been proposed to function as a neurotransmitter or neuromodulator. GHB is used in the treatment of narcolepsy and is a drug of abuse. GHB binds to both GABA(B) receptors and specific high-affinity GHB sites in brain, of which the latter have not been linked unequivocally to function, but are speculated to be GHB receptors. In this study, a series of biaromatic 4-substituted GHB analogues, including 4'-phenethylphenyl, 4'-styrylphenyl, and 4'-benzyloxyphenyl GHB analogues, were synthesized and characterized pharmacologically in a [3H](E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene)acetic acid ([3H]NCS-382) binding assay and in GABA(A) and GABA(B) receptor binding assays. The compounds were selective for the high-affinity GHB binding sites and several displayed Ki values below 100 nM. The affinity of the 4-[4'-(2-iodobenzyloxy)phenyl] GHB analogue 17b was shown to reside predominantly with the R-enantiomer (Ki = 22 nM), which has higher affinity than previously reported GHB ligands.

  2. CB2 cannabinoid receptor agonist enantiomers HU-433 and HU-308: An inverse relationship between binding affinity and biological potency.

    Science.gov (United States)

    Smoum, Reem; Baraghithy, Saja; Chourasia, Mukesh; Breuer, Aviva; Mussai, Naama; Attar-Namdar, Malka; Kogan, Natalya M; Raphael, Bitya; Bolognini, Daniele; Cascio, Maria G; Marini, Pietro; Pertwee, Roger G; Shurki, Avital; Mechoulam, Raphael; Bab, Itai

    2015-07-14

    Activation of the CB2 receptor is apparently an endogenous protective mechanism. Thus, it restrains inflammation and protects the skeleton against age-related bone loss. However, the endogenous cannabinoids, as well as Δ(9)-tetrahydrocannabinol, the main plant psychoactive constituent, activate both cannabinoid receptors, CB1 and CB2. HU-308 was among the first synthetic, selective CB2 agonists. HU-308 is antiosteoporotic and antiinflammatory. Here we show that the HU-308 enantiomer, designated HU-433, is 3-4 orders of magnitude more potent in osteoblast proliferation and osteoclast differentiation culture systems, as well as in mouse models, for the rescue of ovariectomy-induced bone loss and ear inflammation. HU-433 retains the HU-308 specificity for CB2, as shown by its failure to bind to the CB1 cannabinoid receptor, and has no activity in CB2-deficient cells and animals. Surprisingly, the CB2 binding affinity of HU-433 in terms of [(3)H]CP55,940 displacement and its effect on [(35)S]GTPγS accumulation is substantially lower compared with HU-308. A molecular-modeling analysis suggests that HU-433 and -308 have two different binding conformations within CB2, with one of them possibly responsible for the affinity difference, involving [(35)S]GTPγS and cAMP synthesis. Hence, different ligands may have different orientations relative to the same binding site. This situation questions the usefulness of universal radioligands for comparative binding studies. Moreover, orientation-targeted ligands have promising potential for the pharmacological activation of distinct processes.

  3. The interaction of human serum albumin with selected lanthanide and actinide ions: Binding affinities, protein unfolding and conformational changes.

    Science.gov (United States)

    Ali, Manjoor; Kumar, Amit; Kumar, Mukesh; Pandey, Badri N

    2016-04-01

    Human serum albumin (HSA), the most abundant soluble protein in blood plays critical roles in transportation of biomolecules and maintenance of osmotic pressure. In view of increasing applications of lanthanides- and actinides-based materials in nuclear energy, space, industries and medical applications, the risk of exposure with these metal ions is a growing concern for human health. In present study, binding interaction of actinides/lanthanides [thorium: Th(IV), uranium: U(VI), lanthanum: La(III), cerium: Ce(III) and (IV)] with HSA and its structural consequences have been investigated. Ultraviolet-visible, Fourier transform-infrared, Raman, Fluorescence and Circular dichroism spectroscopic techniques were applied to study the site of metal ions interaction, binding affinity determination and the effect of metal ions on protein unfolding and HSA conformation. Results showed that these metal ions interacted with carbonyl (CO..:)/amide(N..-H) groups and induced exposure of aromatic residues of HSA. The fluorescence analysis indicated that the actinide binding altered the microenvironment around Trp214 in the subdomain IIA. Binding affinity of U(VI) to HSA was slightly higher than that of Th(IV). Actinides and Ce(IV) altered the secondary conformation of HSA with a significant decrease of α-helix and an increase of β-sheet, turn and random coil structures, indicating a partial unfolding of HSA. A correlation was observed between metal ion's ability to alter HSA conformation and protein unfolding. Both cationic effects and coordination ability of metal ions seemed to determine the consequences of their interaction with HSA. Present study improves our understanding about the protein interaction of these heavy ions and their impact on its secondary structure. In addition, binding characteristics may have important implications for the development of rational antidote for the medical management of health effects of actinides and lanthanides.

  4. Oxytocin binding sites in bovine mammary tissue

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Xin.

    1989-01-01

    Oxytocin binding sites were identified and characterized in bovine mammary tissue. ({sup 3}H)-oxytocin binding reached equilibrium by 50 min at 20{degree}C and by 8 hr at 4{degree}C. The half-time of displacement at 20{degree}C was approximately 1 hr. Thyrotropin releasing hormone, adrenocorticotropin, angiotensin I, angiotensin II, pentagastrin, bradykinin, xenopsin and L-valyl-histidyl-L-leucyl-L-threonyl-L-prolyl-L-valyl-L-glutamyl-L-lysine were not competitive. In the presence of 10 nM LiCl, addition of oxytocin to dispersed bovine mammary cells, in which phosphatidylinositol was pre-labelled, caused a time and dose-dependent increase in radioactive inositiol monophosphate incorporation. The possibility that there are distinct vasopressin receptors in bovine mammary tissue was investigated. ({sup 3}H)-vasopressin binding reached equilibrium by 40 min at 20{degree}. The half-time of displacement at 20{degree}C was approximately 1 hr. The ability of the peptides to inhibit ({sup 3}H)-vasopressin binding was: (Thr{sup 4},Gly{sup 7})-oxytocin > Arg{sup 8}-vasopressin > (lys{sup 8})-vasopressin > (Deamino{sup 1},D-arg{sup 8})-vasopressin > oxytocin > d (CH{sub 2}){sub 5}Tyr(Me)AVP.

  5. Arabidopsis AtADF1 is Functionally Affected by Mutations on Actin Binding Sites

    Institute of Scientific and Technical Information of China (English)

    Chun-Hai Dong; Wei-Ping Tang; Jia-Yao Liu

    2013-01-01

    The plant actin depolymerizing factor (ADF) binds to both monomeric and filamentous actin,and is directly involved in the depolymerization of actin filaments.To better understand the actin binding sites of the Arabidopsis thaliana L.AtADF1,we generated mutants of AtADF1 and investigated their functions in vitro and in vivo.Analysis of mutants harboring amino acid substitutions revealed that charged residues (Arg98 and Lys100) located at the α-helix 3 and forming an actin binding site together with the N-terminus are essential for both G-and F-actin binding.The basic residues on the β-strand 5 (K82/A) and the α-helix 4 (R135/A,R137/A) form another actin binding site that is important for F-actin binding.Using transient expression of CFP-tagged AtADF1 mutant proteins in onion (Allium cepa) peel epidermal cells and transgenic Arabidopsis thaliana L.plants overexpressing these mutants,we analyzed how these mutant proteins regulate actin organization and affect seedling growth.Our results show that the ADF mutants with a lower affinity for actin filament binding can still be functional,unless the affinity foractin monomers is also affected.The G-actin binding activity of the ADF plays an essential role in actin binding,depolymerization of actin polymers,and therefore in the control of actin organization.

  6. Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

    DEFF Research Database (Denmark)

    Cockburn, Darrell; Wilkens, Casper; Dilokpimol, Adiphol

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical...... for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon...... sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites...

  7. Analyzing binding data.

    Science.gov (United States)

    Motulsky, Harvey J; Neubig, Richard R

    2010-07-01

    Measuring the rate and extent of radioligand binding provides information on the number of binding sites, and their affinity and accessibility of these binding sites for various drugs. This unit explains how to design and analyze such experiments.

  8. Comparison of Transcription Factor Binding Site Models

    KAUST Repository

    Bhuyan, Sharifulislam

    2012-05-01

    Modeling of transcription factor binding sites (TFBSs) and TFBS prediction on genomic sequences are important steps to elucidate transcription regulatory mechanism. Dependency of transcription regulation on a great number of factors such as chemical specificity, molecular structure, genomic and epigenetic characteristics, long distance interaction, makes this a challenging problem. Different experimental procedures generate evidence that DNA-binding domains of transcription factors show considerable DNA sequence specificity. Probabilistic modeling of TFBSs has been moderately successful in identifying patterns from a family of sequences. In this study, we compare performances of different probabilistic models and try to estimate their efficacy over experimental TFBSs data. We build a pipeline to calculate sensitivity and specificity from aligned TFBS sequences for several probabilistic models, such as Markov chains, hidden Markov models, Bayesian networks. Our work, containing relevant statistics and evaluation for the models, can help researchers to choose the most appropriate model for the problem at hand.

  9. High-affinity binding of Chp1 chromodomain to K9 methylated histone H3 is required to establish centromeric heterochromatin.

    Science.gov (United States)

    Schalch, Thomas; Job, Godwin; Noffsinger, Victoria J; Shanker, Sreenath; Kuscu, Canan; Joshua-Tor, Leemor; Partridge, Janet F

    2009-04-10

    In fission yeast, assembly of centromeric heterochromatin requires the RITS complex, which consists of Ago1, Tas3, Chp1, and siRNAs derived from centromeric repeats. Recruitment of RITS to centromeres has been proposed to depend on siRNA-dependent targeting of Ago1 to centromeric sequences. Previously, we demonstrated that methylated lysine 9 of histone H3 (H3K9me) acts upstream of siRNAs during heterochromatin establishment. Our crystal structure of Chp1's chromodomain in complex with a trimethylated lysine 9 H3 peptide reveals extensive sites of contact that contribute to Chp1's high-affinity binding. We found that this high-affinity binding is critical for the efficient establishment of centromeric heterochromatin, but preassembled heterochromatin can be maintained when Chp1's affinity for H3K9me is greatly reduced.

  10. The C-terminal region of laminin beta chains modulates the integrin binding affinities of laminins.

    Science.gov (United States)

    Taniguchi, Yukimasa; Ido, Hiroyuki; Sanzen, Noriko; Hayashi, Maria; Sato-Nishiuchi, Ryoko; Futaki, Sugiko; Sekiguchi, Kiyotoshi

    2009-03-20

    Laminins are major cell-adhesive proteins in basement membranes that are capable of binding to integrins. Laminins consist of three chains (alpha, beta, and gamma), in which three laminin globular modules in the alpha chain and the Glu residue in the C-terminal tail of the gamma chain have been shown to be prerequisites for binding to integrins. However, it remains unknown whether any part of the beta chain is involved in laminin-integrin interactions. We compared the binding affinities of pairs of laminin isoforms containing the beta1 or beta2 chain toward a panel of laminin-binding integrins, and we found that beta2 chain-containing laminins (beta2-laminins) bound more avidly to alpha3beta1 and alpha7X2beta1 integrins than beta1 chain-containing laminins (beta1-laminins), whereas alpha6beta1, alpha6beta4, and alpha7X1beta1 integrins did not show any preference toward beta2-laminins. Because alpha3beta1 contains the "X2-type" variable region in the alpha3 subunit and alpha6beta1 and alpha6beta4 contain the "X1-type" region in the alpha6 subunit, we hypothesized that only integrins containing the X2-type region were capable of discriminating between beta1-laminins and beta2-laminins. In support of this possibility, a putative X2-type variant of alpha6beta1 was produced and found to bind preferentially to beta2-laminins. Production of a series of swap mutants between the beta1 and beta2 chains revealed that the C-terminal 20 amino acids in the coiled-coil domain were responsible for the enhanced integrin binding by beta2-laminins. Taken together, the results provide evidence that the C-terminal region of beta chains is involved in laminin recognition by integrins and modulates the binding affinities of laminins toward X2-type integrins.

  11. Insights into structural features determining odorant affinities to honey bee odorant binding protein 14.

    Science.gov (United States)

    Schwaighofer, Andreas; Pechlaner, Maria; Oostenbrink, Chris; Kotlowski, Caroline; Araman, Can; Mastrogiacomo, Rosa; Pelosi, Paolo; Knoll, Wolfgang; Nowak, Christoph; Larisika, Melanie

    2014-04-18

    Molecular interactions between odorants and odorant binding proteins (OBPs) are of major importance for understanding the principles of selectivity of OBPs towards the wide range of semiochemicals. It is largely unknown on a structural basis, how an OBP binds and discriminates between odorant molecules. Here we examine this aspect in greater detail by comparing the C-minus OBP14 of the honey bee (Apis mellifera L.) to a mutant form of the protein that comprises the third disulfide bond lacking in C-minus OBPs. Affinities of structurally analogous odorants featuring an aromatic phenol group with different side chains were assessed based on changes of the thermal stability of the protein upon odorant binding monitored by circular dichroism spectroscopy. Our results indicate a tendency that odorants show higher affinity to the wild-type OBP suggesting that the introduced rigidity in the mutant protein has a negative effect on odorant binding. Furthermore, we show that OBP14 stability is very sensitive to the position and type of functional groups in the odorant. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Induced binding of proteins by ammonium sulfate in affinity and ion-exchange column chromatography.

    Science.gov (United States)

    Arakawa, Tsutomu; Tsumoto, Kouhei; Ejima, Daisuke; Kita, Yoshiko; Yonezawa, Yasushi; Tokunaga, Masao

    2007-04-10

    In general, proteins bind to affinity or ion-exchange columns at low salt concentrations, and the bound proteins are eluted by raising the salt concentration, changing the solvent pH, or adding competing ligands. Blue-Sepharose is often used to remove bovine serum albumin (BSA) from samples, but when we applied BSA to Blue-Sepharose in 20 mM phosphate, pH 7.0, 50%-60% of the protein flowed through the column; however, complete binding of BSA was achieved by the addition of 2 M ammonium sulfate (AS) to the column equilibration buffer and the sample. The bound protein was eluted by decreasing the AS concentration or by adding 1 M NaCl or arginine. AS at high concentrations resulted in binding of BSA even to an ion-exchange column, Q-Sepharose, at pH 7.0. Thus, although moderate salt concentrations elute proteins from Blue-Sepharose or ion-exchange columns, proteins can be bound to these columns under extreme salting-out conditions. Similar enhanced binding of proteins by AS was observed with an ATP-affinity column.

  13. Determining the ice-binding planes of antifreeze proteins by fluorescence-based ice plane affinity.

    Science.gov (United States)

    Basu, Koli; Garnham, Christopher P; Nishimiya, Yoshiyuki; Tsuda, Sakae; Braslavsky, Ido; Davies, Peter

    2014-01-15

    Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

  14. Prediction of MHC class II binding affinity using SMM-align, a novel stabilization matrix alignment method

    DEFF Research Database (Denmark)

    Nielsen, Morten; Lundegaard, Claus; Lund, Ole

    2007-01-01

    the correct alignment of a peptide in the binding groove a crucial part of identifying the core of an MHC class II binding motif. Here, we present a novel stabilization matrix alignment method, SMM-align, that allows for direct prediction of peptide:MHC binding affinities. The predictive performance...

  15. High-affinity binding of southern African HIV type 1 subtype C envelope protein, gp120, to the CCR5 coreceptor.

    Science.gov (United States)

    Fromme, Bernhard J; Coetsee, Marla; Van Der Watt, Pauline; Chan, Mei-Chi; Sperling, Karin M; Katz, Arieh A; Flanagan, Colleen A

    2008-12-01

    HIV-1 subtype C is the fastest spreading subtype worldwide and predominantly uses the CCR5 coreceptor, showing minimal transition to the X4 phenotype. This raises the possibility that envelope proteins of HIV-1 subtype C have structural features that favor interaction with CCR5. Preference for CCR5 could arise from enhanced affinity of HIV-1 subtype C for CCR5. To test this, we have characterized the interaction of gp120 envelope proteins from HIV-1 subtype C clones with CD4 and CCR5. Recombinant gp120 proteins from isolates of HIV-1 subtypes B and C were expressed, purified, and assessed in a CD4 binding assay and a CCR5 chemokine competition binding assay. All gp120 proteins bound to CD4-expressing cells, except one, 97ZA347ts, which had Arg substituted for the Cys239 in the conserved C2 loop. Reconstitution of Cys239, using site-directed mutagenesis, restored CD4 binding, while introducing Arg or Ser into position 239 of the functional Du151 gp120 protein abrogated CD4 binding. This shows that the Cys228-Cys239 disulfide bond of gp120 is required for high-affinity binding to CD4. Recombinant gp120 proteins from two HIV-1 subtype B clones bound CCR5 in the presence of CD4, while gp120 from the X4-tropic, HxB2, clone did not bind CCR5. gp120 from two functional HIV-1 subtype C clones, Du151 and MOLE1, bound CCR5 with high affinity in the presence of CD4 and Du151 showed significant CCR5 binding in the absence of CD4. A gp120 from a nonfunctional subtype C clone had lower affinity for CCR5. These results indicate that HIV-1 subtype C proteins have high affinity for CCR5 with variable dependence on CD4.

  16. STUDY OF ESTROGEN BINDING SITE ON HUMAN EJACULATED SPERMATOZOA

    Institute of Scientific and Technical Information of China (English)

    CHUJin-Shong; WANGYi-Fei

    1989-01-01

    The specific estrogen binding site for 17β-estradiol has been investigated on human spermatozoa by electron microscopec autoradiography. The results show that the binding sites were distributed over the surface of human spermatozoa: acrosomal cap, equatorial

  17. Effects of cytosine methylation on transcription factor binding sites

    KAUST Repository

    Medvedeva, Yulia A

    2014-03-26

    Background: DNA methylation in promoters is closely linked to downstream gene repression. However, whether DNA methylation is a cause or a consequence of gene repression remains an open question. If it is a cause, then DNA methylation may affect the affinity of transcription factors (TFs) for their binding sites (TFBSs). If it is a consequence, then gene repression caused by chromatin modification may be stabilized by DNA methylation. Until now, these two possibilities have been supported only by non-systematic evidence and they have not been tested on a wide range of TFs. An average promoter methylation is usually used in studies, whereas recent results suggested that methylation of individual cytosines can also be important.Results: We found that the methylation profiles of 16.6% of cytosines and the expression profiles of neighboring transcriptional start sites (TSSs) were significantly negatively correlated. We called the CpGs corresponding to such cytosines " traffic lights" We observed a strong selection against CpG " traffic lights" within TFBSs. The negative selection was stronger for transcriptional repressors as compared with transcriptional activators or multifunctional TFs as well as for core TFBS positions as compared with flanking TFBS positions.Conclusions: Our results indicate that direct and selective methylation of certain TFBS that prevents TF binding is restricted to special cases and cannot be considered as a general regulatory mechanism of transcription. 2013 Medvedeva et al.; licensee BioMed Central Ltd.

  18. High affinity binding of proteins HMG1 and HMG2 to semicatenated DNA loops

    Directory of Open Access Journals (Sweden)

    Strauss François

    2000-10-01

    Full Text Available Abstract Background Proteins HMG1 and HMG2 are two of the most abundant non histone proteins in the nucleus of mammalian cells, and contain a domain of homology with many proteins implicated in the control of development, such as the sex-determination factor Sry and the Sox family of proteins. In vitro studies of interactions of HMG1/2 with DNA have shown that these proteins can bind to many unusual DNA structures, in particular to four-way junctions, with binding affinities of 107 to 109 M-1. Results Here we show that HMG1 and HMG2 bind with a much higher affinity, at least 4 orders of magnitude higher, to a new structure, Form X, which consists of a DNA loop closed at its base by a semicatenated DNA junction, forming a DNA hemicatenane. The binding constant of HMG1 to Form X is higher than 5 × 1012 M-1, and the half-life of the complex is longer than one hour in vitro. Conclusions Of all DNA structures described so far with which HMG1 and HMG2 interact, we have found that Form X, a DNA loop with a semicatenated DNA junction at its base, is the structure with the highest affinity by more than 4 orders of magnitude. This suggests that, if similar structures exist in the cell nucleus, one of the functions of these proteins might be linked to the remarkable property of DNA hemicatenanes to associate two distant regions of the genome in a stable but reversible manner.

  19. Being a binding site: characterizing residue composition of binding sites on proteins.

    Science.gov (United States)

    Iván, Gábor; Szabadka, Zoltán; Grolmusz, Vince

    2007-12-30

    The Protein Data Bank contains the description of more than 45,000 three-dimensional protein and nucleic-acid structures today. Started to exist as the computer-readable depository of crystallographic data complementing printed articles, the proper interpretation of the content of the individual files in the PDB still frequently needs the detailed information found in the citing publication. This fact implies that the fully automatic processing of the whole PDB is a very hard task. We first cleaned and re-structured the PDB data, then analyzed the residue composition of the binding sites in the whole PDB for frequency and for hidden association rules. Main results of the paper: (i) the cleaning and repairing algorithm (ii) redundancy elimination from the data (iii) application of association rule mining to the cleaned non-redundant data set. We have found numerous significant relations of the residue-composition of the ligand binding sites on protein surfaces, summarized in two figures. One of the classical data-mining methods for exploring implication-rules, the association-rule mining, is capable to find previously unknown residue-set preferences of bind ligands on protein surfaces. Since protein-ligand binding is a key step in enzymatic mechanisms and in drug discovery, these uncovered preferences in the study of more than 19,500 binding sites may help in identifying new binding protein-ligand pairs.

  20. Discovery of a cobalt complex with high MEK1 binding affinity.

    Science.gov (United States)

    Li, Hongyue; Zhou, Tongliang; Liu, Hui; Xu, Fengrong; Niu, Yan; Wang, Chao; Liang, Lei; Xu, Ping

    2017-05-15

    A series of Schiff base ligands (L(1)-L(5)) and their cobalt(II) complexes (1-5) were designed and synthesized for MEK1 binding experiment. The biological evaluation results showed that Bis(N,N'-disalicylidene)-3,4-phenylenediamine-cobalt(II) 1 and Bis(N,N'-disalicylidene)-1,2-cyclohexanediamine-cobalt(II) 2 are much more effective than the parent Schiff bases (L(1) and L(2)). Importantly, 2 exhibited MEK1 binding affinity with IC5071nM, which is so far the best result for metal complexes and more potent than U0126 (7.02μM) and AZD6244 (2.20μM). Docking study was used to elucidate the binding modes of complex 2 with MEK1. Thus cobalt(II) complex 2 may be further developed as a novel MEK1 inhibitor. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Substrate binds in the S1 site of the F253A mutant of LeuT, a neurotransmitter sodium symporter homologue

    OpenAIRE

    Wang, Hui; Gouaux, Eric

    2012-01-01

    The controversy about the number of substrate binding sites in the neurotransmitter sodium symporter model protein LeuT is ongoing. Ligand binding assays and crystal structures of the LeuT F253A mutant support the presence of a single high affinity substrate binding site.

  2. Substrate binds in the S1 site of the F253A mutant of LeuT, a neurotransmitter sodium symporter homologue

    OpenAIRE

    Wang, Hui; Gouaux, Eric

    2012-01-01

    The controversy about the number of substrate binding sites in the neurotransmitter sodium symporter model protein LeuT is ongoing. Ligand binding assays and crystal structures of the LeuT F253A mutant support the presence of a single high affinity substrate binding site.

