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Sample records for progesterone specific binding sites

  1. Identification and partial characterization of cytosolic progesterone-binding sites in the filamentous fungus Rhizopus nigricans.

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    Lenasi, H; Hudnik-Plevnik, T

    1996-06-01

    Progesterone and some other steroids have been shown to induce a steroid 11alpha-hydroxylating enzyme system requiring cytochrome P450 in the filamentous fungus Rhizopus nigricans. In the present work, we attempted to find out whether the mycelial cytosol contained progesterone-binding sites (PBS) which could function as receptors for P450-inducing steroids and might, therefore, be included in the induction process. Two types of constitutive PBS, PBS-I and PBS-II, were identified in the cytosol pretreated with dextran-coated charcoal which removed the endogenous ligand. The protein nature of these binding activities was indicated by their susceptibility to trypsin and proteinase K digestion, heat denaturation, and their resistance to DNase. Progesterone binding was rapid, the maximal level being reached after 45 min of incubation at 22 degrees C. At this temperature, dissociation of progesterone from PBS-I proceeded with a t1/2 of 17 min and that from PBS-II with a t1/2 of 133 min. The apparent Kd of PBS-I determined by Scatchard analysis was 2.1-7.0 x 10(-9)M, and Bmax 36-218 fmol/mg protein. Bmax for PBS-II was >400 fmol/mg protein, whereas the value of Kd could not be determined accurately due to the sigmoidal nature of the association kinetics. The biological role of PBS-I in transcriptional regulation is suggested by the observation that this receptor-like protein contains a functional DNA-binding domain. A specific function of PBS-I in the induction of 11alpha-hydroxylase seems to be, however, questionable because of poor correlation between the affinity and the inducing capability of corresponding steroids.

  2. The Sigma-2 Receptor and Progesterone Receptor Membrane Component 1 are Different Binding Sites Derived From Independent Genes

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    Uyen B. Chu

    2015-11-01

    Full Text Available The sigma-2 receptor (S2R is a potential therapeutic target for cancer and neuronal diseases. However, the identity of the S2R has remained a matter of debate. Historically, the S2R has been defined as (1 a binding site with high affinity to 1,3-di-o-tolylguanidine (DTG and haloperidol but not to the selective sigma-1 receptor ligand (+-pentazocine, and (2 a protein of 18–21 kDa, as shown by specific photolabeling with [3H]-Azido-DTG and [125I]-iodoazido-fenpropimorph ([125I]-IAF. Recently, the progesterone receptor membrane component 1 (PGRMC1, a 25 kDa protein, was reported to be the S2R (Nature Communications, 2011, 2:380. To confirm this identification, we created PGRMC1 knockout NSC34 cell lines using the CRISPR/Cas9 technology. We found that in NSC34 cells devoid of or overexpressing PGRMC1, the maximum [3H]-DTG binding to the S2R (Bmax as well as the DTG-protectable [125I]-IAF photolabeling of the S2R were similar to those of wild-type control cells. Furthermore, the affinities of DTG and haloperidol for PGRMC1 (KI = 472 μM and 350 μM, respectively, as determined in competition with [3H]-progesterone, were more than 3 orders of magnitude lower than those reported for the S2R (20–80 nM. These results clarify that PGRMC1 and the S2R are distinct binding sites expressed by different genes.

  3. In situ detection of progesterone binding sites in the plasma membrane of the filamentous fungus Rhizopus nigricans: In situ detekcija vezavnih mest za progesteron v plazemski membrani filamentozne glive Rhizopus nigricans:

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    Breskvar, Katja; Jeraj, Nataša; LENASI, HELENA; Romih, Rok

    2003-01-01

    Steroid hydroxylating enzymes in the fungus Rhizopus nigricans are induced by progesterone and by some other steroids. It is known that in higher organisms steroids exert their nongenomic action via steroid binding proteins located inthe plasma membrane of the cells, thus our aim was to detect progesterone binding sites in R. nigricans plasma membrane. In this report membrane receptors were identified by two independent methods, analysis of progesteronebinding to plasma membrane fraction by c...