  3. An HIV-1 encoded peptide mimics the DNA binding loop of NF-{kappa}B and binds thioredoxin with high affinity

    Energy Technology Data Exchange (ETDEWEB)

    Su Guoping [Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, GA 30602-2352 (United States)]. E-mail: gsu@u.washington.edu; Wang Min [Department of Pathology, Yale University School of Medicine, New Haven, CT 06520-8023 (United States)]. E-mail: wang.min@yale.edu; Taylor, Ethan Will [Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, GA 30602-2352 (United States)]. E-mail: wtaylor@rx.uga.edu

    2005-11-11

    Pro-fs is a human immunodeficiency virus type 1 (HIV-l)-encoded putative selenoprotein, predicted by a theoretical analysis of the viral genome; it is potentially expressed by a -1 frameshift from the protease coding region. Pro-fs has significant sequence similarity to the DNA binding loop of nuclear factor kappa B (NF-{kappa}B), which is known to bind thioredoxin (Trx). We hypothesize that the putative HIV-1 pro-fs gene product functions by mimicry of NF-{kappa}B via binding to Trx. The hypothesis was tested in vitro by co-immunoprecipitation and GST-pull down assays, using a purified mutant pro-fs protein, in which the two potential selenocysteine residues were mutated to cysteines, in order to permit expression in bacteria. Both experiments showed that pro-fs binds to human wild type Trx (Trx-wt) with high affinity. Mutation of the two conserved cysteine residues in the Trx active site redox center to serine (Ser) (Trx-CS) weakened but failed to abolish the interaction. In pro-fs-transfected 293T cells, using confocal microscopy and fluorescence resonance energy transfer (FRET), we have observed that pro-fs localizes in cell nuclei and forms oligomers. Upon stimulation by phorbol 12-myristate 13-acetate (PMA), Trx translocates into cell nuclei. Significant FRET efficiency was detected in the nuclei of PMA-stimulated 293T cells co-expressing fluorescence-tagged pro-fs and Trx-wt or Trx-CS. These results indicate that in living cells the double cysteine mutant of pro-fs binds to both Trx and Trx-CS with high affinity, suggesting that Trx-pro-fs binding is a structurally-specific interaction, involving more of the Trx molecule than just its active site cysteine residues. These results establish the capacity for functional mimicry of the Trx binding ability of the NF-{kappa}B/Rel family of transcription factors by the putative HIV-1 pro-fs protein.

  4. Chloride binding site of neurotransmitter sodium symporters.

    Science.gov (United States)

    Kantcheva, Adriana K; Quick, Matthias; Shi, Lei; Winther, Anne-Marie Lund; Stolzenberg, Sebastian; Weinstein, Harel; Javitch, Jonathan A; Nissen, Poul

    2013-05-21

    Neurotransmitter:sodium symporters (NSSs) play a critical role in signaling by reuptake of neurotransmitters. Eukaryotic NSSs are chloride-dependent, whereas prokaryotic NSS homologs like LeuT are chloride-independent but contain an acidic residue (Glu290 in LeuT) at a site where eukaryotic NSSs have a serine. The LeuT-E290S mutant displays chloride-dependent activity. We show that, in LeuT-E290S cocrystallized with bromide or chloride, the anion is coordinated by side chain hydroxyls from Tyr47, Ser290, and Thr254 and the side chain amide of Gln250. The bound anion and the nearby sodium ion in the Na1 site organize a connection between their coordinating residues and the extracellular gate of LeuT through a continuous H-bond network. The specific insights from the structures, combined with results from substrate binding studies and molecular dynamics simulations, reveal an anion-dependent occlusion mechanism for NSS and shed light on the functional role of chloride binding.

  5. Protein purification-free method of binding affinity determination by microscale thermophoresis.

    Science.gov (United States)

    Khavrutskii, Lyuba; Yeh, Joanna; Timofeeva, Olga; Tarasov, Sergey G; Pritt, Samuel; Stefanisko, Karen; Tarasova, Nadya

    2013-08-15

    Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

  6. Enriching Peptide Libraries for Binding Affinity and Specificity Through Computationally Directed Library Design.

    Science.gov (United States)

    Foight, Glenna Wink; Chen, T Scott; Richman, Daniel; Keating, Amy E

    2017-01-01

    Peptide reagents with high affinity or specificity for their target protein interaction partner are of utility for many important applications. Optimization of peptide binding by screening large libraries is a proven and powerful approach. Libraries designed to be enriched in peptide sequences that are predicted to have desired affinity or specificity characteristics are more likely to yield success than random mutagenesis. We present a library optimization method in which the choice of amino acids to encode at each peptide position can be guided by available experimental data or structure-based predictions. We discuss how to use analysis of predicted library performance to inform rounds of library design. Finally, we include protocols for more complex library design procedures that consider the chemical diversity of the amino acids at each peptide position and optimize a library score based on a user-specified input model.

  7. Contribution of the first K-homology domain of poly(C)-binding protein 1 to its affinity and specificity for C-rich oligonucleotides.

    Science.gov (United States)

    Yoga, Yano M K; Traore, Daouda A K; Sidiqi, Mahjooba; Szeto, Chris; Pendini, Nicole R; Barker, Andrew; Leedman, Peter J; Wilce, Jacqueline A; Wilce, Matthew C J

    2012-06-01

    Poly-C-binding proteins are triple KH (hnRNP K homology) domain proteins with specificity for single stranded C-rich RNA and DNA. They play diverse roles in the regulation of protein expression at both transcriptional and translational levels. Here, we analyse the contributions of individual αCP1 KH domains to binding C-rich oligonucleotides using biophysical and structural methods. Using surface plasmon resonance (SPR), we demonstrate that KH1 makes the most stable interactions with both RNA and DNA, KH3 binds with intermediate affinity and KH2 only interacts detectibly with DNA. The crystal structure of KH1 bound to a 5'-CCCTCCCT-3' DNA sequence shows a 2:1 protein:DNA stoichiometry and demonstrates a molecular arrangement of KH domains bound to immediately adjacent oligonucleotide target sites. SPR experiments, with a series of poly-C-sequences reveals that cytosine is preferred at all four positions in the oligonucleotide binding cleft and that a C-tetrad binds KH1 with 10 times higher affinity than a C-triplet. The basis for this high affinity interaction is finally detailed with the structure determination of a KH1.W.C54S mutant bound to 5'-ACCCCA-3' DNA sequence. Together, these data establish the lead role of KH1 in oligonucleotide binding by αCP1 and reveal the molecular basis of its specificity for a C-rich tetrad.

  8. pH-dependent binding engineering reveals an FcRn affinity threshold that governs IgG recycling.

    Science.gov (United States)

    Borrok, M Jack; Wu, Yanli; Beyaz, Nurten; Yu, Xiang-Qing; Oganesyan, Vaheh; Dall'Acqua, William F; Tsui, Ping

    2015-02-13

    The Fc domain of IgG has been the target of multiple mutational studies aimed at altering the pH-dependent IgG/FcRn interaction to modulate IgG pharmacokinetics. These studies have yielded antibody variants with disparate pharmacokinetic characteristics, ranging from extended in vivo half-life to those exhibiting extremely rapid clearance. To better understand pH-dependent binding parameters that govern these outcomes and limit FcRn-mediated half-life extension, we generated a panel of novel Fc variants with high affinity binding at acidic pH that vary in pH 7.4 affinities and assessed pharmacokinetic outcomes. Pharmacokinetic studies in human FcRn transgenic mice and cynomolgus monkeys showed that multiple variants with increased FcRn affinities at acidic pH exhibited extended serum half-lives relative to the parental IgG. Importantly, the results reveal an underappreciated affinity threshold of neutral pH binding that determines IgG recycling efficiency. Variants with pH 7.4 FcRn affinities below this threshold recycle efficiently and can exhibit increased serum persistence. Increasing neutral pH FcRn affinity beyond this threshold reduced serum persistence by offsetting the benefits of increased pH 6.0 binding. Ultra-high affinity binding to FcRn at both acidic and neutral pH leads to rapid serum clearance.

  9. Determining the binding mode and binding affinity constant of tyrosine kinase inhibitor PD153035 to DNA using optical tweezers

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Chih-Ming [School of Dental Technology, Taipei Medical University, Taipei 110, Taiwan (China); Research Center for Biomedical Implants and Microsurgery Devices, Taipei Medical University, Taipei 110, Taiwan (China); Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu 30043, Taiwan (China); Lee, Yuarn-Jang [Section of Infectious Diseases, Department of Internal Medicine, Taipei Medical University Hospital, Taipei 110, Taiwan (China); Wang, Wei-Ting [School of Dental Technology, Taipei Medical University, Taipei 110, Taiwan (China); Institute of Biomedical Materials and Engineering, Taipei Medical University, Taipei 110, Taiwan (China); Research Center for Biomedical Implants and Microsurgery Devices, Taipei Medical University, Taipei 110, Taiwan (China); Hsu, Chien-Ting [Institute of Biomedical Materials and Engineering, Taipei Medical University, Taipei 110, Taiwan (China); Research Center for Biomedical Implants and Microsurgery Devices, Taipei Medical University, Taipei 110, Taiwan (China); Tsai, Jing-Shin [School of Dental Technology, Taipei Medical University, Taipei 110, Taiwan (China); Institute of Biomedical Materials and Engineering, Taipei Medical University, Taipei 110, Taiwan (China); Research Center for Biomedical Implants and Microsurgery Devices, Taipei Medical University, Taipei 110, Taiwan (China); Wu, Chien-Ming [Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu 30043, Taiwan (China); Ou, Keng-Liang [Institute of Biomedical Materials and Engineering, Taipei Medical University, Taipei 110, Taiwan (China); Research Center for Biomedical Implants and Microsurgery Devices, Taipei Medical University, Taipei 110, Taiwan (China); and others

    2011-01-07

    Research highlights: {yields} PD153035 is a DNA intercalator and intercalation occurs only under very low salt concentration. {yields} The minimum distance between adjacent bound PD153035 {approx} 11 bp. {yields} Binding affinity constant for PD153035 is 1.18({+-}0.09) x 10{sup 4} (1/M). {yields} The change of binding free energy of PD153035-DNA interaction is -5.49 kcal mol{sup -1} at 23 {+-} 0.5 {sup o}C. -- Abstract: Accurately predicting binding affinity constant (K{sub A}) is highly required to determine the binding energetics of the driving forces in drug-DNA interactions. Recently, PD153035, brominated anilinoquinazoline, has been reported to be not only a highly selective inhibitor of epidermal growth factor receptor but also a DNA intercalator. Here, we use a dual-trap optical tweezers to determining K{sub A} for PD153035, where K{sub A} is determined from the changes in B-form contour length (L) of PD153035-DNA complex. Here, L is fitted using a modified wormlike chain model. We found that a noticeable increment in L in 1 mM sodium cacodylate was exhibited. Furthermore, our results showed that K{sub A} = 1.18({+-}0.09) x 10{sup 4} (1/M) at 23 {+-} 0.5 {sup o}C and the minimum distance between adjacent bound PD153035 {approx} 11 bp. We anticipate that by using this approach we can determine the complete thermodynamic profiles due to the presence of DNA intercalators.

  10. Influence of target concentration and background binding on in vitro selection of affinity reagents.

    Directory of Open Access Journals (Sweden)

    Jinpeng Wang

    Full Text Available Nucleic acid-based aptamers possess many useful features that make them a promising alternative to antibodies and other affinity reagents, including well-established chemical synthesis, reversible folding, thermal stability and low cost. However, the selection process typically used to generate aptamers (SELEX often requires significant resources and can fail to yield aptamers with sufficient affinity and specificity. A number of seminal theoretical models and numerical simulations have been reported in the literature offering insights into experimental factors that govern the effectiveness of the selection process. Though useful, these previous models have not considered the full spectrum of experimental factors or the potential impact of tuning these parameters at each round over the course of a multi-round selection process. We have developed an improved mathematical model to address this important question, and report that both target concentration and the degree of non-specific background binding are critical determinants of SELEX efficiency. Although smaller target concentrations should theoretically offer superior selection outcome, we show that the level of background binding dramatically affect the target concentration that will yield maximum enrichment at each round of selection. Thus, our model enables experimentalists to determine appropriate target concentrations as a means for protocol optimization. Finally, we perform a comparative analysis of two different selection methods over multiple rounds of selection, and show that methods with inherently lower background binding offer dramatic advantages in selection efficiency.

  11. C-terminal substitution of MDM2 interacting peptides modulates binding affinity by distinctive mechanisms.

    Directory of Open Access Journals (Sweden)

    Christopher J Brown

    Full Text Available The complex between the proteins MDM2 and p53 is a promising drug target for cancer therapy. The residues 19-26 of p53 have been biochemically and structurally demonstrated to be a most critical region to maintain the association of MDM2 and p53. Variation of the amino acid sequence in this range obviously alters the binding affinity. Surprisingly, suitable substitutions contiguous to this region of the p53 peptides can yield tightly binding peptides. The peptide variants may differ by a single residue that vary little in their structural conformations and yet are characterized by large differences in their binding affinities. In this study a systematic analysis into the role of single C-terminal mutations of a 12 residue fragment of the p53 transactivation domain (TD and an equivalent phage optimized peptide (12/1 were undertaken to elucidate their mechanistic and thermodynamic differences in interacting with the N-terminal of MDM2. The experimental results together with atomistically detailed dynamics simulations provide insight into the principles that govern peptide design protocols with regard to protein-protein interactions and peptidomimetic design.

  12. Ligand binding affinity and changes in the lateral diffusion of receptor for advanced glycation endproducts (RAGE).

    Science.gov (United States)

    Syed, Aleem; Zhu, Qiaochu; Smith, Emily A

    2016-12-01

    The effect of ligand on the lateral diffusion of receptor for advanced glycation endproducts (RAGE), a receptor involved in numerous pathological conditions, remains unknown. Single particle tracking experiments that use quantum dots specifically bound to hemagglutinin (HA)-tagged RAGE (HA-RAGE) are reported to elucidate the effect of ligand binding on HA-RAGE diffusion in GM07373 cell membranes. The ligand used in these studies is methylglyoxal modified-bovine serum albumin (MGO-BSA) containing advanced glycation end products modifications. The binding affinity between soluble RAGE and MGO-BSA increases by 1.8 to 9.7-fold as the percent primary amine modification increases from 24 to 74% and with increasing negative charge on the MGO-BSA. Ligand incubation affects the HA-RAGE diffusion coefficient, the radius of confinement, and duration of confinement. There is, however, no correlation between MGO-BSA ligand binding affinity with soluble RAGE and the extent of the changes in HA-RAGE lateral diffusion. The ligand induced changes to HA-RAGE lateral diffusion do not occur when cholesterol is depleted from the cell membrane, indicating the mechanism for ligand-induced changes to HA-RAGE diffusion is cholesterol dependent. The results presented here serve as a first step in unraveling how ligand influences RAGE lateral diffusion. Copyright © 2016. Published by Elsevier B.V.

  13. Lactobacillus acidophilus binds to MUC3 component of cultured intestinal epithelial cells with highest affinity.

    Science.gov (United States)

    Das, Jugal Kishore; Mahapatra, Rajani Kanta; Patro, Shubhransu; Goswami, Chandan; Suar, Mrutyunjay

    2016-04-01

    Lactobacillus strains have been shown to adhere to the mucosal components of intestinal epithelial cells. However, established in vitro adhesion assays have several drawbacks in assessing the adhesion of new Lactobacillus strains. The present study aimed to compare the adhesion of four different Lactobacillus strains and select the most adherent microbe, based on in silico approach supported by in vitro results. The mucus-binding proteins in Lactobacillus acidophilus, L. plantarum, L. brevis and L. fermentum were identified and their capacities to interact with intestinal mucin were compared by molecular docking analysis. Lactobacillus acidophilus had the maximal affinity of binding to mucin with predicted free energy of -6.066 kcal mol(-1) Further, in vitro experimental assay of adhesion was performed to validate the in silico results. The adhesion of L. acidophilus to mucous secreting colon epithelial HT-29 MTX cells was highest at 12%, and it formed biofilm with maximum depth (Z = 84 μm). Lactobacillus acidophilus was determined to be the most adherent strain in the study. All the Lactobacillus strains tested in this study, displayed maximum affinity of binding to MUC3 component of mucus as compared to other gastrointestinal mucins. These findings may have importance in the design of probiotics and health care management.

  14. A new BODIPY/nanoparticle/Ni affinity system for binding of cytochrome c

    Energy Technology Data Exchange (ETDEWEB)

    Maltas, Esra, E-mail: maltasesra@gmail.com [Selcuk University, Faculty of Science, Department of Chemistry, 42075 Konya (Turkey); Selcuk University, Faculty of Science, Department of Biochemistry, 42075 Konya (Turkey); Kursunlu, Ahmed Nuri [Selcuk University, Faculty of Science, Department of Chemistry, 42075 Konya (Turkey); Arslan, Gulsin [Selcuk University, Faculty of Science, Department of Biochemistry, 42075 Konya (Turkey); Selcuk University, Advanced Research Technology and Application Center, 42075 Konya (Turkey); Ozmen, Mustafa [Selcuk University, Faculty of Science, Department of Chemistry, 42075 Konya (Turkey); Selcuk University, Advanced Research Technology and Application Center, 42075 Konya (Turkey)

    2015-09-15

    Highlights: • BODIPY was synthesized, and then attached to magnetic nanoparticles. • Ni(II) ions were chelated on prepared material. • The binding of cytochrome c to obtained material was studied. - Abstract: In this study, 3,5-{Bis[4,4-difluoro, 8-(2,6-diethyl, 1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene)]}benzoylchloride (BODIPY) was synthesized for the improving of a new immobilized metal affinity supporting material. Firstly, the synthesized BODIPY was immobilized on iron oxide superparamagnetic nanoparticles (SPIONs) and then, Ni(II) ions were chelated with the active terminals of BODIPY on nanoparticles surfaces to prepare an immobilized metal affinity (IMA) adsorbent for protein adsorption. The amount of BODIPY coated on SPIONs was about 29.7 μM at 10 mg nanoparticles. 738 μmol of Ni(II) ions were loaded to 10 mg of the SPIONs/BODIPY. The binding amount of cytochrome c was found to be 170 μg to the SPIONs/BODIPY/Ni at pH 7.4. The binding amount of the molecules on SPIONs was analyzed by using UV–vis, fluorescence and atomic absorption spectroscopy. The characterization of the prepared surfaces was performed by FT-IR, SEM and TEM.

  15. Tuning affinity and reversibility for O2 binding in dinuclear Co(II) complexes

    DEFF Research Database (Denmark)

    Vad, Mads Sørensen; Johansson, Frank Bartnik; Seidler-Egdal, Rune Kirk

    2013-01-01

    of the peroxido O–O stretch in the solid-state resonance Raman spectra at 298 K (830–836 cm−1). Using density functional theory calculations, we conclude that the Co(II) atoms of the deoxy complexes coordinate solvent molecules as auxiliary ligands and that a conformation change of the ligand is involved...... in the reversible O2 binding process. The alternative of five coordination in the deoxy Co(II) complexes is therefore seen as less likely. The crystal structure and p(O2)50% are also reported for the 1-naphthoato-bridged oxy complex [Co2(bpbp)(O2)(C10H7O2)]2+, and the O2 binding affinity in that case is also......The O2 binding affinity of a series of dicobalt(II) complexes can be tuned between p(O2)50% = 2.3 × 10−3 and 700 × 10−3 atm at 40 °C by varying the number of H and Cl atoms in the bridging acetato ligands of [Co2(bpbp)(CH(3−n)ClnCO2)(CH3CN)2]2+, where bpbp− = 2,6-bis(N,N-bis(2-pyridylmethyl...

  16. Binding-affinity predictions of HSP90 in the D3R Grand Challenge 2015 with docking, MM/GBSA, QM/MM, and free-energy simulations

    Science.gov (United States)

    Misini Ignjatović, Majda; Caldararu, Octav; Dong, Geng; Muñoz-Gutierrez, Camila; Adasme-Carreño, Francisco; Ryde, Ulf

    2016-09-01

    We have estimated the binding affinity of three sets of ligands of the heat-shock protein 90 in the D3R grand challenge blind test competition. We have employed four different methods, based on five different crystal structures: first, we docked the ligands to the proteins with induced-fit docking with the Glide software and calculated binding affinities with three energy functions. Second, the docked structures were minimised in a continuum solvent and binding affinities were calculated with the MM/GBSA method (molecular mechanics combined with generalised Born and solvent-accessible surface area solvation). Third, the docked structures were re-optimised by combined quantum mechanics and molecular mechanics (QM/MM) calculations. Then, interaction energies were calculated with quantum mechanical calculations employing 970-1160 atoms in a continuum solvent, combined with energy corrections for dispersion, zero-point energy and entropy, ligand distortion, ligand solvation, and an increase of the basis set to quadruple-zeta quality. Fourth, relative binding affinities were estimated by free-energy simulations, using the multi-state Bennett acceptance-ratio approach. Unfortunately, the results were varying and rather poor, with only one calculation giving a correlation to the experimental affinities larger than 0.7, and with no consistent difference in the quality of the predictions from the various methods. For one set of ligands, the results could be strongly improved (after experimental data were revealed) if it was recognised that one of the ligands displaced one or two water molecules. For the other two sets, the problem is probably that the ligands bind in different modes than in the crystal structures employed or that the conformation of the ligand-binding site or the whole protein changes.

  17. Binding-affinity predictions of HSP90 in the D3R Grand Challenge 2015 with docking, MM/GBSA, QM/MM, and free-energy simulations.

    Science.gov (United States)

    Misini Ignjatović, Majda; Caldararu, Octav; Dong, Geng; Muñoz-Gutierrez, Camila; Adasme-Carreño, Francisco; Ryde, Ulf

    2016-09-01

    We have estimated the binding affinity of three sets of ligands of the heat-shock protein 90 in the D3R grand challenge blind test competition. We have employed four different methods, based on five different crystal structures: first, we docked the ligands to the proteins with induced-fit docking with the Glide software and calculated binding affinities with three energy functions. Second, the docked structures were minimised in a continuum solvent and binding affinities were calculated with the MM/GBSA method (molecular mechanics combined with generalised Born and solvent-accessible surface area solvation). Third, the docked structures were re-optimised by combined quantum mechanics and molecular mechanics (QM/MM) calculations. Then, interaction energies were calculated with quantum mechanical calculations employing 970-1160 atoms in a continuum solvent, combined with energy corrections for dispersion, zero-point energy and entropy, ligand distortion, ligand solvation, and an increase of the basis set to quadruple-zeta quality. Fourth, relative binding affinities were estimated by free-energy simulations, using the multi-state Bennett acceptance-ratio approach. Unfortunately, the results were varying and rather poor, with only one calculation giving a correlation to the experimental affinities larger than 0.7, and with no consistent difference in the quality of the predictions from the various methods. For one set of ligands, the results could be strongly improved (after experimental data were revealed) if it was recognised that one of the ligands displaced one or two water molecules. For the other two sets, the problem is probably that the ligands bind in different modes than in the crystal structures employed or that the conformation of the ligand-binding site or the whole protein changes.

  18. Relative binding affinity does not predict biological response to xenoestrogens in rat endometrial adenocarcinoma cells.