  4. Endogenous progesterone and its cellular binding sites in wheat exposed to drought stress

    Czech Academy of Sciences Publication Activity Database

    Janeczko, A.; Oklešťková, Jana; Siwek, A.; Dziurka, M.; Pociecha, E.; Kocurek, M.; Novák, Ondřej

    2013-01-01

    Roč. 138, č. 2013 (2013), s. 384-394 ISSN 0960-0760 R&D Projects: GA MŠk LK21306 Institutional research plan: CEZ:AV0Z50380511 Keywords : Plants * Wheat * Progesterone Subject RIV: EF - Botanics Impact factor: 4.049, year: 2013

  5. Genome-wide progesterone receptor binding: cell type-specific and shared mechanisms in T47D breast cancer cells and primary leiomyoma cells.

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    Ping Yin

    Full Text Available BACKGROUND: Progesterone, via its nuclear receptor (PR, exerts an overall tumorigenic effect on both uterine fibroid (leiomyoma and breast cancer tissues, whereas the antiprogestin RU486 inhibits growth of these tissues through an unknown mechanism. Here, we determined the interaction between common or cell-specific genome-wide binding sites of PR and mRNA expression in RU486-treated uterine leiomyoma and breast cancer cells. PRINCIPAL FINDINGS: ChIP-sequencing revealed 31,457 and 7,034 PR-binding sites in breast cancer and uterine leiomyoma cells, respectively; 1,035 sites overlapped in both cell types. Based on the chromatin-PR interaction in both cell types, we statistically refined the consensus progesterone response element to G•ACA• • •TGT•C. We identified two striking differences between uterine leiomyoma and breast cancer cells. First, the cis-regulatory elements for HSF, TEF-1, and C/EBPα and β were statistically enriched at genomic RU486/PR-targets in uterine leiomyoma, whereas E2F, FOXO1, FOXA1, and FOXF sites were preferentially enriched in breast cancer cells. Second, 51.5% of RU486-regulated genes in breast cancer cells but only 6.6% of RU486-regulated genes in uterine leiomyoma cells contained a PR-binding site within 5 kb from their transcription start sites (TSSs, whereas 75.4% of RU486-regulated genes contained a PR-binding site farther than 50 kb from their TSSs in uterine leiomyoma cells. RU486 regulated only seven mRNAs in both cell types. Among these, adipophilin (PLIN2, a pro-differentiation gene, was induced via RU486 and PR via the same regulatory region in both cell types. CONCLUSIONS: Our studies have identified molecular components in a RU486/PR-controlled gene network involved in the regulation of cell growth, cell migration, and extracellular matrix function. Tissue-specific and common patterns of genome-wide PR binding and gene regulation may determine the therapeutic effects of antiprogestins in

  6. Drug-binding properties of rat alpha-foetoprotein. Specificities of the phenylbutazone-binding and warfarin-binding sites.

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    Hervé, F; Rajkowski, K M; Martin, M T; Dessen, P; Cittanova, N

    1986-01-01

    Rat alpha-foetoprotein (alpha-FP) strongly binds the drugs warfarin and phenylbutazone, as does albumin; however, the binding sites for the two drugs seemed to be different. This possibility and the specificity of this/these drug-binding site(s) of rat alpha-FP were investigated by competitive protein-binding experiments with a variety of drugs, representing different pharmacological groups, and biomolecules that are strongly bound by the foetal protein and that are suspected to play a specific role during foetal development. The binding mechanisms were further investigated by using comparisons between computer-derived theoretical displacement curves and experimental points in order to distinguish different possible binding models. The results indicate: that warfarin and phenylbutazone are bound at two distinct sites on rat alpha-FP and that a negative modulatory effect is exerted between the two sites; that the degree of specificity of these two drug-binding sites is different, since the warfarin-binding site appears to be specific for the binding of coumarinic and anthranilic drugs whereas that for phenylbutazone is able to bind substances of very varied chemical structure and is more hydrophobic; that the phenylbutazone-binding site is the site that binds oestrogens that thyroid hormones and, probably, fatty acids and bilirubin are bound at (an)other site(s) but exert negative modulatory effects on phenylbutazone binding. The nature of the different binding areas of rat alpha-FP is compared with that of those already proposed for albumin. The potential risks of toxicity of such interactions between drugs and/or biomolecules on foetal development are also discussed. PMID:2434073

  7. Specific interactions of steroids, arylhydrocarbons and flavonoids with progesterone receptors from the cytosol of the fungus Rhizopus nigricans.

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    Lenasi, Helena; Breskvar, Katja

    2004-08-01

    Rhizopus nigricans (R. nigricans) transforms fungitoxic progesterone into the less toxic 11alpha-hydroxyprogesterone which is then able to exit the mycelia into the surrounding water. Hydroxylation of progesterone is an inducible process in which cytosolic progesterone receptors could be involved. In the present study, we characterised receptors with respect to ligand specificity and to their involvement in progesterone induction of hydroxylase. EC(50) values of different ligands (steroids, xenobiotic arylhydrocarbons and natural flavonoids) were determined by competition studies using 40nM ((3)H)progesterone. C21 and C19 3-oxo-4-ene steroids were good competitors (EC(50) of progesterone 2.3 +/- 0.1 x 10(-7)M, EC(50) of androsten-3,17-dione 24 +/- 2 x 10(-7)M). The presence of hydroxyl groups in steroids significantly decreased the affinity for receptors. The arylhydrocarbons alpha-naphthoflavone and ketoconazole exhibited EC(50) values of 0.3 +/- 0.01 x 10(-7)M and 27 +/- 5 x 10(-7)M, respectively, whereas beta-naphthoflavone and benzo(a)pyrene were not able to displace labelled progesterone completely. The competition curves obtained by natural flavonoids also did not reach the bottom level of non-labelled progesterone, indicating the interaction at some allosteric binding site(s) of progesterone receptors. All ligands were examined for their involvement in progesterone-hydroxylase induction. Steroid agonists induced the enzyme in a dose-dependent manner in accordance with their affinity for receptors, whereas arylhydrocarbons and natural flavonoids did not induce the enzyme. The agonistic action of steroids, together with the antagonistic action of alpha-naphthoflavone, strongly suggests the involvement of progesterone receptors in progesterone signalling resulting in the induction of progesterone-hydroxylase.

  8. MONKEY: Identifying conserved transcription-factor binding sitesin multiple alignments using a binding site-specific evolutionarymodel

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    Moses, Alan M.; Chiang, Derek Y.; Pollard, Daniel A.; Iyer, VenkyN.; Eisen, Michael B.

    2004-10-28

    We introduce a method (MONKEY) to identify conserved transcription-factor binding sites in multispecies alignments. MONKEY employs probabilistic models of factor specificity and binding site evolution, on which basis we compute the likelihood that putative sites are conserved and assign statistical significance to each hit. Using genomes from the genus Saccharomyces, we illustrate how the significance of real sites increases with evolutionary distance and explore the relationship between conservation and function.

  9. An Improved Method for Identifying Specific DNA-Protein-Binding Sites In Vitro.

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    Wang, Liangyan; Lu, Huizhi; Wang, Yunguang; Yang, Su; Xu, Hong; Cheng, Kaiying; Zhao, Ye; Tian, Bing; Hua, Yuejin

    2017-03-01

    Binding of proteins to specific DNA sequences is essential for a variety of cellular processes such as DNA replication, transcription and responses to external stimuli. Chromatin immunoprecipitation is widely used for determining intracellular DNA fragments bound by a specific protein. However, the subsequent specific or accurate DNA-protein-binding sequence is usually determined by DNA footprinting. Here, we report an alternative method for identifying specific sites of DNA-protein-binding (designated SSDP) in vitro. This technique is mainly dependent on antibody-antigen immunity, simple and convenient, while radioactive isotope labeling and optimization of partial degradation by deoxyribonuclease (DNase) are avoided. As an example, the specific binding sequence of a target promoter by DdrO (a DNA damage response protein from Deinococcus radiodurans) in vitro was determined by the developed method. The central sequence of the binding site could be easily located using this technique.

  10. Position specific variation in the rate of evolution intranscription factor binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Moses, Alan M.; Chiang, Derek Y.; Kellis, Manolis; Lander, EricS.; Eisen, Michael B.