    Science.gov (United States)

    Strunck, E; Stemmann, N; Hopert, A; Wünsche, W; Frank, K; Vollmer, G

    2000-10-01

    The possible adverse effects of the so-called environmental estrogens have raised considerable concern. Developmental, endocrine and reproductive disorders in wildlife animals have been linked to high exposure to persistent environmental chemicals with estrogen-like activity (xenoestrogens); yet, the potential impact of environmental estrogens on human health is currently under debate also due to lack of data. A battery of in vitro assays exist for identifying compounds with estrogenic activity, but only a few models are available to assess estrogenic potency in a multiparametric analysis. We have recently established the endometrial adenocarcinoma cell line RUCA-I; it enables us to compare estrogenic effects both in vitro and in vivo as these cells are estrogen responsive in vitro and grow estrogen sensitive tumors if inoculated in syngeneic animals in vivo. Here we report in vitro data concerning (a) the relative binding affinity of the selected synthetic chemicals Bisphenol A, nonylphenol, p-tert-octylphenol, and o,p-DDT to the estrogen receptor of RUCA-I cells and (b) the relative potency of these compounds in inducing increased production of complement C3, an endogenous estrogen-responsive gene. Competitive Scatchard analysis revealed that xenoestrogens bound with an at least 1000-fold lower affinity to the estrogen receptor of RUCA-I cells than estradiol itself, thereby exhibiting the following affinity ranking, estradiol>nonylphenol>bisphenol A approximately p-tert-octylphenol>o,p-DDT. Despite these low binding affinities, bisphenol A, nonylphenol and p-tert-octylphenol increased production of complement C3 in a dose dependent manner. Compared with estradiol, only 100-fold higher concentrations were needed for all the compounds to achieve similar levels of induction, except o,p-DDT which was by far less potent. Northern blot analyses demonstrated that the increased production of complement C3 was mediated by an increased transcription. In summary, cultured

  19. Modeling the binding affinity of structurally diverse industrial chemicals to carbon using the artificial intelligence approaches.

    Science.gov (United States)

    Gupta, Shikha; Basant, Nikita; Rai, Premanjali; Singh, Kunwar P

    2015-11-01

    Binding affinity of chemical to carbon is an important characteristic as it finds vast industrial applications. Experimental determination of the adsorption capacity of diverse chemicals onto carbon is both time and resource intensive, and development of computational approaches has widely been advocated. In this study, artificial intelligence (AI)-based ten different qualitative and quantitative structure-property relationship (QSPR) models (MLPN, RBFN, PNN/GRNN, CCN, SVM, GEP, GMDH, SDT, DTF, DTB) were established for the prediction of the adsorption capacity of structurally diverse chemicals to activated carbon following the OECD guidelines. Structural diversity of the chemicals and nonlinear dependence in the data were evaluated using the Tanimoto similarity index and Brock-Dechert-Scheinkman statistics. The generalization and prediction abilities of the constructed models were established through rigorous internal and external validation procedures performed employing a wide series of statistical checks. In complete dataset, the qualitative models rendered classification accuracies between 97.04 and 99.93%, while the quantitative models yielded correlation (R(2)) values of 0.877-0.977 between the measured and the predicted endpoint values. The quantitative prediction accuracies for the higher molecular weight (MW) compounds (class 4) were relatively better than those for the low MW compounds. Both in the qualitative and quantitative models, the Polarizability was the most influential descriptor. Structural alerts responsible for the extreme adsorption behavior of the compounds were identified. Higher number of carbon and presence of higher halogens in a molecule rendered higher binding affinity. Proposed QSPR models performed well and outperformed the previous reports. A relatively better performance of the ensemble learning models (DTF, DTB) may be attributed to the strengths of the bagging and boosting algorithms which enhance the predictive accuracies. The

  20. Structural characterization of a high affinity mononuclear site in the copper(II)-α-synuclein complex.

    Science.gov (United States)

    Bortolus, Marco; Bisaglia, Marco; Zoleo, Alfonso; Fittipaldi, Maria; Benfatto, Maurizio; Bubacco, Luigi; Maniero, Anna Lisa

    2010-12-29

    Human α-Synuclein (aS), a 140 amino acid protein, is the main constituent of Lewy bodies, the cytoplasmatic deposits found in the brains of Parkinson's disease patients, where it is present in an aggregated, fibrillar form. Recent studies have shown that aS is a metal binding protein. Moreover, heavy metal ions, in particular divalent copper, accelerate the aggregation process of the protein. In this work, we investigated the high affinity binding mode of truncated aS (1-99) (aS99) with Cu(II), in a stoichiometric ratio, to elucidate the residues involved in the binding site and the role of copper ions in the protein oligomerization. We used Electron Paramagnetic Resonance spectroscopy on the Cu(II)-aS99 complex at pH 6.5, performing both multifrequency continuous wave experiments and pulsed experiments at X-band. The comparison of 9.5 and 95 GHz data showed that at this pH only one binding mode is present. To identify the nature of the ligands, we performed Electron Spin Echo Envelope Modulation, Hyperfine Sublevel Correlation Spectroscopy, and pulsed Davies Electron-Nuclear Double Resonance (Davies-ENDOR) experiments. We determined that the EPR parameters are typical of a type-II copper complex, in a slightly distorted square planar geometry. Combining the results from the different pulsed techniques, we obtained that the equatorial coordination is {N(Im), N(-), H(2)O, O}, where N(im) is the imino nitrogen of His50, N(-) a deprotonated amido backbone nitrogen that we attribute to His50, H(2)O an exchangeable water molecule, and O an unidentified oxygen ligand. Moreover, we propose that the free amino terminus (Met1) participates in the complex as an axial ligand. The MXAN analysis of the XAS k-edge absorption data allowed us to independently validate the structural features proposed on the basis of the magnetic parameters of the Cu(II)-aS99 complex and then to further refine the quality of the proposed structural model.

  1. Serotonin receptor binding affinities of several hallucinogenic phenylalkylamine and N,N-dimethyltryptamine analogues.

    Science.gov (United States)

    Glennon, R A; Liebowitz, S M; Mack, E C

    1978-08-01

    Hallucinogenic phenylalkylamine and N,N-dimethyltryptamine analogues are known to affect serotonergic systems both in vivo and in vitro. Using a rat stomach fundus model, the 5-HT receptor binding affinities of several of these analogues were determined and compared. The most behaviorally potent analogues examined, DOB, DOM, and 5-methoxy-N,N-dimethyltryptamine, were found to possess rather high affirmities (pA2 = 7.35, 7.12, and 7.08, respectively) for the 5-HT receptors of the model system.

  2. Metal binding affinity and selectivity in metalloproteins: insights from computational studies.

    Science.gov (United States)

    Dudev, Todor; Lim, Carmay

    2008-01-01

    This review highlights insights gained from computational studies on protein-metal recognition. We systematically dissect the various factors governing metal binding affinity and selectivity in proteins starting from (a) the intrinsic properties of the metal and neighboring metal cations (if present), to (b) the primary coordination sphere, (c) the second coordination shell, (d) the protein matrix, (e) the bulk solvent, and (f) competing non-protein ligands from the surrounding biological environment. The results herein reveal the fundamental principles and the molecular bases underlying protein-metal recognition, which serve as a guide to engineer novel metalloproteins with programmed properties.

  3. Copper(II) ions and the Alzheimer's amyloid-β peptide: Affinity and stoichiometry of binding

    Science.gov (United States)

    Tõugu, Vello; Friedemann, Merlin; Tiiman, Ann; Palumaa, Peep

    2014-10-01

    Deposition of amyloid beta (Aβ) peptides into amyloid plaques is the hallmark of Alzheimer's disease. According to the amyloid cascade hypothesis this deposition is an early event and primary cause of the disease, however, the mechanisms that cause this deposition remain elusive. An increasing amount of evidence shows that the interactions of biometals can contribute to the fibrillization and amyloid formation by amyloidogenic peptides. From different anions the copper ions deserve the most attention since it can contribute not only toamyloid formation but also to its toxicity due to the generation of ROS. In this thesis we focus on the affinity and stoichiometry of copper(II) binding to the Aβ molecule.

  4. A structure-based design of new C2- and C13-substituted taxanes: tubulin binding affinities and extended quantitative structure-activity relationships using comparative binding energy (COMBINE) analysis.

    Science.gov (United States)

    Coderch, Claire; Tang, Yong; Klett, Javier; Zhang, Shu-En; Ma, Yun-Tao; Shaorong, Wang; Matesanz, Ruth; Pera, Benet; Canales, Angeles; Jiménez-Barbero, Jesús; Morreale, Antonio; Díaz, J Fernando; Fang, Wei-Shuo; Gago, Federico

    2013-05-14

    Ten novel taxanes bearing modifications at the C2 and C13 positions of the baccatin core have been synthesized and their binding affinities for mammalian tubulin have been experimentally measured. The design strategy was guided by (i) calculation of interaction energy maps with carbon, nitrogen and oxygen probes within the taxane-binding site of β-tubulin, and (ii) the prospective use of a structure-based QSAR (COMBINE) model derived from an earlier series comprising 47 congeneric taxanes. The tubulin-binding affinity displayed by one of the new compounds (CTX63) proved to be higher than that of docetaxel, and an updated COMBINE model provided a good correlation between the experimental binding free energies and a set of weighted residue-based ligand-receptor interaction energies for 54 out of the 57 compounds studied. The remaining three outliers from the original training series have in common a large unfavourable entropic contribution to the binding free energy that we attribute to taxane preorganization in aqueous solution in a conformation different from that compatible with tubulin binding. Support for this proposal was obtained from solution NMR experiments and molecular dynamics simulations in explicit water. Our results shed additional light on the determinants of tubulin-binding affinity for this important class of antitumour agents and pave the way for further rational structural modifications.

  5. Study on Synthesis and Binding Ability of a New Anion Receptor Containing NH Binding Sites

    Institute of Scientific and Technical Information of China (English)

    QIAO,Yan-Hong; LIN,Hai; LIN,Hua-Kuan

    2007-01-01

    A new colorimetric recognition receptor 1 based on the dual capability containing NH binding sites of selectively sensing anionic guest species has been synthesized. Compared with other halide anions, its UV/Vis absorption spectrum in dimethyl sulfoxide showed the response toward the presence of fluoride anion with high selectivity,and also displayed dramatic color changes from colorless to yellow in the presence of TBAF (5 × 10-5 mol/L). The similar UV/Vis absorption spectrum change also occurred when 1 was treated with AcO- while a little change with H2PO-4 and OH-. Receptor 1 has almost not affinity abilities to Cl-, Br- and I-. The binding ability of receptor 1to fluoride with high selectivity over other halides contributes to the anion size and the ability of forming hydrogen bonding. While the different ability of binding with geometrically triangular (AcO-), tetrahedral (H2PO-4 ) and linear (OH-) anions maybe result from their geometry configuration.

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

    Science.gov (United States)

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

    2016-08-31

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

  7. Differential affinity of FLIP and procaspase 8 for FADD's DED binding surfaces regulates DISC assembly.

    Science.gov (United States)

    Majkut, J; Sgobba, M; Holohan, C; Crawford, N; Logan, A E; Kerr, E; Higgins, C A; Redmond, K L; Riley, J S; Stasik, I; Fennell, D A; Van Schaeybroeck, S; Haider, S; Johnston, P G; Haigh, D; Longley, D B

    2014-02-28

    Death receptor activation triggers recruitment of FADD, which via its death effector domain (DED) engages the DEDs of procaspase 8 and its inhibitor FLIP to form death-inducing signalling complexes (DISCs). The DEDs of FADD, FLIP and procaspase 8 interact with one another using two binding surfaces defined by α1/α4 and α2/α5 helices, respectively. Here we report that FLIP has preferential affinity for the α1/α4 surface of FADD, whereas procaspase 8 has preferential affinity for FADD's α2/α5 surface. These relative affinities contribute to FLIP being recruited to the DISC at comparable levels to procaspase 8 despite lower cellular expression. Additional studies, including assessment of DISC stoichiometry and functional assays, suggest that following death receptor recruitment, the FADD DED preferentially engages FLIP using its α1/α4 surface and procaspase 8 using its α2/α5 surface; these tripartite intermediates then interact via the α1/α4 surface of FLIP DED1 and the α2/α5 surface of procaspase 8 DED2.

  8. Structure-based identification of new high-affinity nucleosome binding sequences.

    Science.gov (United States)

    Battistini, Federica; Hunter, Christopher A; Moore, Irene K; Widom, Jonathan

    2012-06-29

    The substrate for the proteins that express genetic information in the cell is not naked DNA but an assembly of nucleosomes, where the DNA is wrapped around histone proteins. The organization of these nucleosomes on genomic DNA is influenced by the DNA sequence. Here, we present a structure-based computational approach that translates sequence information into the energy required to bend DNA into a nucleosome-bound conformation. The calculations establish the relationship between DNA sequence and histone octamer binding affinity. In silico selection using this model identified several new DNA sequences, which were experimentally found to have histone octamer affinities comparable to the highest-affinity sequences known. The results provide insights into the molecular mechanism through which DNA sequence information encodes its organization. A quantitative appreciation of the thermodynamics of nucleosome positioning and rearrangement will be one of the key factors in understanding the regulation of transcription and in the design of new promoter architectures for the purposes of tuning gene expression dynamics.

  9. A cleavable silica-binding affinity tag for rapid and inexpensive protein purification.

    Science.gov (United States)

    Coyle, Brandon L; Baneyx, François

    2014-10-01

    We describe a new affinity purification tag called Car9 that confers proteins to which it is fused micromolar affinity for unmodified silica. When appended to the C-terminus of GFPmut2 through a flexible linker, Car9 promotes efficient adsorption to silica gel and the fusion protein can be released from the particles by incubation with L-lysine. Using a silica gel column and the lysine elution approach in fast protein liquid chromatography (FPLC) mode, Car9-tagged versions of GFPmut2, mCherry and maltose binding protein (MBP) can be recovered from clarified lysates with a purity of 80-90%. Capitalizing on silica's ability to handle large pressure drops, we further show that it is possible to go from cell lysates to purified protein in less than 15 min using a fully disposable device. Finally, we demonstrate that the linker-Car9 region is susceptible to proteolysis by E. coli OmpT and take advantage of this observation to excise the C-terminal extension of GFPmut2-Car9 by incubating purified fusion protein with cells that overproduce the outer membrane protease OmpT. The set of strategies described herein, should reduce the cost of affinity purification by at least 10-fold, cut down purification times to minutes, and allow for the production of proteins with native (or nearly native) termini from their C-terminally-tagged versions.

  10. Characterization of high affinity (/sup 3/H)triazolam binding in rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Earle, M.; Concas, A.; Yamamura, H.I.

    1986-03-01

    The hypnotic Triazolam (TZ), a triazolo (1,4)-benzodiazepine, displays a short physiological half life and has been used for the treatment of insomnia related to anxiety states. Specific binding properties of this recently tritiated TZ were characterized. The authors major objectives were the direct measurement of the temperature dependence and the GABA effect on (/sup 3/H)TZ binding. Saturation studies showed a shift to lower affinity at 37/sup 0/C (K/sub d/ = 0.25 +/- 0.01 nM at O/sup 0/C; K/sub d/ = 1.46 +/- 0.03 nM at 37/sup 0/C) while the B/sub max/ values remained unchanged (1003 +/- 37 fmoles/mg prot. at 0/sup 0/C and 1001 +/- 43 fmoles/mg prot. at 37/sup 0/C). Inhibition studies showed that (/sup 3/H)TZ binding displayed no GABA shift at 0/sup 0/C(K/sub i/ 0.37 +/- 0.03 nM/- GABA and K/sub i/ = 0.55 +/- 0.13 nM/+GABA) but a nearly two-fold shift was apparent at 37/sup 0/C (K/sub i/ = 2.92 +/- 0.2 nM/-GABA; K/sub i/ = 1.37 +/- 0.11 mM/+GABA). These results were also confirmed by saturation studies in the presence or absence of GABA showing a shift to higher affinity in the presence of GABA only at 37/sup 0/C. In Ro 15-1788/(/sup 3/H)TZ competition experiments the presence of GABA did not affect the inhibitory potency of Ro 15-1788 on (/sup 3/H)TZ binding at both temperatures. In conclusion (/sup 3/H)TZ binding showed an extremely high affinity for benzodiazepine receptors. In contrast to reported literature, the findings suggest that TZ interacts with benzodiazepine receptors similar to other benzodiazepine agonists.

  11. Biological effect of varying peptide binding affinity to the BoLA-DRB3*2703 allele

    Directory of Open Access Journals (Sweden)

    Alizadeh Zahra

    2003-06-01

    Full Text Available Abstract MHC class I and II molecules are immunoregulatory cell surface glycoproteins, which selectively bind to and present antigenic peptides to T-lymphocytes. Murine and human studies show that variable peptide binding affinity to MHC II molecules influences Th1/Th2 responses by inducing distinctive cytokine expression. To examine the biological effects of peptide binding affinity to bovine MHC (BoLA, various self peptides (BoLA-DQ and fibrinogen fragments and non-self peptides from ovalbumin (OVA, as well as VP2 and VP4 peptides from foot and mouth disease virus (FMD-V were used to (1 determine binding affinities to the BoLA-DRB3*2703 allele, previously associated with mastitis susceptibility and (2 determine whether peptide binding affinity influences T-lymphocyte function. Peptide binding affinity was determined by a competitive assay using high affinity biotinylated self-peptide incubated with purified BoLA-DRB3*2703 in the presence of various concentrations of competing peptides. The concentrations of non-self peptide required to inhibit self-peptide binding by 50% (IC50 were variable, ranging from 26.92 to > 320 μM. Peptide-specific T-lymphocyte function was determined by measuring DNA synthesis, cell division, and IFN-γ production in cultures of mononuclear cells from a BoLA-DRB3*2703 homozygous cow. When compared to non-stimulated control cultures, differences in lymphocyte function were observed for all of the assessed parameters; however, peptide-binding affinity did not always account for the observed differences in lymphocyte function.

  12. Ligand docking and binding site analysis with PyMOL and Autodock/Vina.

    Science.gov (United States)

    Seeliger, Daniel; de Groot, Bert L

    2010-05-01

    Docking of small molecule compounds into the binding site of a receptor and estimating the binding affinity of the complex is an important part of the structure-based drug design process. For a thorough understanding of the structural principles that determine the strength of a protein/ligand complex both, an accurate and fast docking protocol and the ability to visualize binding geometries and interactions are mandatory. Here we present an interface between the popular molecular graphics system PyMOL and the molecular docking suites Autodock and Vina and demonstrate how the combination of docking and visualization can aid structure-based drug design efforts.

  13. 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.

  14. UO₂²⁺ uptake by proteins: understanding the binding features of the super uranyl binding protein and design of a protein with higher affinity.

    Science.gov (United States)

    Odoh, Samuel O; Bondarevsky, Gary D; Karpus, Jason; Cui, Qiang; He, Chuan; Spezia, Riccardo; Gagliardi, Laura

    2014-12-17

    The capture of uranyl, UO2(2+), by a recently engineered protein (Zhou et al. Nat. Chem. 2014, 6, 236) with high selectivity and femtomolar sensitivity has been examined by a combination of density functional theory, molecular dynamics, and free-energy simulations. It was found that UO2(2+) is coordinated to five carboxylate oxygen atoms from four amino acid residues of the super uranyl binding protein (SUP). A network of hydrogen bonds between the amino acid residues coordinated to UO2(2+) and residues in its second coordination sphere also affects the protein's uranyl binding affinity. Free-energy simulations show how UO2(2+) capture is governed by the nature of the amino acid residues in the binding site, the integrity and strength of the second-sphere hydrogen bond network, and the number of water molecules in the first coordination sphere. Alteration of any of these three factors through mutations generally results in a reduction of the binding free energy of UO2(2+) to the aqueous protein as well as of the difference between the binding free energies of UO2(2+) and other ions (Ca(2+), Cu(2+), Mg(2+), and Zn(2+)), a proxy for the protein's selectivity over these ions. The results of our free-energy simulations confirmed the previously reported experimental results and allowed us to discover a mutant of SUP, specifically the GLU64ASP mutant, that not only binds UO2(2+) more strongly than SUP but that is also more selective for UO2(2+) over other ions. The predictions from the computations were confirmed experimentally.

  15. Decreased angiotensin II binding affinity and binding capacity in the anterior pituitary gland of adult spontaneously hypertensive rats

    Energy Technology Data Exchange (ETDEWEB)

    Gutkind, J.S.; Castren, E.; Saavedra, J.M.

    1988-01-01

    Angiotensin II (ANG) binding sites were quantified in single pituitary glands from 4-week-old and 14-week-old male spontaneously hypertensive rats (SHR) and age-matched male normotensive Wistar-Kyoto (WKY) control rats after incubation with /sup 125/I-(Sar/sup 1/)-ANG, autoradiography with computerized densitometry, and comparison to /sup 125/I-standards. The maximum binding capacity (B/sub max/) decreased while the dissociation constant (K/sub d/) for ANG increased in 14-week-old SHR when compared to age-matched WKY control rats. Conversely, no difference between rat strains was found in 4-week-old animals. Our results suggest that pituitary ANG binding sites may play a role in the pathophysiology of established genetic hypertension.

  16. DBD2BS: connecting a DNA-binding protein with its binding sites.

    Science.gov (United States)

    Chien, Ting-Ying; Lin, Chih-Kang; Lin, Chih-Wei; Weng, Yi-Zhong; Chen, Chien-Yu; Chang, Darby Tien-Hao

    2012-07-01

    By binding to short and highly conserved DNA sequences in genomes, DNA-binding proteins initiate, enhance or repress biological processes. Accurately identifying such binding sites, often represented by position weight matrices (PWMs), is an important step in understanding the control mechanisms of cells. When given coordinates of a DNA-binding domain (DBD) bound with DNA, a potential function can be used to estimate the change of binding affinity after base substitutions, where the changes can be summarized as a PWM. This technique provides an effective alternative when the chromatin immunoprecipitation data are unavailable for PWM inference. To facilitate the procedure of predicting PWMs based on protein-DNA complexes or even structures of the unbound state, the web server, DBD2BS, is presented in this study. The DBD2BS uses an atom-level knowledge-based potential function to predict PWMs characterizing the sequences to which the query DBD structure can bind. For unbound queries, a list of 1066 DBD-DNA complexes (including 1813 protein chains) is compiled for use as templates for synthesizing bound structures. The DBD2BS provides users with an easy-to-use interface for visualizing the PWMs predicted based on different templates and the spatial relationships of the query protein, the DBDs and the DNAs. The DBD2BS is the first attempt to predict PWMs of DBDs from unbound structures rather than from bound ones. This approach increases the number of existing protein structures that can be exploited when analyzing protein-DNA interactions. In a recent study, the authors showed that the kernel adopted by the DBD2BS can generate PWMs consistent with those obtained from the experimental data. The use of DBD2BS to predict PWMs can be incorporated with sequence-based methods to discover binding sites in genome-wide studies. Available at: http://dbd2bs.csie.ntu.edu.tw/, http://dbd2bs.csbb.ntu.edu.tw/, and http://dbd2bs.ee.ncku.edu.tw.

  17. Acylated heptapeptide binds albumin with high affinity and application as tag furnishes long-acting peptides

    Science.gov (United States)

    Zorzi, Alessandro; Middendorp, Simon J.; Wilbs, Jonas; Deyle, Kaycie; Heinis, Christian

    2017-07-01

    The rapid renal clearance of peptides in vivo limits this attractive platform for the treatment of a broad range of diseases that require prolonged drug half-lives. An intriguing approach for extending peptide circulation times works through a `piggy-back' strategy in which peptides bind via a ligand to the long-lived serum protein albumin. In accordance with this strategy, we developed an easily synthesized albumin-binding ligand based on a peptide-fatty acid chimera that has a high affinity for human albumin (Kd=39 nM). This ligand prolongs the elimination half-life of cyclic peptides in rats 25-fold to over seven hours. Conjugation to a peptide factor XII inhibitor developed for anti-thrombotic therapy extends the half-life from 13 minutes to over five hours, inhibiting coagulation for eight hours in rabbits. This high-affinity albumin ligand could potentially extend the half-life of peptides in human to several days, substantially broadening the application range of peptides as therapeutics.

  18. Bicarbonate increases binding affinity of Vibrio cholerae ToxT to virulence gene promoters.

    Science.gov (United States)

    Thomson, Joshua J; Withey, Jeffrey H

    2014-11-01

    The major Vibrio cholerae virulence gene transcription activator, ToxT, is responsible for the production of the diarrhea-inducing cholera toxin (CT) and the major colonization factor, toxin coregulated pilus (TCP). In addition to the two primary virulence factors mentioned, ToxT is responsible for the activation of accessory virulence genes, such as aldA, tagA, acfA, acfD, tcpI, and tarAB. ToxT activity is negatively modulated by bile and unsaturated fatty acids found in the upper small intestine. Conversely, previous work identified another intestinal signal, bicarbonate, which enhances the ability of ToxT to activate production of CT and TCP. The work presented here further elucidates the mechanism for the enhancement of ToxT activity by bicarbonate. Bicarbonate was found to increase the activation of ToxT-dependent accessory virulence promoters in addition to those that produce CT and TCP. Bicarbonate is taken up into the V. cholerae cell, where it positively affects ToxT activity by increasing DNA binding affinity for the virulence gene promoters that ToxT activates regardless of toxbox configuration. The increase in ToxT binding affinity in the presence of bicarbonate explains the elevated level of virulence gene transcription.

  19. Cellulose affinity purification of fusion proteins tagged with fungal family 1 cellulose-binding domain.

    Science.gov (United States)

    Sugimoto, Naohisa; Igarashi, Kiyohiko; Samejima, Masahiro

    2012-04-01

    N- or C-terminal fusions of red-fluorescent protein (RFP) with various fungal cellulose-binding domains (CBDs) belonging to carbohydrate binding module (CBM) family 1 were expressed in a Pichia pastoris expression system, and the resulting fusion proteins were used to examine the feasibility of large-scale affinity purification of CBD-tagged proteins on cellulose columns. We found that RFP fused with CBD from Trichoderma reesei CBHI (CBD(Tr)(CBHI)) was expressed at up to 1.2g/l in the culture filtrate, which could be directly injected into the cellulose column. The fusion protein was tightly adsorbed on the cellulose column in the presence of a sufficient amount of ammonium sulfate and was efficiently eluted with pure water. Bovine serum albumin (BSA) was not captured under these conditions, whereas both BSA and the fusion protein were adsorbed on a phenyl column, indicating that the cellulose column can be used for the purification of not only hydrophilic proteins but also for hydrophobic proteins. Recovery of various fusion proteins exceeded 80%. Our results indicate that protein purification by expression of a target protein as a fusion with a fungal family 1 CBD tag in a yeast expression system, followed by affinity purification on a cellulose column, is simple, effective and easily scalable.

  20. Advances and applications of binding affinity prediction methods in drug discovery.

    Science.gov (United States)

    Parenti, Marco Daniele; Rastelli, Giulio

    2012-01-01

    Nowadays, the improvement of R&D productivity is the primary commitment in pharmaceutical research, both in big pharma and smaller biotech companies. To reduce costs, to speed up the discovery process and to increase the chance of success, advanced methods of rational drug design are very helpful, as demonstrated by several successful applications. Among these, computational methods able to predict the binding affinity of small molecules to specific biological targets are of special interest because they can accelerate the discovery of new hit compounds. Here we provide an overview of the most widely used methods in the field of binding affinity prediction, as well as of our own work in developing BEAR, an innovative methodology specifically devised to overtake some limitations in existing approaches. The BEAR method was successfully validated against different biological targets, and proved its efficacy in retrieving active compounds from virtual screening campaigns. The results obtained so far indicate that BEAR may become a leading tool in the drug discovery pipeline. We primarily discuss advantages and drawbacks of each technique and show relevant examples and applications in drug discovery.

  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. Allostery between two binding sites in the ion channel subunit TRIP8b confers binding specificity to HCN channels.

    Science.gov (United States)

    Lyman, Kyle A; Han, Ye; Heuermann, Robert J; Cheng, Xiangying; Kurz, Jonathan E; Lyman, Reagan E; Van Veldhoven, Paul P; Chetkovich, Dane M

    2017-09-08

    Tetratricopeptide repeat (TPR) domains are ubiquitous structural motifs that mediate protein-protein interactions. For example, the TPR domains in the peroxisomal import receptor PEX5 enable binding to a range of type 1 peroxisomal targeting signal (PTS1) motifs. A homolog of PEX5, tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), binds to and functions as an auxiliary subunit of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Given the similarity between TRIP8b and PEX5, this difference in function raises the question of what mechanism accounts for their binding specificity. In this report, we found that the cyclic nucleotide-binding domain (CNBD) and the C-terminus of the HCN channel are critical for conferring specificity to TRIP8b binding. We show that TRIP8b binds the HCN CNBD through a 37-residue domain and the HCN C-terminus through the TPR domains. Using a combination of fluorescence polarization and co-immunoprecipitation based assays, we establish that binding at either site increases affinity at the other. Thus, allosteric coupling of the TRIP8b TPR domains both promotes binding to HCN channels and limits binding to PTS1 substrates. These results raise the possibility that other TPR domains may similarly be influenced by allosteric mechanisms as a general feature of protein-protein interactions. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  3. Exploring the interplay between experimental methods and the performance of predictors of binding affinity change upon mutations in protein complexes.

    Science.gov (United States)

    Geng, Cunliang; Vangone, Anna; Bonvin, Alexandre M J J

    2016-08-01

    Reliable prediction of binding affinity changes (ΔΔG) upon mutations in protein complexes relies not only on the performance of computational methods but also on the availability and quality of experimental data. Binding affinity changes can be measured by various experimental methods with different accuracies and limitations. To understand the impact of these on the prediction of binding affinity change, we present the Database of binding Affinity Change Upon Mutation (DACUM), a database of 1872 binding affinity changes upon single-point mutations, a subset of the SKEMPI database (Moal,I.H. and Fernández-Recio,J. Bioinformatics, 2012;28:2600-2607) extended with information on the experimental methods used for ΔΔG measurements. The ΔΔG data were classified into different data sets based on the experimental method used and the position of the mutation (interface and non-interface). We tested the prediction performance of the original HADDOCK score, a newly trained version of it and mutation Cutoff Scanning Matrix (Pires,D.E.V., Ascher,D.B. and Blundell,T.L. Bioinformatics 2014;30:335-342), one of the best reported ΔΔG predictors so far, on these various data sets. Our results demonstrate a strong impact of the experimental methods on the performance of binding affinity change predictors for protein complexes. This underscores the importance of properly considering and carefully choosing experimental methods in the development of novel binding affinity change predictors. The DACUM database is available online at https://github.com/haddocking/DACUM.

  4. Determinants of binding affinity and specificity for the interaction of TEM-1 and SME-1 beta-lactamase with beta-lactamase inhibitory protein.

    Science.gov (United States)

    Zhang, Zhen; Palzkill, Timothy

    2003-11-14

    The hydrolysis of beta-lactam antibiotics by class A beta-lactamases is a common cause of bacterial resistance to these agents. The beta-lactamase inhibitory protein (BLIP) is able to bind and inhibit several class A beta-lactamases, including TEM-1 beta-lactamase and SME-1 beta-lactamase. Although the TEM-1 and SME-1 enzymes share 33% amino acid sequence identity and a similar fold, they differ substantially in surface electrostatic properties and the conformation of a loop-helix region that BLIP binds. Alanine-scanning mutagenesis was performed to identify the residues on BLIP that contribute to its binding affinity for each of these enzymes. The results indicate that the sequence requirements for binding are similar for both enzymes with most of the binding free energy provided by two patches of aromatic residues on the surface of BLIP. Polar residues such as several serines in the interface do not make significant contributions to affinity for either enzyme. In addition, the specificity of binding is significantly altered by mutation of two charged residues, Glu73 and Lys74, that are buried in the structure of the TEM-1.BLIP complex as well as by residues located on two loops that insert into the active site pocket. Based on the results, a E73A/Y50A double mutant was constructed that exhibited a 220,000-fold change in binding specificity for the TEM-1 versus SME-1 enzymes.

  5. Marked reduction in the number of platelet-tritiated imipramine binding sites in geriatric depression

    Energy Technology Data Exchange (ETDEWEB)

    Nemeroff, C.B.; Knight, D.L.; Krishnan, R.R.; Slotkin, T.A.; Bissette, G.; Melville, M.L.; Blazer, D.G.

    1988-10-01

    The number (Bmax) and affinity (Kd) of platelet-tritiated imipramine binding sites was determined in young and middle-aged controls 50 years of age and younger (n = 25), elderly normal controls over 60 years of age (n = 18), patients who fulfilled DSM-III criteria for major depression who were under 50 years of age (n = 29), patients who fulfilled DSM-III criteria for major depression who were 60 years of age and older (n = 19), and patients who fulfilled both DSM-III criteria for primary degenerative dementia and National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association criteria for probable Alzheimer's disease (n = 13). Both groups of depressed patients (under 50 and over 60 years of age) exhibited significant reductions (decreases 42%) in the number of platelet-tritiated imipramine binding sites with no change in affinity, when compared with their age-matched controls. There was little overlap in Bmax values between the elderly depressed patients and their controls. The patients with probable Alzheimer's disease showed no alteration in platelet-tritiated imipramine binding. There was no statistically significant relationship between postdexamethasone plasma cortisol concentrations and tritiated imipramine binding. These results indicate that platelet-tritiated imipramine binding may have potential utility as a diagnostic adjunct in geriatric depression, and moreover that the reduction in the number of platelet-tritiated imipramine binding sites is not due to hypercortisolemia.

  6. Comparison of binding parameters of sigma 1 and sigma 2 binding sites in rat and guinea pig brain membranes: novel subtype-selective trishomocubanes.

    Science.gov (United States)

    Nguyen, V H; Kassiou, M; Johnston, G A; Christie, M J

    1996-09-12

    Comparisons of binding parameters of [3H](+)-pentazocine and [3H]1,3-di-o-tolylguanidine (DTG) at sigma binding sites in guinea pig and rat brain membranes demonstrated that [3H](+)-pentazocine binds to a single high-affinity site, whereas [3H]DTG binds to two high-affinity sites in both species. The Kd values of the radioligands were similar in both types of membranes. However, the density of sigma 1 sites in guinea pig was significantly higher than that of rat. Novel trishomocubanes were tested for their affinities at sigma 1 and sigma 2 binding sites in guinea pig brain membranes using [3H](+)-pentazocine and [3H]DTG as the radioligands. N-(4-Phenylbutyl)-3-hydroxy-4- azahexacyclo[5.4.1.0(2,6).0(3,10).0(5,9).0(8,11)]dodecane (ANSTO-14) showed the highest affinity for the sigma 1 site (Ki = 9.4 nM) and 19-fold sigma 1/sigma 2 selectivity, as a result of increasing the alkyl chain between the cubane moiety and the aromatic ring. N-(3'-Fluorophenyl)methyl- 3-hydroxy-4-azahexacyclo[5.4.1.0(2,6).0(3,10).0(5,9).0(8,11]dodeca ne (ANSTO-19), displayed the highest affinity for sigma 2 sites (Ki = 19.6 nM) and 8-fold sigma 2/sigma 1 selectivity due to a fluoro substitution in the meta position of the aromatic ring. These represent structurally novel lead compounds, especially for the development of selective sigma 2 receptor ligands.

  7. Surface binding sites in amylase have distinct roles in recognition of starch structure motifs and degradation.

    Science.gov (United States)

    Cockburn, Darrell; Nielsen, Morten M; Christiansen, Camilla; Andersen, Joakim M; Rannes, Julie B; Blennow, Andreas; Svensson, Birte

    2015-04-01

    Carbohydrate converting enzymes often possess extra substrate binding regions that enhance their activity. These can be found either on separate domains termed carbohydrate binding modules or as so-called surface binding sites (SBSs) situated on the catalytic domain. SBSs are common in starch degrading enzymes and critically important for their function. The affinity towards a variety of starch granules as well as soluble poly- and oligosaccharides of barley α-amylase 1 (AMY1) wild-type and mutants of two SBSs (SBS1 and SBS2) was investigated using Langmuir binding analysis, confocal laser scanning microscopy, affinity gel electrophoresis and surface plasmon resonance to unravel functional roles of the SBSs. SBS1 was critical for binding to different starch types as Kd increased by 7-62-fold or was not measurable upon mutation. By contrast SBS2 was particularly important for binding to soluble polysaccharides and oligosaccharides with α-1,6 linkages, suggesting that branch points are key structural elements in recognition by SBS2. Mutation at both SBS1 and SBS2 eliminated binding to all starch granule types tested. Taken together, the findings indicate that the two SBSs act in concert to localize AMY1 to the starch granule surface and that SBS2 works synergistically with the active site in the degradation of amylopectin.

  8. Substrate and drug binding sites in LeuT.

    Science.gov (United States)

    Nyola, Ajeeta; Karpowich, Nathan K; Zhen, Juan; Marden, Jennifer; Reith, Maarten E; Wang, Da-Neng

    2010-08-01

    LeuT is a member of the neurotransmitter/sodium symporter family, which includes the neuronal transporters for serotonin, norepinephrine, and dopamine. The original crystal structure of LeuT shows a primary leucine-binding site at the center of the protein. LeuT is inhibited by different classes of antidepressants that act as potent inhibitors of the serotonin transporter. The newly determined crystal structures of LeuT-antidepressant complexes provide opportunities to probe drug binding in the serotonin transporter, of which the exact position remains controversial. Structure of a LeuT-tryptophan complex shows an overlapping binding site with the primary substrate site. A secondary substrate binding site was recently identified, where the binding of a leucine triggers the cytoplasmic release of the primary substrate. This two binding site model presents opportunities for a better understanding of drug binding and the mechanism of inhibition for mammalian transporters.

  9. Single-Nucleotide Mutation Matrix: A New Model for Predicting the NF-κB DNA Binding Sites

    OpenAIRE

    Wenxin Du; Jing Gao; Tingting Wang; Jinke Wang

    2014-01-01

    In this study, we established a single nucleotide mutation matrix (SNMM) model based on the relative binding affinities of NF-κB p50 homodimer to a wild-type binding site (GGGACTTTCC) and its all single-nucleotide mutants detected with the double-stranded DNA microarray. We evaluated this model by scoring different groups of 10-bp DNA sequences with this model and analyzing the correlations between the scores and the relative binding affinities detected with three wet experiments, including t...

  10. The binding sites for benztropines and dopamine in the dopamine transporter overlap

    DEFF Research Database (Denmark)

    Jensen, Heidi Bisgaard; Larsen, M Andreas B; Mazier, Sonia

    2011-01-01

    Analogs of benztropines (BZTs) are potent inhibitors of the dopamine transporter (DAT) but are less effective than cocaine as behavioral stimulants. As a result, there have been efforts to evaluate these compounds as leads for potential medication for cocaine addiction. Here we use computational...... the pocket, including(2) Val152(3.46) to Ala or Ile, Ser422(8.60) to Ala and Asn157(3.51) to Cys or Ala, resulted in decreased affinity for BZT and the analog JHW007, as assessed in [(3)H]dopamine uptake inhibition assays and/or [(3)H]CFT competition binding assay. A putative polar interaction of one...... with a larger decrease in the affinity for BZT than for JHW007. Summarized, our data suggest that BZTs display a classical competitive binding mode with binding sites overlapping those of cocaine and dopamine....

  11. The enigmatic drug binding site for sodium channel inhibitors.

    Science.gov (United States)

    Mike, Arpad; Lukacs, Peter

    2010-11-01

    Local anesthetics have been in clinical use since 1884, and different aspects of the local anesthetic binding site have been studied in enormous detail. In spite of all these efforts, some of the most fundamental questions--such as which exact residues constitute the binding site, how many binding sites exist, do local anesthetics share their binding site(s) with other sodium channel inhibitors, and what are the mechanisms of inhibition--are still largely unanswered. We review accumulated data on the "local anesthetic receptor"and discuss controversial points, such as possible mechanisms of inhibition, the possibility of additional binding sites, the orientation of S6 helices, and the internal vs. external position of the anticonvulsant binding site. We describe the four following specific groups of functionally important residues: i) conserved asparagines six residues below the hinge residues; we propose that they are oriented toward the external surface of S6 helices, and have a critical role in the coupling of voltage sensors to gating, ii) residues lining the inner vestibule and constructing the "orthodox" binding site, iii) residues around the outer vestibule, which have been proposed to constitute an alternative external binding site, and iv) residues determining external access for quaternary amine inhibitors, such as QX314. We conclude that sodium channel inhibitors must be heterogenous in terms of binding sites and inhibition mechanisms, and propose that this heterogeneity should be taken into consideration during drug development.

  12. Characterization of the cation binding sites of the purple membrane. Electron spin resonance and flash photolysis studies

    Energy Technology Data Exchange (ETDEWEB)

    Dunach, M.; Seigneuret, M.; Rigaud, J.L.; Padros, E.

    1987-02-24

    The binding of Mn/sup 2 +/ and La/sup 3 +/ to the blue membrane prepared by deionization of the Halobacterium halobium purple membrane has been studied by electron spin resonance (ESR) spectroscopy, visible absorption spectroscopy, and flash photolysis. ESR studies indicated that 10 Mn/sup 2 +/ binding sites are present per bacteriorhodopsin monomer. Five high- and medium-affinity sites, normally occupied by Ca/sup 2 +/ and Mg/sup 2 +/ in the purple membrane, as well as five low-affinity sites were found. Proteolysis and chemical modification experiments indicated that the low-affinity sites are located on the bacteriorhodopsin C-terminal segment, while the high- and medium-affinity sites involve other carboxyl groups of the protein. Competition experiments indicated that La/sup 2 +/ binds much more strongly than Mn/sup 2 +/ to these sites. Visible absorption spectroscopy and flash photolysis experiments indicated that binding of Mn/sup 2 +/ or La/sup 3 +/ regenerates both the purple color and formation of the M/sub 4//sup 12/ intermediate. The effect occurs progressively as cations bind to the high- and medium-affinity sites, bound La/sup 3 +/ being more effective than bound Mn/sup 2 +/. It is suggested that divalent cations support both the purple color and proton-pumping activity by rendering less negative the surface potential of the purple membrane. This process may promote deprotonation of the counterion of the retinal Schiff base and possibly of other functional groups. On the other hand, it is proposed that the inhibitory effect of La/sup 3 +/ is mainly due to binding to a site distinct from those of divalent cations.

  13. Sulfated polymannuroguluronate inhibits Tat-induced SLK cell adhesion via a novel binding site, a KKR spatial triad

    Institute of Scientific and Technical Information of China (English)

    Yan-lin WU; Jing AI; Jing-ming ZHAO; Bing XIONG; Xiao-jie XIN; Mei-yu GENG; Xian-liang XIN; Han-dong JIANG

    2011-01-01

    Aim: Sulfated polymannuroguluronate (SPMG), a candidate anti-AIDS drug, inhibited HIV replication and interfered with HIV entry into host T lymphocytes. SPMG has high binding affinity for the transactivating factor of the HIV-1 virus (Tat) via its basic domain. However, deletion or substitution of the basic domain affected, but did not completely eliminated Tat-SPMG interactions. Here, we sought to identify other SPMG binding sites in addition to the basic domain.Methods: The potential SPMG binding sites were determined using molecular simulation and a surface plasmon resonance (SPR) based competitive inhibition assay. The effect of SPMG on Tat induced adhesion was evaluated using a cell adhesion assay. Results: The KKR domain, a novel high-affinity heparin binding site, was identified, which consisted of a triad of Lys12, Lys41, and Arg78. The KKR domain, spatially enclosed SPMG binding site on Tat, functions as another binding domain for SPMG. Further func- tional evaluation demonstrated that SPMG inhibits Tat-mediated SLK cell adhesion by directly binding to the KKR region.Conclusion: The KKR domain is a novel high-affinity binding domain for SPMG. Our findings provide important new insights into the molecular mechanisms of SPMG and a potential therapeutic intervention for Tat-induced cell adhesion.

  14. Interaction of Palmitic Acid with Metoprolol Succinate at the Binding Sites of Bovine Serum Albumin

    Directory of Open Access Journals (Sweden)

    Mashiur Rahman

    2014-12-01

    Full Text Available Purpose: The aim of this study was to characterize the binding profile as well as to notify the interaction of palmitic acid with metoprolol succinate at its binding site on albumin. Methods: The binding of metoprolol succinate to bovine serum albumin (BSA was studied by equilibrium dialysis method (ED at 27°C and pH 7.4, in order to have an insight in the binding chemistry of the drug to BSA in presence and absence of palmitic acid. The study was carried out using ranitidine as site-1 and diazepam as site-2 specific probe. Results: Different analysis of binding of metoprolol succinate to bovine serum albumin suggested two sets of association constants: high affinity association constant (k1 = 11.0 x 105 M-1 with low capacity (n1 = 2 and low affinity association (k2 = 4.0×105 M-1 constant with high capacity (n2 = 8 at pH 7.4 and 27°C. During concurrent administration of palmitic acid and metoprolol succinate in presence or absence of ranitidine or diazepam, it was found that palmitic acid displaced metoprolol succinate from its binding site on BSA resulting reduced binding of metoprolol succinate to BSA. The increment in free fraction of metoprolol succinate was from 26.27% to 55.08% upon the addition of increased concentration of palmitic acid at a concentration of 0×10-5 M to 16×10-5 M. In presence of ranitidine and diazepam, palmitic acid further increases the free fraction of metoprolol succinate from 33.05% to 66.95% and 40.68% to 72.88%, respectively. Conclusion: This data provided the evidence of interaction at higher concentration of palmitic acid at the binding sites on BSA, which might change the pharmacokinetic properties of metoprolol succinate.

  15. To be, or not to be two sites: that is the question about LeuT substrate binding.

    Science.gov (United States)

    Reyes, Nicolas; Tavoulari, Sotiria

    2011-10-01

    Transport proteins of the neurotransmitter sodium symporter (NSS) family regulate the extracellular concentration of several neurotransmitters in the central nervous system. The only member of this family for which atomic-resolution structural data are available is the prokaryotic homologue LeuT. This protein has been used as a model system to study the molecular mechanism of transport of the NSS family. In this Journal Club, we discuss two strikingly different LeuT transport mechanisms: one involving a single high-affinity substrate binding site and one recently proposed alternative involving two high-affinity substrate binding sites that are allosterically coupled.