    2003-08-28

    The binding sites of sequence specific transcription factors are an important and relatively well-understood class of functional non-coding DNAs. Although a wide variety of experimental and computational methods have been developed to characterize transcription factor binding sites, they remain difficult to identify. Comparison of non-coding DNA from related species has shown considerable promise in identifying these functional non-coding sequences, even though relatively little is known about their evolution. Here we analyze the genome sequences of the budding yeasts Saccharomyces cerevisiae, S. bayanus, S. paradoxus and S. mikataeto study the evolution of transcription factor binding sites. As expected, we find that both experimentally characterized and computationally predicted binding sites evolve slower than surrounding sequence, consistent with the hypothesis that they are under purifying selection. We also observe position-specific variation in the rate of evolution within binding sites. We find that the position-specific rate of evolution is positively correlated with degeneracy among binding sites within S. cerevisiae. We test theoretical predictions for the rate of evolution at positions where the base frequencies deviate from background due to purifying selection and find reasonable agreement with the observed rates of evolution. Finally, we show how the evolutionary characteristics of real binding motifs can be used to distinguish them from artifacts of computational motif finding algorithms. As has been observed for protein sequences, the rate of evolution in transcription factor binding sites varies with position, suggesting that some regions are under stronger functional constraint than others. This variation likely reflects the varying importance of different positions in the formation of the protein-DNA complex. The characterization of the pattern of evolution in known binding sites will likely contribute to the effective use of comparative

  11. Position specific variation in the rate of evolution in transcription factor binding sites

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    Kellis Manolis

    2003-08-01

    Full Text Available Abstract Background The binding sites of sequence specific transcription factors are an important and relatively well-understood class of functional non-coding DNAs. Although a wide variety of experimental and computational methods have been developed to characterize transcription factor binding sites, they remain difficult to identify. Comparison of non-coding DNA from related species has shown considerable promise in identifying these functional non-coding sequences, even though relatively little is known about their evolution. Results Here we analyse the genome sequences of the budding yeasts Saccharomyces cerevisiae, S. bayanus, S. paradoxus and S. mikatae to study the evolution of transcription factor binding sites. As expected, we find that both experimentally characterized and computationally predicted binding sites evolve slower than surrounding sequence, consistent with the hypothesis that they are under purifying selection. We also observe position-specific variation in the rate of evolution within binding sites. We find that the position-specific rate of evolution is positively correlated with degeneracy among binding sites within S. cerevisiae. We test theoretical predictions for the rate of evolution at positions where the base frequencies deviate from background due to purifying selection and find reasonable agreement with the observed rates of evolution. Finally, we show how the evolutionary characteristics of real binding motifs can be used to distinguish them from artefacts of computational motif finding algorithms. Conclusion As has been observed for protein sequences, the rate of evolution in transcription factor binding sites varies with position, suggesting that some regions are under stronger functional constraint than others. This variation likely reflects the varying importance of different positions in the formation of the protein-DNA complex. The characterization of the pattern of evolution in known binding sites will

  12. Pharmacological specificity of some psychotomimetic and antipsychotic agents for the sigma and PCP binding sites

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    Itzhak, Y.

    1988-01-01

    The pharmacological specificity of representative psychotomimetic agents such a phencyclidine (PCP) analogs, opiate benzomorphans and several antipsychotic agents was assessed for the sigma and PCP binding sites. In a series of binding experiments, in rat brain membranes, sigma and PCP binding sites were labeled with (/sup 3/H)-1-(1-(3-hydroxyphenyl) cyclohexyl) piperidine ((/sup 3/H)PCP-3-OH), (+)(/sup 3/H)-N-allylnormetazocine ((+)(/sup 3/H)SKF 10047) and (+) (/sup 3/H)-3-(3-hydroxy-phenyl)-N-(1-propyl) piperidine and ((+)(/sup 3/H)-3-PPP). PCP analogs inhibit potently high affinity (/sup 3/H)PCP-3-OH binding and (+)(/sup 3/H)SKF 10047 binding, moderately the low affinity binding component of (/sup 3/H)PCP-3-OH and very weakly (+) (/sup 3/H)-3-PPP binding. (+)SKF 10047 and cyclazocine are potent to moderate inhibitors of (+)(/sup 3/H)SKF 10047, high affinity (/sup 3/H)PCP-3-OH and (+)(/sup 3/H)-3-PCP-3-OH binding. The antipsychotic agents display high affinity for (+)(/sup 3/H)-3-PPP binding sites, moderate affinity for (+)(/sup 3/H)SKF 10047 sites and have no effect on either the high or low affinity (/sup 3/H)PCP-3-OH binding. 20 references, 3 figures, 2 tables.