  16. Identifying ligand binding sites and poses using GPU-accelerated Hamiltonian replica exchange molecular dynamics.

    Science.gov (United States)

    Wang, Kai; Chodera, John D; Yang, Yanzhi; Shirts, Michael R

    2013-12-01

    We present a method to identify small molecule ligand binding sites and poses within a given protein crystal structure using GPU-accelerated Hamiltonian replica exchange molecular dynamics simulations. The Hamiltonians used vary from the physical end state of protein interacting with the ligand to an unphysical end state where the ligand does not interact with the protein. As replicas explore the space of Hamiltonians interpolating between these states, the ligand can rapidly escape local minima and explore potential binding sites. Geometric restraints keep the ligands from leaving the vicinity of the protein and an alchemical pathway designed to increase phase space overlap between intermediates ensures good mixing. Because of the rigorous statistical mechanical nature of the Hamiltonian exchange framework, we can also extract binding free energy estimates for all putative binding sites. We present results of this methodology applied to the T4 lysozyme L99A model system for three known ligands and one non-binder as a control, using an implicit solvent. We find that our methodology identifies known crystallographic binding sites consistently and accurately for the small number of ligands considered here and gives free energies consistent with experiment. We are also able to analyze the contribution of individual binding sites to the overall binding affinity. Our methodology points to near term potential applications in early-stage structure-guided drug discovery.

  17. Discovery and validation of information theory-based transcription factor and cofactor binding site motifs.

    Science.gov (United States)

    Lu, Ruipeng; Mucaki, Eliseos J; Rogan, Peter K

    2016-11-28

    Data from ChIP-seq experiments can derive the genome-wide binding specificities of transcription factors (TFs) and other regulatory proteins. We analyzed 765 ENCODE ChIP-seq peak datasets of 207 human TFs with a novel motif discovery pipeline based on recursive, thresholded entropy minimization. This approach, while obviating the need to compensate for skewed nucleotide composition, distinguishes true binding motifs from noise, quantifies the strengths of individual binding sites based on computed affinity and detects adjacent cofactor binding sites that coordinate with the targets of primary, immunoprecipitated TFs. We obtained contiguous and bipartite information theory-based position weight matrices (iPWMs) for 93 sequence-specific TFs, discovered 23 cofactor motifs for 127 TFs and revealed six high-confidence novel motifs. The reliability and accuracy of these iPWMs were determined via four independent validation methods, including the detection of experimentally proven binding sites, explanation of effects of characterized SNPs, comparison with previously published motifs and statistical analyses. We also predict previously unreported TF coregulatory interactions (e.g. TF complexes). These iPWMs constitute a powerful tool for predicting the effects of sequence variants in known binding sites, performing mutation analysis on regulatory SNPs and predicting previously unrecognized binding sites and target genes.

  18. Radiolabelling of phoneutria nigriventer spider toxin (Tx1): a tool to study its binding site

    Energy Technology Data Exchange (ETDEWEB)

    Santos, Raquel Gouvea dos [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN), Belo Horizonte, MG (Brazil); Diniz, Carlos Roberto; Nascimento, Marta Cordeiro [FUNED, Belo Horizonte, MG (Brazil); Lima, Maria Elena de [Minas Gerais Univ., Belo Horizonte, MG (Brazil). Dept. de Bioquimica e Imunologia

    1996-07-01

    The neurotoxin Tx1, isolated from the venom of the South American spider Phoneutria nigriventer produces tail elevation and spastic paralysis of posterior limbs after intracerebral ventricular injection in mice. Tx1 also produces ileum contraction in bioassay. We have investigated the binding of radioiodinated-Tx1 ({sup 125} I-Tx1) on the preparation of myenteric plexus-longitudinal muscle membrane from guinea pig ileum (MPLM) as a tool to characterize the interaction of this neurotoxin with its site. The neurotoxin Tx1 was radioiodinated with Na{sup 125} I by the lactoperoxidase method. {sup 125} I-Tx1 specifically binds to a single class of noninteracting binding sites of high affinity (Kd= 3.5 x 10{sup -10} M) and low capacity (1.2 pmol/mg protein). The specific binding increased in parallel with the protein concentration. In competition experiments the ligands of ionic channels used (sodium, potassium and calcium) did not affect the binding of {sup 125} I-Tx1 to MPLM neither did the cholinergic ligands (hemicholinium-3, hexamethonium, d-tubocurarine and atropine). Another neurotoxin (Tx2-6, one of the isoforms of Tx2 pool) decreased toxin with MPLM and showed that toxin has a specific and saturable binding site in guinea pig ileum and this binding site appears to be related to the Tx2 site. (author)

  19. Propofol Shares the Binding Site with Isoflurane and Sevoflurane on Leukocyte Function-Associated Antigen-1

    Science.gov (United States)

    Yuki, Koichi; Bu, Weiming; Xi, Jin; Shimaoka, Motomu; Eckenhoff, Roderic

    2013-01-01

    Background We previously demonstrated that propofol interacted with the leukocyte adhesion molecule leukocyte function–associated antigen-1 (LFA-1) and inhibited the production of interleukin-2 via LFA-1 in a dependent manner. However, the binding site(s) of propofol on LFA-1 remains unknown. Methods First, the inhibition of LFA-1's ligand binding by propofol was confirmed in an ELISA-type assay. The binding site of propofol on LFA-1 was probed with a photolabeling experiment using a photoactivatable propofol analog called azi-propofol-m. The adducted residues of LFA-1 by this compound were determined using liquid chromatography–mass spectrometry. In addition, the binding of propofol to the ligand-binding domain of LFA-1 was examined using 1-aminoanthracene (1-AMA) displacement assay. Furthermore, the binding site(s) of 1-AMA and propofol on LFA-1 was studied using the docking program GLIDE. Results We demonstrated that propofol impaired the binding of LFA-1 to its ligand intercellular adhesion molecule-1. The photolabeling experiment demonstrated that the adducted residues were localized in the allosteric cavity of the ligand-binding domain of LFA-1 called “lovastatin site. ” The shift of fluorescence spectra was observed when 1-AMA was coincubated with the low-affinity conformer of LFA-1 ligand-binding domain (wild-type [WT] αL I domain), not with the high-affinity conformer, suggesting that 1-AMA bound only to WT αL I domain. In the 1-AMA displacement assay, propofol decreased 1-AMA fluorescence signal (at 520 nm), suggesting that propofol competed with 1-AMA and bound to the WT αL I domain. The docking simulation demonstrated that both 1-AMA and propofol bound to the lovastatin site, which agreed with the photolabeling experiment. Conclusions We demonstrated that propofol bound to the lovastatin site in LFA-1. Previously we showed that the volatile anesthetics isoflurane and sevoflurane bound to this site. Taken together, the lovastatin site is an

  20. Defining the bacteroides ribosomal binding site.

    Science.gov (United States)

    Wegmann, Udo; Horn, Nikki; Carding, Simon R

    2013-03-01

    The human gastrointestinal tract, in particular the colon, hosts a vast number of commensal microorganisms. Representatives of the genus Bacteroides are among the most abundant bacterial species in the human colon. Bacteroidetes diverged from the common line of eubacterial descent before other eubacterial groups. As a result, they employ unique transcription initiation signals and, because of this uniqueness, they require specific genetic tools. Although some tools exist, they are not optimal for studying the roles and functions of these bacteria in the human gastrointestinal tract. Focusing on translation initiation signals in Bacteroides, we created a series of expression vectors allowing for different levels of protein expression in this genus, and we describe the use of pepI from Lactobacillus delbrueckii subsp. lactis as a novel reporter gene for Bacteroides. Furthermore, we report the identification of the 3' end of the 16S rRNA of Bacteroides ovatus and analyze in detail its ribosomal binding site, thus defining a core region necessary for efficient translation, which we have incorporated into the design of our expression vectors. Based on the sequence logo information from the 5' untranslated region of other Bacteroidales ribosomal protein genes, we conclude that our findings are relevant to all members of this order.

  1. Recognition of oxidatively modified bases within the biotin-binding site of avidin.

    Science.gov (United States)

    Conners, Rebecca; Hooley, Elizabeth; Clarke, Anthony R; Thomas, Siân; Brady, R Leo

    2006-03-17

    Oxidative damage of DNA results in the formation of many products, including 8-oxodeoxyguanosine, which has been used as a marker to quantify DNA damage. Earlier studies have demonstrated that avidin, a protein prevalent in egg-white and which has high affinity for the vitamin biotin, binds to 8-oxodeoxyguanosine and related bases. In this study, we have determined crystal structures of avidin in complex with 8-oxodeoxyguanosine and 8-oxodeoxyadenosine. In each case, the base is observed to bind within the biotin-binding site of avidin. However, the mode of association between the bases and the protein varies and, unlike in the avidin:biotin complex, complete ordering of the protein in this region does not accompany binding. Fluorescence studies indicate that in solution the individual bases, and a range of oligonucleotides, bind to avidin with micromolar affinity. Only one of the modes of binding observed is consistent with recognition of oxidised purines when incorporated within a DNA oligomer, and from this structure a model is proposed for the selective binding of avidin to DNA containing oxidatively damaged deoxyguanosine. These studies illustrate the molecular basis by which avidin might act as a marker of DNA damage, although the low levels of binding observed are inconsistent with the recognition of oxidised purines forming a major physiological role for avidin.

  2. Neuroimaging of the serotonin reuptake site requires high-affinity ligands.

    Science.gov (United States)

    Elfving, Betina; Madsen, Jacob; Knudsen, Gitte M

    2007-11-01

    Numerous attempts have been made to develop suitable radiolabeled tracers for positron emission tomography or single photon emission computed tomography imaging of the serotonin transporter (SERT), but most often, negative outcomes are reported. The aim of this study is to define characteristics of a good SERT radioligand and to investigate species differences. We examined seven different selective serotonin reuptake inhibitors (SSRIs) and that except for one all have been previously tested as emission tomography ligands. The outcome of the ligands as emission tomography tracers was compared in relation with receptor density (Bmax) and/or ligand affinity (Kd) in rat and monkey cerebrum and cerebellum (reference region) membranes. [3H]-(S)-Citalopram and [3H]-(+)-McN5652 display statistically significantly lower affinity, whereas [3H]paroxetine displays statistically significantly higher affinity for SERT in monkey cortex when compared with the rat cerebrum. The affinity of [3H]MADAM, [123I]ADAM, and [11C]DASB for SERT obtained with rat cerebrum and monkey cortex are similar. In monkey cortex, Kd and Bmax could not be determined with [3H]fluoxetine. Of the seven SSRIs, [3H]-(S)-citalopram, [3H]MADAM, and [11C]DASB displayed significant specific binding to SERT in monkey cerebellum, with Bmax cortex:cerebellum ratios being 17, 3, and 4, respectively. In rat brain tissue the ratios were 12, 6, and 3, respectively. In conclusion, it can be estimated that imaging of the human SERT in a high-density region requires radioligands with Kd values between 0.03 and a maximum of 0.3 nM (at 37 degrees C). The differential specific cerebellar binding raises the question of the suitability of cerebellum as a reference region for nonspecific binding.

  3. Computational Estimates of Binding Affinities for Estrogen Receptor Isoforms in Rainbow Trout

    CERN Document Server

    Shyu, Conrad

    2009-01-01

    Molecular dynamics simulations are performed to determine the binding affinities between the hormone 17 beta-estradiol (E2) and different estrogen receptor (ER) isoforms in the rainbow trout (Oncorhynchus mykiss). Previous studies have demonstrated that a recent, unique gene duplication of the ER alpha subtype created two isoforms ER alpha 1 and ER alpha 2, and an early secondary split of ER beta produced two distinct isoforms of ER beta 1 and ER beta 2 based on the phylogenetic analysis. The objective of our computational studies is to provide insight into the underlying evolutionary selection pressure on the ER isoforms. Our results show that E2 binds preferentially to ER alpha 1. This finding corresponds to the experimental results as the ERs evolved from gene duplication events are frequently free from selective pressure and should exhibit no deleterious effects. The E2, however, only binds slightly better to ER beta 2. Both isoforms remain competitive. This finding reflects the fact that since ER beta 2 ...

  4. The N-terminus of TDP-43 promotes its oligomerization and enhances DNA binding affinity

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Chung-ke [Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan (China); Wu, Tzong-Huah [Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan (China); Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Institute of Biochemistry, Academia Sinica, Taipei 115, Taiwan (China); Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 300, Taiwan (China); Wu, Chu-Ya [Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan (China); Graduate Institute of Engineering, National Taiwan University of Science and Technology, Taipei 106, Taiwan (China); Chiang, Ming-hui; Toh, Elsie Khai-Woon [Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan (China); Hsu, Yin-Chih; Lin, Ku-Feng [Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan (China); Liao, Yu-heng [Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan (China); Huang, Tai-huang, E-mail: bmthh@gate.sinica.edu.tw [Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan (China); Department of Physics, National Taiwan Normal University, Taipei 106, Taiwan (China); Huang, Joseph Jen-Tse, E-mail: jthuang@chem.sinica.edu.tw [Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan (China)

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer The N-terminus of TDP-43 contains an independently folded structural domain (NTD). Black-Right-Pointing-Pointer The structural domains of TDP-43 are arranged in a beads-on-a-string fashion. Black-Right-Pointing-Pointer The NTD promotes TDP-43 oligomerization in a concentration-dependent manner. Black-Right-Pointing-Pointer The NTD may assist nucleic acid-binding activity of TDP-43. -- Abstract: TDP-43 is a DNA/RNA-binding protein associated with different neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-U). Here, the structural and physical properties of the N-terminus on TDP-43 have been carefully characterized through a combination of nuclear magnetic resonance (NMR), circular dichroism (CD) and fluorescence anisotropy studies. We demonstrate for the first time the importance of the N-terminus in promoting TDP-43 oligomerization and enhancing its DNA-binding affinity. An unidentified structural domain in the N-terminus is also disclosed. Our findings provide insights into the N-terminal domain function of TDP-43.

  5. The Dac-tag, an affinity tag based on penicillin-binding protein 5.

    Science.gov (United States)

    Lee, David Wei; Peggie, Mark; Deak, Maria; Toth, Rachel; Gage, Zoe Olivia; Wood, Nicola; Schilde, Christina; Kurz, Thimo; Knebel, Axel

    2012-09-01

    Penicillin-binding protein 5 (PBP5), a product of the Escherichia coli gene dacA, possesses some β-lactamase activity. On binding to penicillin or related antibiotics via an ester bond, it deacylates and destroys them functionally by opening the β-lactam ring. This process takes several minutes. We exploited this process and showed that a fragment of PBP5 can be used as a reversible and monomeric affinity tag. At ambient temperature (e.g., 22°C), a PBP5 fragment binds rapidly and specifically to ampicillin Sepharose. Release can be facilitated either by eluting with 10mM ampicillin or in a ligand-free manner by incubation in the cold (1-10°C) in the presence of 5% glycerol. The "Dac-tag", named with reference to the gene dacA, allows the isolation of remarkably pure fusion protein from a wide variety of expression systems, including (in particular) eukaryotic expression systems.

  6. Recognition and binding of β-lactam antibiotics to bovine serum albumin by frontal affinity chromatography in combination with spectroscopy and molecular docking.

    Science.gov (United States)

    Li, Qian; Zhang, Tianlong; Bian, Liujiao

    2016-03-01

    Serum albumins are the most abundant carrier proteins in blood plasma and participate in the binding and transportation of various exogenous and endogenous compounds in the body. This work was designed to investigate the recognition and binding of three typical β-lactam antibiotics including penicillin G (Pen G), penicillin V (Pen V) and cefalexin (Cef) with bovine serum albumin (BSA) by frontal affinity chromatography in combination with UV-vis absorption spectra, fluorescence emission spectra, binding site marker competitive experiment and molecular docking under simulated physiological conditions. The results showed that a BSA only bound with one antibiotic molecule in the binding process, and the binding constants for Pen G-BSA, Pen V-BSA and Cef-BSA complexes were 4.22×10(1), 4.86×10(2) and 3.32×10(3) (L/mol), respectively. All the three β-lactam antibiotics were mainly inserted into the subdomain IIA (binding site 1) of BSA by hydrogen bonds and Van der Waals forces. The binding capacity between the antibiotics and BSA was closely related to the functional groups and flexibility of side chains in antibiotics. This study provided an important insight into the molecular recognition and binding interaction of BSA with β-lactam antibiotics, which may be a useful guideline for the innovative clinical medications and new antibiotic designs with effective pharmacological properties.

  7. Substrate binds in the S1 site of the F253A mutant of LeuT, a neurotransmitter sodium symporter homologue

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Hui; Gouaux, Eric (Oregon HSU)

    2012-10-10

    LeuT serves as the model protein for understanding the relationships between structure, mechanism and pharmacology in neurotransmitter sodium symporters (NSSs). At the present time, however, there is a vigorous debate over whether there is a single high-affinity substrate site (S1) located at the original, crystallographically determined substrate site or whether there are two high-affinity substrates sites, one at the primary or S1 site and the other at a second site (S2) located at the base of the extracellular vestibule. In an effort to address the controversy over the number of high-affinity substrate sites in LeuT, one group studied the F253A mutant of LeuT and asserted that in this mutant substrate binds exclusively to the S2 site and that 1 mM clomipramine entirely ablates substrate binding to the S2 site. Here we study the binding of substrate to the F253A mutant of LeuT using ligand binding and X-ray crystallographic methods. Both experimental methods unambiguously show that substrate binds to the S1 site of the F253A mutant and that binding is retained in the presence of 1 mM clomipramine. These studies, in combination with previous work, are consistent with a mechanism ofr LeuT that involves a single high-affinity substrate binding site.

  8. Substrate binds in the S1 site of the F253A mutant of LeuT, a neurotransmitter sodium symporter homologue.

    Science.gov (United States)

    Wang, Hui; Gouaux, Eric

    2012-09-01

    LeuT serves as the model protein for understanding the relationships between structure, mechanism and pharmacology in neurotransmitter sodium symporters (NSSs). At the present time, however, there is a vigorous debate over whether there is a single high-affinity substrate site (S1) located at the original, crystallographically determined substrate site or whether there are two high-affinity substrates sites, one at the primary or S1 site and the other at a second site (S2) located at the base of the extracellular vestibule. In an effort to address the controversy over the number of high-affinity substrate sites in LeuT, one group studied the F253A mutant of LeuT and asserted that in this mutant substrate binds exclusively to the S2 site and that 1 mM clomipramine entirely ablates substrate binding to the S2 site. Here we study the binding of substrate to the F253A mutant of LeuT using ligand binding and X-ray crystallographic methods. Both experimental methods unambiguously show that substrate binds to the S1 site of the F253A mutant and that binding is retained in the presence of 1 mM clomipramine. These studies, in combination with previous work, are consistent with a mechanism for LeuT that involves a single high-affinity substrate binding site.

  9. Protein function annotation by local binding site surface similarity.

    Science.gov (United States)

    Spitzer, Russell; Cleves, Ann E; Varela, Rocco; Jain, Ajay N

    2014-04-01

    Hundreds of protein crystal structures exist for proteins whose function cannot be confidently determined from sequence similarity. Surflex-PSIM, a previously reported surface-based protein similarity algorithm, provides an alternative method for hypothesizing function for such proteins. The method now supports fully automatic binding site detection and is fast enough to screen comprehensive databases of protein binding sites. The binding site detection methodology was validated on apo/holo cognate protein pairs, correctly identifying 91% of ligand binding sites in holo structures and 88% in apo structures where corresponding sites existed. For correctly detected apo binding sites, the cognate holo site was the most similar binding site 87% of the time. PSIM was used to screen a set of proteins that had poorly characterized functions at the time of crystallization, but were later biochemically annotated. Using a fully automated protocol, this set of 8 proteins was screened against ∼60,000 ligand binding sites from the PDB. PSIM correctly identified functional matches that predated query protein biochemical annotation for five out of the eight query proteins. A panel of 12 currently unannotated proteins was also screened, resulting in a large number of statistically significant binding site matches, some of which suggest likely functions for the poorly characterized proteins.

  10. Relative binding affinities of chlorophylls in peridinin-chlorophyll-protein reconstituted with heterochlorophyllous mixtures.

    Science.gov (United States)

    Brotosudarmo, T H P; Mackowski, S; Hofmann, E; Hiller, R G; Bräuchle, C; Scheer, H

    2008-01-01

    Peridinin-chlorophyll-protein (PCP), containing differently absorbing chlorophyll derivatives, are good models with which to study energy transfer among monomeric chlorophylls (Chls) by both bulk and single-molecule spectroscopy. They can be obtained by reconstituting the N-terminal domain of the protein (N-PCP) with peridinin and chlorophyll mixtures. Upon dimerization of these "half-mers", homo- and heterochlorophyllous complexes are generated, that correspond structurally to monomeric protomers of native PCP from Amphidinium carterae. Heterochlorophyllous complexes contain two different Chls in the two halves of the complete structure. Here, we report reconstitution of N-PCP with binary mixtures of Chl a, Chl b, and [3-acetyl]-Chl a. The ratios of the pigments were varied in the reconstitution mixture, and relative binding constants were determined from quantification of these pigments in the reconstituted PCPs. We find higher affinities for both Chl b and [3-acetyl]-Chl a than for the native pigment, Chl a.

  11. A complex water network contributes to high-affinity binding in an antibody–antigen interface

    Directory of Open Access Journals (Sweden)

    S.F. Marino

    2016-03-01

    Full Text Available This data article presents an analysis of structural water molecules in the high affinity interaction between a potent tumor growth inhibiting antibody (fragment, J22.9-xi, and the tumor marker antigen CD269 (B cell maturation antigen, BCMA. The 1.89 Å X-ray crystal structure shows exquisite details of the binding interface between the two molecules, which comprises relatively few, mostly hydrophobic, direct contacts but many indirect interactions over solvent waters. These are partly or wholly buried in, and therefore part of, the interface. A partial description of the structure is included in an article on the tumor inhibiting effects of the antibody: “Potent anti-tumor response by targeting B cell maturation antigen (BCMA in a mouse model of multiple myeloma”, Mol. Oncol. 9 (7 (2015 pp. 1348–58.

  12. Quantitative distribution of angiotensin II binding sites in rat brain by autoradiography

    Energy Technology Data Exchange (ETDEWEB)

    Saavedra, J.M.; Israel, A.; Plunkett, L.M.; Kurihara, M.; Shigematsu, K.; Correa, F.M.

    1986-07-01

    Angiotensin II binding sites were localized and quantified in individual brain nuclei from single rats by incubation of tissue sections with 1 nM /sup 125/I-(Sar1)-angiotensin II, (/sup 3/H)-Ultrofilm autoradiography, computerized microdensitometry and comparison with /sup 125/I-standards. High angiotensin II binding was present in the circumventricular organs (organon vasculosum laminae terminalis, organon subfornicalis and area postrema), in selected hypothalamic nuclei (nuclei suprachiasmatis, periventricularis and paraventricularis) and in the nucleus tractus olfactorii lateralis, the nucleus preopticus medianus, the dorsal motor nucleus of the vagus and the nucleus tractus solitarii. High affinity (KA from 0.3 to 1.5 X 10(9) M-1) angiotensin II binding sites were demonstrated in the organon subfornicalis, the nucleus tractus solitarii and the area postrema after incubation of consecutive sections from single rat brains with /sup 125/I-(Sar1)-angiotensin II in concentrations from 100 pM to 5 nM. These results demonstrate and characterize brain binding sites for angiotensin II of variable high affinity binding both inside and outside the blood-brain barrier.

  13. Recognition of anesthetic barbiturates by a protein binding site: a high resolution structural analysis.

    Directory of Open Access Journals (Sweden)

    Simon Oakley

    Full Text Available Barbiturates potentiate GABA actions at the GABA(A receptor and act as central nervous system depressants that can induce effects ranging from sedation to general anesthesia. No structural information has been available about how barbiturates are recognized by their protein targets. For this reason, we tested whether these drugs were able to bind specifically to horse spleen apoferritin, a model protein that has previously been shown to bind many anesthetic agents with affinities that are closely correlated with anesthetic potency. Thiopental, pentobarbital, and phenobarbital were all found to bind to apoferritin with affinities ranging from 10-500 µM, approximately matching the concentrations required to produce anesthetic and GABAergic responses. X-ray crystal structures were determined for the complexes of apoferritin with thiopental and pentobarbital at resolutions of 1.9 and 2.0 Å, respectively. These structures reveal that the barbiturates bind to a cavity in the apoferritin shell that also binds haloalkanes, halogenated ethers, and propofol. Unlike these other general anesthetics, however, which rely entirely upon van der Waals interactions and the hydrophobic effect for recognition, the barbiturates are recognized in the apoferritin site using a mixture of both polar and nonpolar interactions. These results suggest that any protein binding site that is able to recognize and respond to the chemically and structurally diverse set of compounds used as general anesthetics is likely to include a versatile mixture of both polar and hydrophobic elements.

  14. Predicted metal binding sites for phytoremediation

    OpenAIRE

    Sharma, Ashok; Roy, Sudeep; Tripathi, Kumar Parijat; Roy, Pratibha; Mishra, Manoj; Khan, Feroz; Meena, Abha

    2009-01-01

    Metal ion binding domains are found in proteins that mediate transport, buffering or detoxification of metal ions. The objective of the study is to design and analyze metal binding motifs against the genes involved in phytoremediation. This is being done on the basis of certain pre-requisite amino-acid residues known to bind metal ions/metal complexes in medicinal and aromatic plants (MAP's). Earlier work on MAP's have shown that heavy metals accumulated by aromatic and medicinal plants do no...

  15. Putative cholesterol-binding sites in human immunodeficiency virus (HIV) coreceptors CXCR4 and CCR5.

    Science.gov (United States)

    Zhukovsky, Mikhail A; Lee, Po-Hsien; Ott, Albrecht; Helms, Volkhard

    2013-04-01

    Using molecular docking, we identified a cholesterol-binding site in the groove between transmembrane helices 1 and 7 near the inner membrane-water interface of the G protein-coupled receptor CXCR4, a coreceptor for HIV entry into cells. In this docking pose, the amino group of lysine K67 establishes a hydrogen bond with the hydroxyl group of cholesterol, whereas tyrosine Y302 stacks with cholesterol by its aromatic side chain, and a number of residues form hydrophobic contacts with cholesterol. Sequence alignment showed that a similar putative cholesterol-binding site is also present in CCR5, another HIV coreceptor. We suggest that the interaction of cholesterol with these putative cholesterol-binding sites in CXCR4 and CCR5 is responsible for the presence of these receptors in lipid rafts, for the effect of cholesterol on their conformational stability and function, and for the role that cell cholesterol plays in the cell entry of HIV strains that use these membrane proteins as coreceptors. We propose that mutations of residues that are involved in cholesterol binding will make CXCR4 and CCR5 insensitive to membrane cholesterol content. Cholesterol-binding sites in HIV coreceptors are potential targets for steroid drugs that bind to CXCR4 and CCR5 with higher binding affinity than cholesterol, but do not stabilize the native conformation of these proteins.

  16. Structure-affinity properties of a high-affinity ligand of FKBP12 studied by molecular simulations of a binding intermediate.

    Directory of Open Access Journals (Sweden)

    Lilian Olivieri

    Full Text Available With a view to explaining the structure-affinity properties of the ligands of the protein FKBP12, we characterized a binding intermediate state between this protein and a high-affinity ligand. Indeed, the nature and extent of the intermolecular contacts developed in such a species may play a role on its stability and, hence, on the overall association rate. To find the binding intermediate, a molecular simulation protocol was used to unbind the ligand by gradually decreasing the biasing forces introduced. The intermediate was subsequently refined with 17 independent stochastic boundary molecular dynamics simulations that provide a consistent picture of the intermediate state. In this state, the core region of the ligand remains stable, notably because of the two anchoring oxygen atoms that correspond to recurrent motifs found in all FKBP12 ligand core structures. Besides, the non-core regions participate in numerous transient intermolecular and intramolecular contacts. The dynamic aspect of most of the contacts seems important both for the ligand to retain at least a part of its configurational entropy and for avoiding a trapped state along the binding pathway. Since the transient and anchoring contacts contribute to increasing the stability of the intermediate, as a corollary, the dissociation rate constant [Formula: see text] of this intermediate should be decreased, resulting in an increase of the affinity constant [Formula: see text]. The present results support our previous conclusions and provide a coherent rationale for explaining the prevalence in high-affinity ligands of (i the two oxygen atoms found in carbonyl or sulfonyl groups of dissimilar core structures and of (ii symmetric or pseudo-symmetric mobile groups of atoms found as non-core moieties. Another interesting aspect of the intermediate is the distortion of the flexible 80 s loop of the protein, mainly in its tip region, that promotes the accessibility to the bound state.

  17. Comparing the effects of Fe(III) and Cu(II) on the binding affinity of erlotinib to bovine serum albumin using spectroscopic methods

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Yan [The State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002 (China); Chen, Mingmao [Institute of Biomedical and Pharmaceutical Technology, Fuzhou University, Fuzhou, Fujian 350002 (China); Song, Ling, E-mail: songling@fjirsm.ac.cn [The State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002 (China)

    2013-02-15

    The interactions between erlotinib (ET) and bovine serum albumin (BSA) in the absence and presence of Cu(II) and Fe(III) in aqueous solution were investigated by using fluorescence, circular dichroism and three-dimensional (3D) fluorescence spectroscopic methods under simulative physiological conditions. Erlotinib effectively quenched the intrinsic fluorescence of BSA with slight redshifts in the absence and presence of Cu(II) and Fe(III). Cu(II) decreased the binding affinity and reduced the binding sites of erlotinib to BSA, while Fe(III) increased the binding affinity and binding sites of erlotinib to BSA. The negative values of {Delta}H and {Delta}S illustrate that the binding is mainly driven by the hydrogen bond and van der Waals force. The conformation of BSA was changed through ET binding in the presence of Cu(II) and Fe(III), which was revealed by circular dichroism, synchronous fluorescence and 3D fluorescence spectroscopic methods. The results indicate that the binding capability of erlotinib to BSA is affected by the types of metal ions. Highlights: Black-Right-Pointing-Pointer Interaction of erlotinib (ET) with BSA in the presence of Cu(II) and Fe(III) was studied. Black-Right-Pointing-Pointer Using various spectroscopic methods such as UV, CD, fluorescence and three-dimensional (3D) fluorescence. Black-Right-Pointing-Pointer Effects of metal ions on the binding activity of ET to BSA have not been reported. Black-Right-Pointing-Pointer Ternary system Cu(II)/Fe(III)-ET-BSA induced the conformational change of BSA.

  18. Molecular cloning, expression profile, odorant affinity, and stability of two odorant-binding proteins in Macrocentrus cingulum Brischke (Hymenoptera: Braconidae).

    Science.gov (United States)

    Ahmed, Tofael; Zhang, Tiantao; Wang, Zhenying; He, Kanglai; Bai, Shuxiong

    2017-02-01

    The polyembryonic endoparasitoid wasp Macrocentrus cingulum Brischke (Hymenoptera: Braconidae) is deployed successfully as a biocontrol agent for corn pest insects from the Lepidopteran genus Ostrinia in Europe and throughout Asia, including Japan, Korea, and China. The odorants are recognized, bound, and solubilized by odorant-binding protein (OBP) in the initial biochemical recognition steps in olfaction that transport them across the sensillum lymph to initiate behavioral response. In the present study, we examine the odorant-binding effects on thermal stability of McinOBP2, McinOBP3, and their mutant form that lacks the third disulfide bonds. Real-time PCR experiments indicate that these two are expressed mainly in adult antennae, with expression levels differing by sex. Odorant-binding affinities of aldehydes, terpenoids, and aliphatic alcohols were measured with circular dichroism spectroscopy based on changes in the thermal stability of the proteins upon their affinities to odorants. The obtained results reveal higher affinity of trans-caryophelle, farnesene, and cis-3-Hexen-1-ol exhibits to both wild and mutant McinOBP2 and McinOBP3. Although conformational flexibility of the mutants and shape of binding cavity make differences in odorant affinity between the wild-type and mutant, it suggested that lacking the third disulfide bond in mutant proteins may have chance to incorrect folded structures that reduced the affinity to these odorants. In addition, CD spectra clearly indicate proteins enriched with α-helical content.

  19. Protein-protein interactions: general trends in the relationship between binding affinity and interfacial buried surface area.

    Science.gov (United States)

    Chen, Jieming; Sawyer, Nicholas; Regan, Lynne

    2013-04-01

    Protein-protein interactions play key roles in many cellular processes and their affinities and specificities are finely tuned to the functions they perform. Here, we present a study on the relationship between binding affinity and the size and chemical nature of protein-protein interfaces. Our analysis focuses on heterodimers and includes curated structural and thermodynamic data for 113 complexes. We observe a direct correlation between binding affinity and the amount of surface area buried at the interface. For a given amount of surface area buried, the binding affinity spans four orders of magnitude in terms of the dissociation constant (Kd ). Across the entire dataset, we observe no obvious relationship between binding affinity and the chemical composition of the interface. We also calculate the free energy per unit surface area buried, or "surface energy density," of each heterodimer. For interfacial surface areas between 500 and 2000 Å(2) , the surface energy density decreases as the buried surface area increases. As the buried surface area increases beyond about 2000 Å(2) , the surface energy density levels off to a constant value. We believe that these analyses and data will be useful for researchers with an interest in understanding, designing or inhibiting protein-protein interfaces.

  20. 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.

  1. Evaluation of drug-muscarinic receptor affinities using cell membrane chromatography and radioligand binding assay in guinea pig jejunum membrane

    Institute of Scientific and Technical Information of China (English)

    Bing-xiang YUAN; Jin HOU; Lang-chong HE; Guang-de YANG

    2005-01-01

    Aim: To study if cell membrane chromatography (CMC) could reflect drug-receptor interaction and evaluate the affinity and competitive binding to muscarinic acetylcholine receptor (mAChR). Methods: The cell membrane stationary phase(CMSP) was prepared by immobilizing guinea pig jejunum cell membrane on the surface of a silica carrier, and was used for the rapid on-line chromatographic evaluation of ligand binding affinities to mAChR. The affinity to mAChR was also evaluated from radioligand binding assays (RBA) using the same jejunum membrane preparation. Results: The capacity factor (k') profiles in guinea pig jejunum CMSP were: (-)QNB (15.4)>(+)QNB (11.5)>atropine (5.35)>pirenzepine(5.26)>4-DAMP (4.45)>AF-DX 116 (4.18)>pilocarpine (3.93)>acetylcholine(1.31). These results compared with the affinity rank orders obtained from radioligand binding assays indicated that there wasa positive correlation (r2=0.8525, P<0.0001) between both data sets. Conclusion: The CMC method can be used to evaluate drug-receptor affinities for drug candidates.

  2. Affinity of ceftobiprole for penicillin-binding protein 2b in Streptococcus pneumoniae strains with various susceptibilities to penicillin.

    Science.gov (United States)

    Davies, Todd A; He, Wenping; Bush, Karen; Flamm, Robert K

    2010-10-01

    Wild-type penicillin-binding protein (PBP) 2b from penicillin-susceptible Streptococcus pneumoniae had high affinity for ceftobiprole and penicillin (50% inhibitory concentrations [IC(50)s] of ≤0.15 μg/ml) but not ceftriaxone (IC(50) of >8 μg/ml). In clinical isolates, ceftobiprole and PBP 2b affinities were reduced 15- to 30-fold with a Thr-446-Ala substitution and further still with an additional Ala-619-Gly PBP 2b substitution. Ceftobiprole remained active (MICs of ≤1 μg/ml) against all strains tested and behaved more like penicillin than ceftriaxone with respect to PBP 2b binding.

  3. Identification of Aptamer-Binding Sites in Hepatitis C Virus Envelope Glycoprotein E2

    Directory of Open Access Journals (Sweden)

    Fan Chen

    2015-01-01

    Full Text Available Hepatitis C Virus (HCV encodes two envelope glycoproteins, E1 and E2. Our previous work selected a specific aptamer ZE2, which could bind to E2 with high affinity, with a great potential for developing new molecular probes as an early diagnostic reagents or therapeutic drugs targeting HCV. In this study, the binding sites between E2 and aptamer ZE2 were further explored. E2 was truncated to 15 peptides (P1 to P15 and these peptides were used to detect the affinity with ZE2 by ELISA respectively. The peptide with high affinity was then further truncated, detected and compared with six kinds of HCV genotypes. The basic amino acid in 500 aa bound to ZE2 with high affinity, while acidic amino acid in 501 aa reduced the reaction between E2 and ZE2. The results showed the 500 aa and 501 aa of E2 were the key sites that bound to ZE2.

  4. Structure and localisation of drug binding sites on neurotransmitter transporters.

    Science.gov (United States)

    Ravna, Aina W; Sylte, Ingebrigt; Dahl, Svein G

    2009-10-01

    The dopamine (DAT), serotontin (SERT) and noradrenalin (NET) transporters are molecular targets for different classes of psychotropic drugs. The crystal structure of Aquifex aeolicus LeuT(Aa) was used as a template for molecular modeling of DAT, SERT and NET, and two putative drug binding sites (pocket 1 and 2) in each transporter were identified. Cocaine was docked into binding pocket 1 of DAT, corresponding to the leucine binding site in LeuT(Aa), which involved transmembrane helices (TMHs) 1, 3, 6 and 8. Clomipramine was docked into binding pocket 2 of DAT, involving TMHs 1, 3, 6, 10 and 11, and extracellular loops 4 and 6, corresponding to the clomipramine binding site in a crystal structure of a LeuT(Aa)-clomipramine complex. The structures of the proposed cocaine- and tricyclic antidepressant-binding sites may be of particular interest for the design of novel DAT interacting ligands.

  5. Frequent gain and loss of functional transcription factor binding sites.

    Directory of Open Access Journals (Sweden)

    Scott W Doniger

    2007-05-01

    Full Text Available Cis-regulatory sequences are not always conserved across species. Divergence within cis-regulatory sequences may result from the evolution of species-specific patterns of gene expression or the flexible nature of the cis-regulatory code. The identification of functional divergence in cis-regulatory sequences is therefore important for both understanding the role of gene regulation in evolution and annotating regulatory elements. We have developed an evolutionary model to detect the loss of constraint on individual transcription factor binding sites (TFBSs. We find that a significant fraction of functionally constrained binding sites have been lost in a lineage-specific manner among three closely related yeast species. Binding site loss has previously been explained by turnover, where the concurrent gain and loss of a binding site maintains gene regulation. We estimate that nearly half of all loss events cannot be explained by binding site turnover. Recreating the mutations that led to binding site loss confirms that these sequence changes affect gene expression in some cases. We also estimate that there is a high rate of binding site gain, as more than half of experimentally identified S. cerevisiae binding sites are not conserved across species. The frequent gain and loss of TFBSs implies that cis-regulatory sequences are labile and, in the absence of turnover, may contribute to species-specific patterns of gene expression.

  6. CaFE: a tool for binding affinity prediction using end-point free energy methods.

    Science.gov (United States)

    Liu, Hui; Hou, Tingjun

    2016-07-15

    Accurate prediction of binding free energy is of particular importance to computational biology and structure-based drug design. Among those methods for binding affinity predictions, the end-point approaches, such as MM/PBSA and LIE, have been widely used because they can achieve a good balance between prediction accuracy and computational cost. Here we present an easy-to-use pipeline tool named Calculation of Free Energy (CaFE) to conduct MM/PBSA and LIE calculations. Powered by the VMD and NAMD programs, CaFE is able to handle numerous static coordinate and molecular dynamics trajectory file formats generated by different molecular simulation packages and supports various force field parameters. CaFE source code and documentation are freely available under the GNU General Public License via GitHub at https://github.com/huiliucode/cafe_plugin It is a VMD plugin written in Tcl and the usage is platform-independent. tingjunhou@zju.edu.cn. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  7. Towards full Quantum-Mechanics-based Protein-Ligand Binding Affinities.

    Science.gov (United States)

    Ehrlich, Stephan; Göller, Andreas H; Grimme, Stefan

    2017-01-29

    Computational methods play a key role in modern drug design in the pharmaceutical industry but are mostly based on force fields, which are limited in accuracy when describing non-classical binding effects, proton transfer, or metal coordination. Here, we propose a general fully quantum mechanical (QM) scheme for the computation of protein-ligand affinities. It works on a single protein cutout (of about 1000 atoms) and evaluates all contributions (interaction energy, solvation, thermostatistical) to absolute binding free energy on the highest feasible QM level. The methodology is tested on two different protein targets: activated serine protease factor X (FXa) and tyrosine-protein kinase 2 (TYK2). We demonstrate that the geometry of the model systems can be efficiently energy-minimized by using general purpose graphics processing units, resulting in structures that are close to the co-crystallized protein-ligand structures. Our best calculations at a hybrid DFT level (PBEh-3c composite method) for the FXa ligand set result in an overall mean absolute deviation as low as 2.1 kcal mol(-1) . Though very encouraging, an analysis of outliers indicates that the structure optimization level, conformational sampling, and solvation treatment require further improvement.

  8. Significance of affinity and cooperativity in oxygen binding to hemoglobin of horse fetal and maternal blood.

    Science.gov (United States)

    Zhang, Yan; Kobayashi, Keiko; Sasagawa, Keisuke; Imai, Kiyohiro; Kobayashi, Michiyori

    2003-09-01

    The physiological significance of the position and shape of the oxygen equilibrium curve (OEC) of horse hemoglobin (Hb) is considered from the viewpoint of oxygen (O2) transport efficiency and the effectiveness of the Bohr effect. In horse fetal and maternal bloods, their physiological O2 affinities are nearly optimized with respect to the effectiveness of the Bohr shift occurring at the O2 release site, when it is measured by the change in O2 saturation per unit change in P50. With relatively low cooperativity (n=2.69) of horse Hb under physiological conditions, the effectiveness of the Bohr shift for fetal blood at O2 uptake site and maternal blood at O2 release site is high. These facts imply that the position and the cooperativity of horse Hb OEC are optimized to receive maximal benefit from the double Bohr shift. Before exercise, the position of the OEC for adult mares is nearly optimized for the effectiveness of the Bohr shift occurring at the O2 release site, whereas, at maximal exercise, the position of the OEC tends to become advantageous for O2 transport efficiency.

  9. The novel alpha 2-adrenoceptor agonist [3H]mivazerol binds to non-adrenergic binding sites in human striatum membranes that are distinct from imidazoline receptors.

    Science.gov (United States)

    Flamez, A; Gillard, M; De Backer, J P; Vauquelin, G; Noyer, M

    1997-07-01

    The alpha 2 adrenergic agonist [3H]mivazerol labelled two populations of binding sites in membranes from the human striatum. Forty per cent of the sites labelled by 3 nM [3H]mivazerol corresponded to alpha 2 adrenergic receptors as they displayed a high affinity for (-)-adrenaline and for rauwolscine. The remaining binding was displaced by mivazerol with a pIC50 of 6.5 +/- 0.1. These sites displayed higher affinity for dexmedetomidine (pIC50 = 7.1 +/- 0.1), but much lower affinity for clonidine (pIC50 < 5.0) and for idazoxan (pIC50 = 5.1 +/- 0.1). Mivazerol also showed low affinity for the [3H]clonidine-labelled I1 imidazoline receptors and for the [3H]idazoxan-labelled I2 receptors (pIC50 = 5.1 and 3.9, respectively). These results suggest that the non-adrenergic [3H]mivazerol binding sites are distinct from the imidazoline receptors in the human striatum.

  10. The Binding of Four Licorice Flavonoids to Bovine Serum Albumin by Multi-Spectroscopic and Molecular Docking Methods: Structure-Affinity Relationship

    Science.gov (United States)

    Hou, J.; Liang, Q.; Shao, S.

    2017-03-01

    Flavanones are the main compound of licorice, and the C'-4 position substitution is a significant structural feature for their biological activity. The ability of three selected flavanones (liquiritigenin, liquiritin, and liquiritin apioside) bearing different substituents (hydroxyl groups, glucose, and glucose-apiose sugar moiety) at the C'-4 position and a chalcone ( isoliquiritigenin, an isomer of liquiritigenin) to bind bovine serum albumin (BSA) was studied by multispectroscopic and molecular docking methods under physiological conditions. The binding mechanism of fl avonoids to BSA can be explained by the formation of a flavonoids-BSA complex, and the binding affinity is the strongest for isoliquiritigenin, followed by liquiritin apioside, liquiritin, and liquiritigenin. The thermodynamic analysis and the molecular docking indicated that the interaction between flavonoids and BSA was dominated by the hydrophobic force and hydrogen bonds. The competitive experiments as well as the molecular docking results suggested the most possible binding site of licorice flavonoids on BSA at subdomain IIA. These results revealed that the basic skeleton structure and the substituents at the C'-4 position of flavanones significantly affect the structure-affinity relationships of the licorice flavonoid binding to BSA.

  11. Identification and characterization of anion binding sites in RNA

    Energy Technology Data Exchange (ETDEWEB)

    Kieft, Jeffrey S.; Chase, Elaine; Costantino, David A.; Golden, Barbara L. (Purdue); (Colorado)

    2010-05-24

    Although RNA molecules are highly negatively charged, anions have been observed bound to RNA in crystal structures. It has been proposed that anion binding sites found within isolated RNAs represent regions of the molecule that could be involved in intermolecular interactions, indicating potential contact points for negatively charged amino acids from proteins or phosphate groups from an RNA. Several types of anion binding sites have been cataloged based on available structures. However, currently there is no method for unambiguously assigning anions to crystallographic electron density, and this has precluded more detailed analysis of RNA-anion interaction motifs and their significance. We therefore soaked selenate into two different types of RNA crystals and used the anomalous signal from these anions to identify binding sites in these RNA molecules unambiguously. Examination of these sites and comparison with other suspected anion binding sites reveals features of anion binding motifs, and shows that selenate may be a useful tool for studying RNA-anion interactions.

  12. Identification of Calcium binding sites on calsequestrin 1 and its implications to polymerization

    Science.gov (United States)

    Kumar, Amit; Chakravarty, Harapriya; Bal, Naresh C.; Balaraju, Tuniki; Jena, Nivedita; Misra, Gauri; Bal, Chandralata; Pieroni, Enrico; Periasamy, Muthu; Sharon, Ashoke

    2013-01-01

    Biophysical studies have shown that each molecule of calsequestrin 1 (CASQ1) can bind about 70–80 Ca2+ ions. However, the nature of Ca2+-binding sites has not yet been fully characterized. In this study, we employed in-silico approaches to identify the Ca2+ binding sites and to understand the molecular basis of CASQ1-Ca2+ recognition. We built the protein model by extracting the atomic coordinates for the back-to-back dimeric unit from the recently solved hexameric CASQ1 structure (PDB id: 3UOM) and adding the missing C-terminal residues (aa350–364). Using this model we performed extensive 30 ns molecular dynamics simulations exposed to wide range of Ca2+ concentrations ([Ca2+]). Our results show that the Ca2+-binding sites on CASQ1 differ both in affinity and geometry. The high affinity Ca2+-binding sites share a similar geometry and interestingly, majority of them were found to be induced by increased [Ca2+]. We also found that the system undergoes maximal Ca2+-binding to the CAS (consecutive aspartate stretch at the C-terminus) before the rest of the CASQ1 surface becomes saturated. Simulated data shows that the CASQ1 back-to-back stacking is progressively stabilized by emergence of an increasing number of hydrophobic interactions with increasing [Ca2+]. Further, this study shows that the CAS domain assumes a compact structure with increase in Ca2+ binding, which suggests that the CAS domain might function as a Ca2+-sensor that may be a novel structural motif to sense metal. We propose the term “Dn-motif” for the CAS domain. PMID:23629537

  13. On-column entrapment of alpha1-acid glycoprotein for studies of drug-protein binding by high-performance affinity chromatography.