  13. Conformational and physicochemical DNA features specific for transcription factor binding sites.

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    Ponomarenko, J V; Ponomarenko, M P; Frolov, A S; Vorobyev, D G; Overton, G C; Kolchanov, N A

    1999-01-01

    A reliable recognition of transcription factor binding sites is essential for analysis of regulatory genomic sequences. The experimental data make evident an important role of DNA conformational features for site functioning. However, Internet-available tools for revealing conformational and physicochemical DNA features significant for the site functioning and subsequent use of these features for site recognition have not been developed up to now. We suggest an approach for revealing significant conformational and physicochemical properties of functional sites implemented in the database B-DNA-VIDEO. This database is designed to study the sets of various transcription factor binding sites, providing evidence that transcription factor binding sites are characterized by specific sets of significant conformational and physicochemical DNA properties. For a fixed site, by using the B-DNA features selected for this site recognition, the C-program recognizing this site may be generated, control tested and stored in the database B-DNA-VIDEO. Each B-DNA-VIDEO entry links to the Web-applet recognizing the site, whose significant B-DNA features are stored in this entry as the 'site recognition programs'. The pairwise linked entry-applet pairs are compiled within the B-DNA-VIDEO system, which is simultaneously the database and the program tools package applicable immediately for recognizing the sites stored in the database. Indeed, this is the novelty. Hence, B-DNA-VIDEO is the Web resource of both 'searching for static data' and 'active computation' type, that is why it was called an 'activated database'. B-DNA-VIDEO is available at http://wwwmgs.bionet.nsc.ru/systems/BDNAVideo/ and the mirror site at http://www.cbil.upenn.edu/mgs/systems/c onsfreq/.

  14. Specific binding sites for alcohols and anesthetics on ligand-gated ion channels

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    Mascia, Maria Paola; Trudell, James R.; Harris, R. Adron

    2000-01-01

    Ligand-gated ion channels are a target for inhaled anesthetics and alcohols in the central nervous system. The inhibitory strychnine-sensitive glycine and γ-aminobutyric acid type A receptors are positively modulated by anesthetics and alcohols, and site-directed mutagenesis techniques have identified amino acid residues important for the action of volatile anesthetics and alcohols in these receptors. A key question is whether these amino acids are part of an alcohol/anesthetic-binding site. In the present study, we used an alkanethiol anesthetic to covalently label its binding site by mutating selected amino acids to cysteine. We demonstrated that the anesthetic propanethiol, or alternatively, propyl methanethiosulfonate, covalently binds to cysteine residues introduced into a specific second transmembrane site in glycine receptor and γ-aminobutyric acid type A receptor subunits and irreversibly enhances receptor function. Moreover, upon permanent occupation of the site by propyl disulfide, the usual ability of octanol, enflurane, and isoflurane to potentiate the function of the ion channels was lost. This approach provides strong evidence that the actions of anesthetics in these receptors are due to binding at a single site. PMID:10908659

  15. Cell-type specificity of ChIP-predicted transcription factor binding sites

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    Håndstad Tony

    2012-08-01

    Full Text Available Abstract Background Context-dependent transcription factor (TF binding is one reason for differences in gene expression patterns between different cellular states. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq identifies genome-wide TF binding sites for one particular context—the cells used in the experiment. But can such ChIP-seq data predict TF binding in other cellular contexts and is it possible to distinguish context-dependent from ubiquitous TF binding? Results We compared ChIP-seq data on TF binding for multiple TFs in two different cell types and found that on average only a third of ChIP-seq peak regions are common to both cell types. Expectedly, common peaks occur more frequently in certain genomic contexts, such as CpG-rich promoters, whereas chromatin differences characterize cell-type specific TF binding. We also find, however, that genotype differences between the cell types can explain differences in binding. Moreover, ChIP-seq signal intensity and peak clustering are the strongest predictors of common peaks. Compared with strong peaks located in regions containing peaks for multiple transcription factors, weak and isolated peaks are less common between the cell types and are less associated with data that indicate regulatory activity. Conclusions Together, the results suggest that experimental noise is prevalent among weak peaks, whereas strong and clustered peaks represent high-confidence binding events that often occur in other cellular contexts. Nevertheless, 30-40% of the strongest and most clustered peaks show context-dependent regulation. We show that by combining signal intensity with additional data—ranging from context independent information such as binding site conservation and position weight matrix scores to context dependent chromatin structure—we can predict whether a ChIP-seq peak is likely to be present in other cellular contexts.