    Science.gov (United States)

    Anguizola, Jeanethe; Bi, Cong; Koke, Michelle; Jackson, Abby; Hage, David S

    2016-08-01

    An on-column approach for protein entrapment was developed to immobilize alpha1-acid glycoprotein (AGP) for drug-protein binding studies based on high-performance affinity chromatography. Soluble AGP was physically entrapped by using microcolumns that contained hydrazide-activated porous silica and by employing mildly oxidized glycogen as a capping agent. Three on-column entrapment methods were evaluated and compared to a previous slurry-based entrapment method. The final selected method was used to prepare 1.0 cm × 2.1 mm I.D. affinity microcolumns that contained up to 21 (±4) μg AGP and that could be used over the course of more than 150 sample applications. Frontal analysis and zonal elution studies were performed on these affinity microcolumns to examine the binding of various drugs with the entrapped AGP. Site-selective competition studies were also conducted for these drugs. The results showed good agreement with previous observations for these drug-protein systems and with binding constants that have been reported in the literature. The entrapment method developed in this study should be useful for future work in the area of personalized medicine and in the high-throughput screening of drug interactions with AGP or other proteins. Graphical abstract On-column protein entrapment using a hydrazide-activated support and oxidized glycogen as a capping agent.

  14. Drug binding to the inactivated state is necessary but not sufficient for high-affinity binding to human ether-à-go-go-related gene channels.

    Science.gov (United States)

    Perrin, Mark J; Kuchel, Philip W; Campbell, Terence J; Vandenberg, Jamie I

    2008-11-01

    Drug block of the human ether-à-go-go-related gene K(+) channel (hERG) is the most common cause of acquired long QT syndrome, a disorder of cardiac repolarization that may result in ventricular tachycardia and sudden cardiac death. We investigated the open versus inactivated state dependence of drug block by using hERG mutants N588K and N588E, which shift the voltage dependence of inactivation compared with wild-type but in which the mutated residue is remote from the drug-binding pocket in the channel pore. Four high-affinity drugs (cisapride, dofetilide, terfenadine, and astemizole) demonstrated lower affinity for the inactivation-deficient N588K mutant hERG channel compared with N588E and wild-type hERG. Three of four low-affinity drugs (erythromycin, perhexiline, and quinidine) demonstrated no preference for N588E over N588K channels, whereas dl-sotalol was an example of a low-affinity state-dependent blocker. All five state-dependent blockers showed an even lower affinity for S620T mutant hERG (no inactivation) compared with N588K mutant hERG (greatly reduced inactivation). Computer modeling indicates that the reduced affinity for S620T compared with N588K and wild-type channels can be explained by the relative kinetics of drug block and unblock compared with the kinetics of inactivation and recovery from inactivation. We were also able to calculate, for the first time, the relative affinities for the inactivated versus the open state, which for the drugs tested here ranged from 4- to 70-fold. Our results show that preferential binding to the inactivated state is necessary but not sufficient for high-affinity binding to hERG channels.

  15. Specific binding of Bacillus thuringiensis Cry2A insecticidal proteins to a common site in the midgut of Helicoverpa species.

    Science.gov (United States)

    Hernández-Rodríguez, Carmen Sara; Van Vliet, Adri; Bautsoens, Nadine; Van Rie, Jeroen; Ferré, Juan

    2008-12-01

    For a long time, it has been assumed that the mode of action of Cry2A toxins was unique and different from that of other three-domain Cry toxins due to their apparent nonspecific and unsaturable binding to an unlimited number of receptors. However, based on the homology of the tertiary structure among three-domain Cry toxins, similar modes of action for all of them are expected. To confirm this hypothesis, binding assays were carried out with (125)I-labeled Cry2Ab. Saturation assays showed that Cry2Ab binds in a specific and saturable manner to brush border membrane vesicles (BBMVs) of Helicoverpa armigera. Homologous-competition assays with (125)I-Cry2Ab demonstrated that this toxin binds with high affinity to binding sites in H. armigera and Helicoverpa zea midgut. Heterologous-competition assays showed a common binding site for three toxins belonging to the Cry2A family (Cry2Aa, Cry2Ab, and Cry2Ae), which is not shared by Cry1Ac. Estimation of K(d) (dissociation constant) values revealed that Cry2Ab had around 35-fold less affinity than Cry1Ac for BBMV binding sites in both insect species. Only minor differences were found regarding R(t) (concentration of binding sites) values. This study questions previous interpretations from other authors performing binding assays with Cry2A toxins and establishes the basis for the mode of action of Cry2A toxins.

  16. Specific Binding of Bacillus thuringiensis Cry2A Insecticidal Proteins to a Common Site in the Midgut of Helicoverpa Species▿

    Science.gov (United States)

    Hernández-Rodríguez, Carmen Sara; Van Vliet, Adri; Bautsoens, Nadine; Van Rie, Jeroen; Ferré, Juan

    2008-01-01

    For a long time, it has been assumed that the mode of action of Cry2A toxins was unique and different from that of other three-domain Cry toxins due to their apparent nonspecific and unsaturable binding to an unlimited number of receptors. However, based on the homology of the tertiary structure among three-domain Cry toxins, similar modes of action for all of them are expected. To confirm this hypothesis, binding assays were carried out with 125I-labeled Cry2Ab. Saturation assays showed that Cry2Ab binds in a specific and saturable manner to brush border membrane vesicles (BBMVs) of Helicoverpa armigera. Homologous-competition assays with 125I-Cry2Ab demonstrated that this toxin binds with high affinity to binding sites in H. armigera and Helicoverpa zea midgut. Heterologous-competition assays showed a common binding site for three toxins belonging to the Cry2A family (Cry2Aa, Cry2Ab, and Cry2Ae), which is not shared by Cry1Ac. Estimation of Kd (dissociation constant) values revealed that Cry2Ab had around 35-fold less affinity than Cry1Ac for BBMV binding sites in both insect species. Only minor differences were found regarding Rt (concentration of binding sites) values. This study questions previous interpretations from other authors performing binding assays with Cry2A toxins and establishes the basis for the mode of action of Cry2A toxins. PMID:18931285

  17. Neuropeptide Y receptor binding sites in rat brain: differential autoradiographic localizations with sup 125 I-peptide YY and sup 125 I-neuropeptide Y imply receptor heterogeneity

    Energy Technology Data Exchange (ETDEWEB)

    Lynch, D.R.; Walker, M.W.; Miller, R.J.; Snyder, S.H. (Johns Hopkins Univ. School of Medicine, Baltimore, MD (USA))

    1989-08-01

    Neuropeptide Y (NPY) receptor binding sites have been localized in the rat brain by in vitro autoradiography using picomolar concentrations of both 125I-NPY and 125I-peptide YY (PYY) and new evidence provided for differentially localized receptor subtypes. Equilibrium binding studies using membranes indicate that rat brain contains a small population of high-affinity binding sites and a large population of moderate-affinity binding sites. 125I-PYY (10 pM) is selective for high-affinity binding sites (KD = 23 pM), whereas 10 pM 125I-NPY labels both high- and moderate-affinity sites (KD = 54 pM and 920 pM). The peptide specificity and affinity of these ligands in autoradiographic experiments match those seen in homogenates. Binding sites for 125I-PYY are most concentrated in the lateral septum, stratum oriens, and radiatum of the hippocampus, amygdala, piriform cortex, entorhinal cortex, several thalamic nuclei, including the reuniens and lateral posterior nuclei, and substantia nigra, pars compacta, and pars lateralis. In the brain stem, 125I-PYY sites are densest in a variety of nuclei on the floor of the fourth ventricle, including the pontine central grey, the supragenual nucleus, and the area postrema. 125I-NPY binding sites are found in similar areas, but relative levels of NPY binding and PYY binding differ regionally, suggesting differences in sites labeled by the two ligands. These receptor localizations resemble the distribution of endogenous NPY in some areas, but others, such as the hypothalamus, contain NPY immunoreactivity but few binding sites.

  18. Autoradiographic characterization of L-(/sup 3/H)glutamate binding sites in the central nervous system

    Energy Technology Data Exchange (ETDEWEB)

    Greenamyre, J.T.

    1986-01-01

    A quantitative autoradiographic technique was developed to study L-(/sup 3/H(glutamate binding in sections of central nervous system tissue. This technique circumvented some problems associated with conventional receptor binding methodologies and allowed direct assessment of regional distribution, numbers and affinities of glutamate binding sites. The sensitivity and high degree of anatomical resolution attainable by autoradiography obviated the need for pooled samples of microdissected specimens. Under assay conditions, (/sup 4/H)glutamate bound rapidly and reversibly to sections of rat brain and was not metabolized appreciably. The distribution of glutamate binding sites corresponded to the projection areas of putative glutamatergic pathways. Thus, there was heavy glutamate binding in regions where there is evidence for glutamatergic innervation and little binding in nuclei which apparently do not receive glutamatergic input. Scatchard and Hill plots suggested that glutamate was interacting with a single population of sites; however, competition studies revealed binding site heterogeneity. Anatomical and pharmacological evidence suggested that the NMDA-, high affinity quisqualate-, and kainate-sensitive glutamate binding sites may correspond to physiologically-defined NMDA, quisqualate and kainate receptors.

  19. Structure and binding efficiency relations of QB site inhibitors of photosynthetic reaction centres.

    Science.gov (United States)

    Husu, Ivan; Magyar, Melinda; Szabó, Tibor; Fiser, Béla; Gómez-Bengoa, Enrique; Nagy, László

    2015-04-01

    Many herbicides employed in agriculture and also some antibiotics bind to a specific site of the reaction centre protein (RC) blocking the photosynthetic electron transport. Crystal structures showed that all these compounds bind at the secondary ubiquinone (QB) site albeit to slightly different places. Different herbicide molecules have different binding affinities (evaluated as inhibition constants, KI, and binding enthalpy values, ΔHbind). The action of inhibitors depends on the following parameters: (i) herbicide molecular structure; (ii) interactions between herbicide and quinone binding site; (iii) protein environment. In our investigations KI and ΔHbind were determined for several inhibitors. Bound herbicide structures were optimized and their intramolecular charge distributions were calculated. Experimental and calculated data were compared to those available from databank crystal structures. We can state that the herbicide inhibition efficiency depends on steric and electronic, i.e. geometry of binding with the protein and molecular charge distribution, respectively. Apolar bulky groups on N-7 atom of the inhibitor molecule (like t-buthyl in terbutryn) are preferable for establishing stronger interactions with QB site, while such substituents are not recommended on N-8. The N-4,7,8 nitrogen atoms maintain a larger electron density so that more effective H-bonds are formed between the inhibitor and the surrounding amino acids of the protein.

  20. 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.

  1. A novel high throughput screening assay for binding affinities of perfluoroalkyl iodide for estrogen receptor alpha and beta isoforms.

    Science.gov (United States)

    Song, Wenting; Zhao, Lixia; Sun, Zhendong; Yang, Xiaoxi; Zhou, Qunfang; Jiang, Guibin

    2017-12-01

    Contaminants of emerging concern are continuously increasing, which makes it important to develop high throughput screening techniques for the evaluation of their potential biological effects, especially endocrine disrupting effects, which would directly influence the population dynamics in environment. A novel competitive binding assay based on enzyme fragmentation complementation technology was established to screen the binding affinities of emerging chemicals for estrogen receptor (ER) α or β isoforms. Exogenous compounds could compete with the fragment (ED-ES) of genetically engineered β-galactosidase enzyme (β-gal) for the binding to ERα or β, thus quantitatively altering the formation of enzymatically active β-gal and the hydrolysis of luminescent substrate. According to the monitoring of luminescence curves and the optimization of ERα or β concentrations, it was found that luminescent signals were sustainably emitted for 9h, and 40nM ERα or β in the system would lead to the most sensitive luminescence response. Using 17β-estrodiol (E2) and genistein as the representative estrogenic hormones, their binding affinities for ERα and β were evaluated. The results were consistent with those determined by traditional methods, which confirmed the reliability of this competitive binding assay based on β-gal. Four polyfluorinated iodine alkanes (PFIs) with specific structural characteristics in iodine substitution and carbon chain length were screened, and the results showed diverse binding affinities and different preferences of these chemicals to ERα or β isoforms. The binding affinities of PFIs for ERα were consistent with the result from MVLN transcriptional reporter assay. Overall, the competitive binding assay presented in this study provided a promising alternative to high throughput screening of emerging chemicals with estrogenic effects, which would be important in explanation of their potential toxicological effects and human exposure risks

  2. Viral reverse transcriptases show selective high affinity binding to DNA-DNA primer-templates that resemble the polypurine tract.

    Directory of Open Access Journals (Sweden)

    Gauri R Nair

    Full Text Available Previous results using a SELEX (Systematic Evolution of Ligands by Exponential Enrichment-based approach that selected DNA primer-template duplexes binding with high affinity to HIV reverse transcriptase (RT showed that primers mimicking the 3' end, and in particular the six nt terminal G tract, of the RNA polypurine tract (PPT; HIV PPT: 5'-AAAAGAAAAGGGGGG-3' were preferentially selected. In this report, two viral (Moloney murine leukemia virus (MuLV and avian myeloblastosis virus (AMV and one retrotransposon (Ty3 RTs were used for selection. Like HIV RT, both viral RTs selected duplexes with primer strands mimicking the G tract at the PPT 3' end (AMV PPT: 5'-AGGGAGGGGGA-3'; MuLV PPT: 5'-AGAAAAAGGGGGG-3'. In contrast, Ty3, whose PPT lacks a G tract (5'-GAGAGAGAGGAA-3' showed no selective binding to any duplex sequences. Experiments were also conducted with DNA duplexes (termed DNA PPTs mimicking the RNA PPT-DNA duplex of each virus and a control duplex with a random DNA sequence. Retroviral RTs bound with high affinity to all viral DNA PPT constructs, with HIV and MuLV RTs showing comparable binding to the counterpart DNA PPT duplexes and reduced affinity to the AMV DNA PPT. AMV RT showed similar behavior with a modest preference for its own DNA PPT. Ty3 RT showed no preferential binding for its own or any other DNA PPT and viral RTs bound the Ty3 DNA PPT with relatively low affinity. In contrast, binding affinity of HIV RT to duplexes containing the HIV RNA PPT was less dependent on the G tract, which is known to be pivotal for efficient extension. We hypothesize that the G tract on the RNA PPT helps shift the binding orientation of RT to the 3' end of the PPT where extension can occur.

  3. Aluminium fluoride and magnesium, activators of heterotrimeric GTP-binding proteins, affect high-affinity binding of the fungal toxin fusicoccin to the fusicoccin-binding protein in oat root plasma membranes.

    NARCIS (Netherlands)

    de Boer, A.H.; Van der Molen, G.W.; Prins, H.B.A.; Korthout, H.A.A.J.; van der Hoeven, P.C.J.

    1994-01-01

    The fusicoccin-binding protein was solubilised from purified oat root plasma membranes. The solubilised protein retained full binding activity, provided that protease inhibitors were included. Sodium fluoride reduced the high-affinity [H-3]fusicoccin binding to almost zero in a concentration-depende

  4. The Caulobacter crescentus chromosome replication origin evolved two classes of weak DnaA binding sites.

    Science.gov (United States)

    Taylor, James A; Ouimet, Marie-Claude; Wargachuk, Richard; Marczynski, Gregory T

    2011-10-01

    The Caulobacter crescentus replication initiator DnaA and essential response regulator CtrA compete to control chromosome replication. The C. crescentus replication origin (Cori) contains five strong CtrA binding sites but only two apparent DnaA boxes, termed G-boxes (with a conserved second position G, TGATCCACA). Since clusters of DnaA boxes typify bacterial replication origins, this discrepancy suggested that C. crescentus DnaA recognizes different DNA sequences or compensates with novel DNA-binding proteins. We searched for novel DNA sites by scanning mutagenesis of the most conserved Cori DNA. Autonomous replication assays showed that G-boxes and novel W-boxes (TCCCCA) are essential for replication. Further analyses showed that C. crescentus DnaA binds G-boxes with moderate and W-boxes with very weak affinities significantly below DnaA's capacity for high-affinity Escherichia coli-boxes (TTATCCACA). Cori has five conserved W-boxes. Increasing W-box affinities increases or decreases autonomous replication depending on their strategic positions between the G-boxes. In vitro, CtrA binding displaces DnaA from proximal G-boxes and from distal W-boxes implying CtrA-DnaA competition and DnaA-DnaA cooperation between G-boxes and W-boxes. Similarly, during cell cycle progression, CtrA proteolysis coincides with DnaA binding to Cori. We also observe highly conserved W-boxes in other replication origins lacking E. coli-boxes. Therefore, strategically weak DnaA binding can be a general means of replication control. © 2011 Blackwell Publishing Ltd.

  5. A conformation-specific interhelical salt bridge in the K+ binding site of gastric H,K-ATPase

    NARCIS (Netherlands)

    Koenderink, J.B.; Swarts, H.G.P.; Willems, P.; Krieger, E.; Pont, J.J.H.H.M. de

    2004-01-01

    Homology modeling of gastric H, K-ATPase based on the E-2 model of sarcoplasmic reticulum Ca2+-ATPase (Toyoshima, C., and Nomura, H. (2002) Nature 392, 835-839) revealed the presence of a single high-affinity binding site for K+ and an E-2 form-specific salt bridge between Glu(820) (M6) and Lys(791)

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

    Science.gov (United States)

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

    2015-09-01

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

  7. A computational method for designing diverse linear epitopes including citrullinated peptides with desired binding affinities to intravenous immunoglobulin.

    Science.gov (United States)

    Patro, Rob; Norel, Raquel; Prill, Robert J; Saez-Rodriguez, Julio; Lorenz, Peter; Steinbeck, Felix; Ziems, Bjoern; Luštrek, Mitja; Barbarini, Nicola; Tiengo, Alessandra; Bellazzi, Riccardo; Thiesen, Hans-Jürgen; Stolovitzky, Gustavo; Kingsford, Carl

    2016-04-08

    Understanding the interactions between antibodies and the linear epitopes that they recognize is an important task in the study of immunological diseases. We present a novel computational method for the design of linear epitopes of specified binding affinity to Intravenous Immunoglobulin (IVIg). We show that the method, called Pythia-design can accurately design peptides with both high-binding affinity and low binding affinity to IVIg. To show this, we experimentally constructed and tested the computationally constructed designs. We further show experimentally that these designed peptides are more accurate that those produced by a recent method for the same task. Pythia-design is based on combining random walks with an ensemble of probabilistic support vector machines (SVM) classifiers, and we show that it produces a diverse set of designed peptides, an important property to develop robust sets of candidates for construction. We show that by combining Pythia-design and the method of (PloS ONE 6(8):23616, 2011), we are able to produce an even more accurate collection of designed peptides. Analysis of the experimental validation of Pythia-design peptides indicates that binding of IVIg is favored by epitopes that contain trypthophan and cysteine. Our method, Pythia-design, is able to generate a diverse set of binding and non-binding peptides, and its designs have been experimentally shown to be accurate.

  8. Localization of the Substrate-binding Site in the Homodimeric Mannitol Transporter, EIImtl, of Escherichia coli*

    Science.gov (United States)

    Opačić, Milena; Vos, Erwin P. P.; Hesp, Ben H.; Broos, Jaap

    2010-01-01

    The mannitol transporter from Escherichia coli, EIImtl, belongs to a class of membrane proteins coupling the transport of substrates with their chemical modification. EIImtl is functional as a homodimer, and it harbors one high affinity mannitol-binding site in the membrane-embedded C domain (IICmtl). To localize this binding site, 19 single Trp-containing mutants of EIImtl were biosynthetically labeled with 5-fluorotryptophan (5-FTrp) and mixed with azi-mannitol, a substrate analog acting as a Förster resonance energy transfer (FRET) acceptor. Typically, for mutants showing FRET, only one 5-FTrp was involved, whereas the 5-FTrp from the other monomer was too distant. This proves that the mannitol-binding site is asymmetrically positioned in dimeric IICmtl. Combined with the available two-dimensional projection maps of IICmtl, it is concluded that a second resting binding site is present in this transporter. Active transport of mannitol only takes place when EIImtl becomes phosphorylated at Cys384 in the cytoplasmic B domain. Stably phosphorylated EIImtl mutants were constructed, and FRET experiments showed that the position of mannitol in IICmtl remains the same. We conclude that during the transport cycle, the phosphorylated B domain has to move to the mannitol-binding site, located in the middle of the membrane, to phosphorylate mannitol. PMID:20522557

  9. Localization of the substrate-binding site in the homodimeric mannitol transporter, EIImtl, of Escherichia coli.

    Science.gov (United States)

    Opacić, Milena; Vos, Erwin P P; Hesp, Ben H; Broos, Jaap

    2010-08-13

    The mannitol transporter from Escherichia coli, EII(mtl), belongs to a class of membrane proteins coupling the transport of substrates with their chemical modification. EII(mtl) is functional as a homodimer, and it harbors one high affinity mannitol-binding site in the membrane-embedded C domain (IIC(mtl)). To localize this binding site, 19 single Trp-containing mutants of EII(mtl) were biosynthetically labeled with 5-fluorotryptophan (5-FTrp) and mixed with azi-mannitol, a substrate analog acting as a Förster resonance energy transfer (FRET) acceptor. Typically, for mutants showing FRET, only one 5-FTrp was involved, whereas the 5-FTrp from the other monomer was too distant. This proves that the mannitol-binding site is asymmetrically positioned in dimeric IIC(mtl). Combined with the available two-dimensional projection maps of IIC(mtl), it is concluded that a second resting binding site is present in this transporter. Active transport of mannitol only takes place when EII(mtl) becomes phosphorylated at Cys(384) in the cytoplasmic B domain. Stably phosphorylated EII(mtl) mutants were constructed, and FRET experiments showed that the position of mannitol in IIC(mtl) remains the same. We conclude that during the transport cycle, the phosphorylated B domain has to move to the mannitol-binding site, located in the middle of the membrane, to phosphorylate mannitol.

  10. The selectivity of different external binding sites for quaternary ammonium ions in cloned potassium channels.

    Science.gov (United States)

    Jarolimek, W; Soman, K V; Brown, A M; Alam, M

    1995-09-01

    Tetraethylammonium (TEA) is thought to be the most effective quaternary ammonium (QA) ion blocker at the external site of K+ channels, and small changes to the TEA ion reduce its potency. To examine the properties of the external QA receptor, we applied a variety of QA ions to excised patches from human embryonic kidney cells or Xenopus oocytes transfected with the delayed rectifying K+ channels Kv 2.1 and Kv 3.1. In outside-out patches of Kv 3.1, the relative potencies were TEA > tetrapropylammonium (TPA) > tetrabutylammonium (TBA). In contrast to Kv 3.1, the relative potencies in Kv 2.1 were TBA > TEA > TPA. In Kv 3.1 and Kv 2.1, external tetrapentylammonium (TPeA) blocked K+ currents in a fast, reversible and, in contrast to TEA, time-dependent manner. The external binding of TPeA appeared to be voltage independent, unlike the effects of TPeA applied to inside-out patches. External n-alkyl-triethylammonium compounds (C8, C10 chain length) had a lower affinity than TEA in Kv 3.1, but a higher affinity than TEA in Kv 2.1. In Kv 3.1, the decrease in QA affinity was large when one or two methyl groups were substituted for ethyl groups in TEA, but minor when propyl groups replaced ethyl groups. Changes in the free energy of binding could be correlated to changes in the free energy of hydration of TEA derivatives calculated by continuum methodology. These results reveal a substantial hydrophobic component of external QA ion binding to Kv 2.1, and to a lesser degree to Kv 3.1, in addition to the generally accepted electrostatic interactions. The chain length of hydrophobic TEA derivatives affects the affinity for the hydrophobic binding site, whereas the hydropathy of QA ions determines the electrostatic interaction energy.

  11. Soluble T cell receptor Vβ domains engineered for high-affinity binding to staphylococcal or streptococcal superantigens.

    Science.gov (United States)

    Sharma, Preeti; Wang, Ningyan; Kranz, David M

    2014-01-28

    Staphylococcus aureus and group A Streptococcus secrete a collection of toxins called superantigens (SAgs), so-called because they stimulate a large fraction of an individual's T cells. One consequence of this hyperactivity is massive cytokine release leading to severe tissue inflammation and, in some cases, systemic organ failure and death. The molecular basis of action involves the binding of the SAg to both a T cell receptor (TCR) on a T cell and a class II product of the major histocompatibility complex (MHC) on an antigen presenting cell. This cross-linking leads to aggregation of the TCR complex and signaling. A common feature of SAgs is that they bind with relatively low affinity to the variable region (V) of the beta chain of the TCR. Despite this low affinity binding, SAgs are very potent, as each T cell requires only a small fraction of their receptors to be bound in order to trigger cytokine release. To develop high-affinity agents that could neutralize the activity of SAgs, and facilitate the development of detection assays, soluble forms of the Vβ regions have been engineered to affinities that are up to 3 million-fold higher for the SAg. Over the past decade, six different Vβ regions against SAgs from S. aureus (SEA, SEB, SEC3, TSST-1) or S. pyogenes (SpeA and SpeC) have been engineered for high-affinity using yeast display and directed evolution. Here we review the engineering of these high-affinity Vβ proteins, structural features of the six different SAgs and the Vβ proteins, and the specific properties of the engineered Vβ regions that confer high-affinity and specificity for their SAg ligands.

  12. Affinity of ceftaroline and other beta-lactams for penicillin-binding proteins from Staphylococcus aureus and Streptococcus pneumoniae.

    Science.gov (United States)

    Kosowska-Shick, K; McGhee, P L; Appelbaum, P C

    2010-05-01

    We compared the affinities of ceftaroline for all penicillin-binding proteins (PBPs) with those of ceftriaxone and cefotaxime in 6 Staphylococcus aureus and 7 Streptococcus pneumoniae isolates with various resistance phenotypes. Ceftaroline MICs were pneumoniae. Ceftaroline affinities for penicillin-susceptible S. pneumoniae strains were in the order PBP2X and -3 > PBP1A, -1B, and -2A > PBP2B, and ceftaroline had >or=4-fold higher 50% inhibitory concentrations (IC(50)s) (0.1 to 4 microg/ml) for PBP2X, -2A, -2B, and -3 than those for the other cephalosporins tested. Among 3 penicillin-resistant S. pneumoniae strains, ceftaroline had a high affinity for PBP2X (IC(50), 0.1 to 1 microg/ml), a primary target for cephalosporin PBP binding activity, and high affinities for PBP2B (IC(50), 0.5 to 4 microg/ml) and PBP1A (IC(50), 0.125 to 0.25 microg/ml) as well, both of which are also known as major targets for PBP binding activity of cephalosporins. Ceftaroline PBP affinities in methicillin-susceptible S. aureus strains were greater than or equal to those of the 3 other beta-lactams tested. Ceftaroline bound to PBP2a in methicillin-resistant S. aureus (IC(50), 0.01 to 1 microg/ml) with up to 256-fold-higher affinity than those of other agents. Ceftaroline demonstrated very good PBP affinity against all S. aureus and S. pneumoniae strains tested, including resistant isolates.

  13. Soluble T Cell Receptor Vβ Domains Engineered for High-Affinity Binding to Staphylococcal or Streptococcal Superantigens

    Directory of Open Access Journals (Sweden)

    Preeti Sharma

    2014-01-01

    Full Text Available Staphylococcus aureus and group A Streptococcus secrete a collection of toxins called superantigens (SAgs, so-called because they stimulate a large fraction of an individual’s T cells. One consequence of this hyperactivity is massive cytokine release leading to severe tissue inflammation and, in some cases, systemic organ failure and death. The molecular basis of action involves the binding of the SAg to both a T cell receptor (TCR on a T cell and a class II product of the major histocompatibility complex (MHC on an antigen presenting cell. This cross-linking leads to aggregation of the TCR complex and signaling. A common feature of SAgs is that they bind with relatively low affinity to the variable region (V of the beta chain of the TCR. Despite this low affinity binding, SAgs are very potent, as each T cell requires only a small fraction of their receptors to be bound in order to trigger cytokine release. To develop high-affinity agents that could neutralize the activity of SAgs, and facilitate the development of detection assays, soluble forms of the Vβ regions have been engineered to affinities that are up to 3 million-fold higher for the SAg. Over the past decade, six different Vβ regions against SAgs from S. aureus (SEA, SEB, SEC3, TSST-1 or S. pyogenes (SpeA and SpeC have been engineered for high-affinity using yeast display and directed evolution. Here we review the engineering of these high-affinity Vβ proteins, structural features of the six different SAgs and the Vβ proteins, and the specific properties of the engineered Vβ regions that confer high-affinity and specificity for their SAg ligands.

  14. A quantitative model for the in vivo assessment of drug binding sites with positron emission tomography

    Energy Technology Data Exchange (ETDEWEB)

    Mintun, M.A.; Raichle, M.E.; Kilbourn, M.R.; Wooten, G.F.; Welch, M.J.

    1984-03-01

    We propose an in vivo method for use with positron emission tomography (PET) that results in a quantitative characterization of neuroleptic binding sites using radiolabeled spiperone. The data are analyzed using a mathematical model that describes transport, nonspecific binding, and specific binding in the brain. The model demonstrates that the receptor quantities Bmax (i.e., the number of binding sites) and KD-1 (i.e., the binding affinity) are not separably ascertainable with tracer methodology in human subjects. We have, therefore, introduced a new term, the binding potential, equivalent to the product BmaxKD-1, which reflects the capacity of a given tissue, or region of a tissue, for ligand-binding site interaction. The procedure for obtaining these measurements is illustrated with data from sequential PET scans of baboons after intravenous injection of carrier-added (18F)spiperone. From these data we estimate the brain tissue nonspecific binding of spiperone to be in the range of 94.2 to 95.3%, and the regional brain spiperone permeability (measured as the permeability-surface area product) to be in the range of 0.025 to 0.036 cm3/(s X ml). The binding potential of the striatum ranged from 17.4 to 21.6; these in vivo estimates compare favorably to in vitro values in the literature. To our knowledge this represents the first direct evidence that PET can be used to characterize quantitatively, locally and in vivo, drug binding sites in brain. The ability to make such measurements with PET should permit the detailed investigation of diseases thought to result from disorders of receptor function.

  15. Cooperativity between calmodulin-binding sites in Kv7.2 channels.

    Science.gov (United States)

    Alaimo, Alessandro; Alberdi, Araitz; Gomis-Perez, Carolina; Fernández-Orth, Juncal; Gómez-Posada, Juan Camilo; Areso, Pilar; Villarroel, Alvaro

    2013-01-01

    Among the multiple roles assigned to calmodulin (CaM), controlling the surface expression of Kv7.2 channels by binding to two discontinuous sites is a unique property of this Ca(2+) binding protein. Mutations that interfere with CaM binding or the sequestering of CaM prevent this M-channel component from exiting the endoplasmic reticulum (ER), which reduces M-current density in hippocampal neurons, enhancing excitability and offering a rational mechanism to explain some forms of benign familial neonatal convulsions (BFNC). Previously, we identified a mutation (S511D) that impedes CaM binding while allowing the channel to exit the ER, hinting that CaM binding may not be strictly required for Kv7.2 channel trafficking to the plasma membrane. Alternatively, this interaction with CaM might escape detection and, indeed, we now show that the S511D mutant contains functional CaM-binding sites that are not detected by classical biochemical techniques. Surface expression and function is rescued by CaM, suggesting that free CaM in HEK293 cells is limiting and reinforcing the hypothesis that CaM binding is required for ER exit. Within the CaM-binding domain formed by two sites (helix A and helix B), we show that CaM binds to helix B with higher apparent affinity than helix A, both in the presence and absence of Ca(2+), and that the two sites cooperate. Hence, CaM can bridge two binding domains, anchoring helix A of one subunit to helix B of another subunit, in this way influencing the function of Kv7.2 channels.

  16. Metal-ligand binding affinity vs reactivity: qualitative studies in Rh(I)-catalyzed asymmetric ring-opening reactions.

    Science.gov (United States)

    Tsui, Gavin Chit; Dougan, Patrick; Lautens, Mark

    2013-06-01

    Rh(I)-catalyzed asymmetric ring opening (ARO) of oxabenzonorbornadiene is used as a model system to qualitatively study reactions involving multiple metal-ligand interactions. The key feature of this approach is the use of product ee as an indicator to quickly gain important information such as the relative ligand binding affinity and relative reactivity of catalysts.

  17. Mammalian cell-based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity

    NARCIS (Netherlands)

    Martin, Brent R; Giepmans, Ben N G; Adams, Stephen R; Tsien, Roger Y

    2005-01-01

    Membrane-permeant biarsenical dyes such as FlAsH and ReAsH fluoresce upon binding to genetically encoded tetracysteine motifs expressed in living cells, yet spontaneous nonspecific background staining can prevent detection of weakly expressed or dilute proteins. If the affinity of the tetracysteine

  18. Differential thermodynamic driving force of first- and second-generation antihistamines to determine their binding affinity for human H1 receptors.

    Science.gov (United States)

    Hishinuma, Shigeru; Sugawara, Kenta; Uesawa, Yoshihiro; Fukui, Hiroyuki; Shoji, Masaru

    2014-09-15

    Differential binding sites for first- and second-generation antihistamines were indicated on the basis of the crystal structure of human histamine H1 receptors. In this study, we evaluated differences between the thermodynamic driving forces of first- and second-generation antihistamines for human H1 receptors and their structural determinants. The binding enthalpy and entropy of 20 antihistamines were estimated with the van't Hoff equation using their dissociation constants obtained from their displacement curves against the binding of [(3)H]mepyramine to membrane preparations of Chinese hamster ovary cells expressing human H1 receptors at various temperatures from 4°C to 37°C. Structural determinants of antihistamines for their thermodynamic binding properties were assessed by quantitative structure-activity relationship (QSAR) analyses. We found that entropy-dependent binding was more evident in second- than first-generation antihistamines, resulting in enthalpy-entropy compensation between the binding forces of first- and second-generation antihistamines. QSAR analyses indicated that enthalpy-entropy compensation was determined by the sum of degrees, maximal electrostatic potentials, water-accessible surface area and hydrogen binding acceptor count of antihistamines to regulate their affinity for receptors. In conclusion, it was revealed that entropy-dependent hydrophobic interaction was more important in the binding of second-generation antihistamines, even though the hydrophilicity of second-generation antihistamines is generally increased. Furthermore, their structural determinants responsible for enthalpy-entropy compensation were explored by QSAR analyses. These findings may contribute to understanding the fundamental mechanisms of how the affinity of ligands for their receptors is regulated.

  19. Binding of (3H)dihydroergocryptine to an alpha-adrenergic site in the stalk median eminence of the steer

    Energy Technology Data Exchange (ETDEWEB)

    Chen, H.T.; Roberts, J.M.; Weiner, R.I.

    1981-12-01

    Dihydroergocryptine (DHE), a potent dopamine agonist and alpha-adrenergic antagonist, has been used as a radioligand to characterize both dopamine and alpha-adrenergic receptors. In the present study, the binding of (3H)DHE to particulate fractions of the steer stalk median eminence was characterized using a filtration assay. Specific binding was defined by the presence of 10 microM phentolamine or by an iterative nonlinear hyperbolic curve-fitting program. Scatchard analysis of equilibrium isotherms of specific binding defined a single high affinity (Kd . 1.78 +/- 0.22 nM), saturable (maximum binding, 481 +/- 39 fmol/mg protein), stereoselective binding site. The Kd, calculated from the ratio of the rate constants k2 and k1, was 2.8 +/- 0.14 nM. The rank order of potency of agonists to compete for (3H)DHE binding (l-epinephrine greater than l-norepinephrine greater than dopamine greater than l-isoproterenol) was consistent with interactions at an alpha-adrenergic site. The rank order of potency of alpha-antagonists (phentolamine greater than yohimbine greater than prazosin) suggested that this was an alpha 2-adrenergic receptor. The affinity of dopamine agonists for the (3H)DHE-binding site was 10-fold lower relative to their potency at known dopamine receptors, while the affinity of dopaminergic antagonists was 100-fold lower. Furthermore, Scatchard analysis of specific (3H)DHE binding in the presence of a concentration of spiperone which should saturate dopamine receptors, only decreased the number of binding sites by 9%. These data demonstrate the presence of large numbers of alpha-adrenergic receptors in the stalk median eminence of the steer. Only a small number of dopaminergic binding sites for (3H)DHE appeared to be present.

  20. Assessment of algorithms for inferring positional weight matrix motifs of transcription factor binding sites using protein binding microarray data.

    Directory of Open Access Journals (Sweden)

    Yaron Orenstein

    Full Text Available The new technology of protein binding microarrays (PBMs allows simultaneous measurement of the binding intensities of a transcription factor to tens of thousands of synthetic double-stranded DNA probes, covering all possible 10-mers. A key computational challenge is inferring the binding motif from these data. We present a systematic comparison of four methods developed specifically for reconstructing a binding site motif represented as a positional weight matrix from PBM data. The reconstructed motifs were evaluated in terms of three criteria: concordance with reference motifs from the literature and ability to predict in vivo and in vitro bindings. The evaluation encompassed over 200 transcription factors and some 300 assays. The results show a tradeoff between how the methods perform according to the different criteria, and a dichotomy of method types. Algorithms that construct motifs with low information content predict PBM probe ranking more faithfully, while methods that produce highly informative motifs match reference motifs better. Interestingly, in predicting high-affinity binding, all methods give far poorer results for in vivo assays compared to in vitro assays.

  1. Curcumin binds in silico to anti-cancer drug target enzyme MMP-3 (human stromelysin-1) with affinity comparable to two known inhibitors of the enzyme.

    Science.gov (United States)

    Jerah, Ahmed; Hobani, Yahya; Kumar, B Vinod; Bidwai, Anil

    2015-01-01

    In silico interaction of curcumin with the enzyme MMP-3 (human stromelysin-1) was studied by molecular docking using AutoDock 4.2 as the docking software application. AutoDock 4.2 software serves as a valid and acceptable docking application to study the interactions of small compounds with proteins. Interactions of curcumin with MMP-3 were compared to those of two known inhibitors of the enzyme, PBSA and MPPT. The calculated free energy of binding (ΔG binding) shows that curcumin binds with affinity comparable to or better than the two known inhibitors. Binding interactions of curcumin with active site residues of the enzyme are also predicted. Curcumin appears to bind in an extendended conformation making extensive VDW contacts in the active site of the enzyme. Hydrogen bonding and pi-pi interactions with key active site residues is also observed. Thus, curcumin can be considered as a good lead compound in the development of new inhibitors of MMP-3 which is a potential target of anticancer drugs. The results of these studies can serve as a starting point for further computational and experimental studies.

  2. In vitro and in vivo characterisation of [3H]ANSTO-14 binding to the sigma 1 binding sites.

    Science.gov (United States)

    Nguyen, V H; Mardon, K; Kassiou, M; Christie, M D

    1999-02-01

    N-(4-phenylbutyl)-3-hydroxy-4-azahexacyclo[5.4.1.0(2,6).0(3, 10).0(5,9) .0(8,11)]dodecane (ANSTO-14) showed the highest activity for the sigma 1 site (Ki = 9.4 nM) and 19-fold sigma 1/sigma 2 selectivity. The present study showed that [3H]ANSTO-14 binds to a single high-affinity site in guinea pig brain membranes with an equilibrium Ki of 8.0 +/- 0.3 nM, in good agreement with the kinetic studies (Kd = 13.3 +/- 5.4 nM, n = 4), and a Bmax of 3.199 +/- 105 fmol/mg protein (n = 4). The in vivo biodistribution of [3H]ANSTO-14 showed a high uptake in the diencephalon. Pretreatment of rats with sigma ligands including (+)-pentazocine (sigma 1), ANSTO-14 (sigma 1), and DTG (sigma 1 and sigma 2) did not significantly reduce radiotracer uptake in the brain, but did in the spleen. A labelled metabolite was found in the liver and brain. Due to its insensitivity to sigma ligands, the accumulation of [3H]ANSTO-14 in the brain indicates high nonspecific binding. Therefore, [3H]ANSTO-14 is a suitable ligand for labelling sigma 1 sites in vitro but is not suitable for brain imaging of sigma binding sites in vivo.

  3. Kinetic studies show that Ca2+ and Tb3+ have different binding preferences toward the four Ca2+-binding sites of calmodulin.

    Science.gov (United States)

    Wang, C L; Leavis, P C; Gergely, J

    1984-12-18

    The stepwise addition of Tb3+ to calmodulin yields a large tyrosine-sensitized Tb3+ luminescence enhancement as the third and fourth ions bind to the protein [Wang, C.-L. A., Aquaron, R. R., Leavis, P. C., & Gergely, J. (1982) Eur. J. Biochem. 124, 7-12]. Since the only tyrosine residues in calmodulin are located within binding sites III and IV, these results suggest that Tb3+ binds first to sites I and II. Recent NMR studies have provided evidence that Ca2+, on the other hand, binds preferentially to sites III and IV. Kinetic studies using a stopped-flow apparatus also show that the preferential binding of Ca2+ and lanthanide ions is different. Upon rapid mixing of 2Ca-calmodulin with two Tb3+ ions, there was a small and rapid tyrosine fluorescence change, but no Tb3+ luminescence was observed, indicating that Tb3+ binds to sites I and II but not sites III and IV. When two Tb3+ ions are mixed with 2Dy-calmodulin, Tb3+ luminescence rises rapidly as Tb3+ binds to the empty sites III and IV, followed by a more gradual decrease (k = 0.4 s-1 as the ions redistribute themselves over the four sites. These results indicate that (i) both Tb3+ and Dy3+ prefer binding to sites I and II of calmodulin and (ii) the binding of Tb3+ to calmodulin is not impeded by the presence of two Ca2+ ions initially bound to the protein. Thus, the Ca2+ and lanthanide ions must exhibit opposite preferences for the four sites of calmodulin: sites III and IV are the high-affinity sites for Ca2+, whereas Tb3+ and Dy3+ prefer sites I and II.

  4. Affinity Capillary Electrophoresis:Study of the Binding of HIV-1 gp41 with a Membrane Protein (P45) on the Human B Cell Line,Raji

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    Affinity capillary electrophoresis has been used to study the interaction between a membrane protein (P45) isolated from the Human B cell line, Raji, and rsgp41. P45, rsgp41 and the complexes were well resolved. The entire separation was achieved in less than 3min. Formations of two kinds of stable P45-rsgp41 complexes were confirmed based on migration time comparison; the binding equilibrium was achieved as soon as two proteins were mixed. The results indicate that the interaction between P45 and rsgp41 is strong with a fast association rate and a slow dissociation rate, and there are at least two kinds of binding sites with different binding constants between P45 and rsgp41.

  5. Fast and accurate determination of the relative binding affinities of small compounds to HIV-1 protease using non-equilibrium work.

    Science.gov (United States)

    Ngo, Son Tung; Hung, Huynh Minh; Nguyen, Minh Tho

    2016-12-05

    The fast pulling ligand (FPL) out of binding cavity using non-equilibrium molecular dynamics (MD) simulations was demonstrated to be a rapid, accurate and low CPU demand method for the determination of the relative binding affinities of a large number of HIV-1 protease (PR) inhibitors. In this approach, the ligand is pulled out of the binding cavity of the protein using external harmonic forces, and the work of pulling force corresponds to the relative binding affinity of HIV-1 PR inhibitor. The correlation coefficient between the pulling work and the experimental binding free energy of R=-0.95 shows that FPL results are in good agreement with experiment. It is thus easier to rank the binding affinities of HIV-1 PR inhibitors, that have similar binding affinities because the mean error bar of pulling work amounts to δW=7%. The nature of binding is discovered using the FPL approach. © 2016 Wiley Periodicals, Inc.

  6. Effects of neonatal. gamma. -ray irradiation on rat hippocampus: Pt. 2; Development of excitatory amino acid binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Dessi, F.; Represa, A.; Ben-Ari, Y. (Institut National de la Sante et de la Recherche Medicale (INSERM), 75 - Paris (France))

    1991-01-01

    In the rat, neonatal irradiation produces a destruction of denate granule cells and prevents the development of the mossy fibre-CA3 pyramidal cell synapse. The developmental increase of high affinity kainate binding sites in the stratum lucidum was reduced on the irradiated side as compared with the control side. This suggests that a proportion of high affinity kainate binding sites is associated with mossy fibres. In contrast, the development profile of N-methyl-D-aspartate binding sites, which are associated with associational and commissural synapses in CA3, was not affected by irradiation. The role that afferent fibres may play in the development of pyramidal cells is discussed in connection with the modulatory effects of glutamate receptors on the development of neurons. (author).

  7. Mapping of barley alpha-amylases and outer subsite mutants reveals dynamic high-affinity subsites and barriers in the long substrate binding cleft

    DEFF Research Database (Denmark)

    Kandra, L.; Abou Hachem, Maher; Gyemant, G.;

    2006-01-01

    as binding barriers. Barley a-amylase I mutants Y105A and T212Y at subsite -6 and +4 resulted in release or anchoring of bound substrate, thus modifying the affinities of other high-affinity subsites (-2 and +2) and barriers. The double mutant Y105A-T212Y displayed a hybrid subsite affinity profile...

  8. Development of affinity technology for isolating individual human chromosomes by third strand binding

    Energy Technology Data Exchange (ETDEWEB)

    Fresco, Jacques R.

    2003-06-01

    The overall goal was to explore whether nucleic acid third strands could be used to bind with very high specificity to specific targets within whole genomes. Towards this end conditions had to be found to keep erroneous binding to an absolute minimum. The goal to use third strands (linked to magnetic beads) to ''capture'' large particles such as plasmids, cosmids, and whole chromosomes from complex mixtures was partially met; their use to serve as cytogenetic probes of metaphase chromosomes and to deliver reactive reagents to unique target sites on chromosomes in vivo for the purpose of mutagenizing specific base pairs was fully met; and their use as cytogenetic probes of chromosomal DNA in sections of formalin-fixed, paraffin-embedded tissue has been met since the DOE support was terminated.

  9. Serotriflin, a CRISP family protein with binding affinity for small serum protein-2 in snake serum.

    Science.gov (United States)

    Aoki, Narumi; Sakiyama, Akie; Kuroki, Kimiko; Maenaka, Katsumi; Kohda, Daisuke; Deshimaru, Masanobu; Terada, Shigeyuki

    2008-04-01

    Habu (Trimeresurus flavoviridis) serum contains 3 small serum