  16. Site-specific chromatin immunoprecipitation: a selective method to individually analyze neighboring transcription factor binding sites in vivo

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    Schuch Ronaldo

    2012-02-01

    Full Text Available Abstract Background Transcription factors (TFs and their binding sites (TFBSs play a central role in the regulation of gene expression. It is therefore vital to know how the allocation pattern of TFBSs affects the functioning of any particular gene in vivo. A widely used method to analyze TFBSs in vivo is the chromatin immunoprecipitation (ChIP. However, this method in its present state does not enable the individual investigation of densely arranged TFBSs due to the underlying unspecific DNA fragmentation technique. This study describes a site-specific ChIP which aggregates the benefits of both EMSA and in vivo footprinting in only one assay, thereby allowing the individual detection and analysis of single binding motifs. Findings The standard ChIP protocol was modified by replacing the conventional DNA fragmentation, i. e. via sonication or undirected enzymatic digestion (by MNase, through a sequence specific enzymatic digestion step. This alteration enables the specific immunoprecipitation and individual examination of occupied sites, even in a complex system of adjacent binding motifs in vivo. Immunoprecipitated chromatin was analyzed by PCR using two primer sets - one for the specific detection of precipitated TFBSs and one for the validation of completeness of the enzyme digestion step. The method was established exemplary for Sp1 TFBSs within the egfr promoter region. Using this site-specific ChIP, we were able to confirm four previously described Sp1 binding sites within egfr promoter region to be occupied by Sp1 in vivo. Despite the dense arrangement of the Sp1 TFBSs the improved ChIP method was able to individually examine the allocation of all adjacent Sp1 TFBS at once. The broad applicability of this site-specific ChIP could be demonstrated by analyzing these SP1 motifs in both osteosarcoma cells and kidney carcinoma tissue. Conclusions The ChIP technology is a powerful tool for investigating transcription factors in vivo, especially

  17. Progesterone

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    ... cancer. Progesterone is also used to bring on menstruation (period) in women of childbearing age who have ... in the uterus. It works to bring on menstruation by replacing the natural progesterone that some women ...

  18. Astrocytes cultured from specific brain regions differ in their expression of adrenergic binding sites.

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    Ernsberger, P; Iacovitti, L; Reis, D J

    1990-05-28

    We sought to characterize regional heterogeneity of astrocytes using adrenergic receptor sites as cellular markers. Primary cultures made from 6 regions of neonatal rat brain consisted almost exclusively of astrocytes. Membranes from astrocytes cultured 1-3 weeks were prepared for radioligand binding assays of beta- and alpha 2-adrenergic sites using the ligands [3H]dihydroalprenolol and [3H]p-aminoclonidine, respectively. Receptor expression was not affected by time in culture. Astrocytes from different brain regions varied up to 3-fold with respect to number but not affinity for both classes of adrenergic binding site with a rank order of cerebral cortex = superior colliculus greater than hippocampus = ventral midbrain greater than or equal to caudate nucleus greater than or equal to hypothalamus. Binding to beta- and alpha 2-adrenergic receptors was positively correlated across brain regions. Astrocytic receptor binding in each region did not correspond to total receptor levels assessed by quantitative autoradiography. We conclude that: (a) astrocytes are markedly heterogeneous between major brain regions with respect to expression of adrenergic binding sites; (b) regional variations in the density of adrenergic binding sites in brain reflect, in part, local specialization of astrocytes; and (c) a substantial proportion of the adrenergic binding sites in some brain regions may be on astrocytes.

  19. Highly specific sites of prolactin binding in benign and malignant breast tissue.

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    Agarwal, P K; Tandon, S; Agrawal, A K; Kumar, S

    1989-12-01

    An immunocytochemical method involving the application of polyclonal antisera to human prolactin (PRL) followed by a highly sensitive and a modified version of dinitrophenyl (DNP) hapten sandwich staining procedure using anti-DNP IgM monoclonal antibody has been used to detect PRL binding in benign and malignant breast tissue. The technique was applied to 5 microns thick sections of paraffin embedded formalin fixed tissue. Out of 107 breast biopsies 40 were carcinomas, 41 were fibroadenomas, 18 were benign cystic disease and 8 were gynaecomastia. In cases of carcinoma positive staining was observed in 82.5% cases whereas in fibroadenoma the positivity was in 57% cases only. The positive reaction in fibroadenoma was mainly due to the presence of apocrine metaplasia associated with the tumour. Also PRL was present in greater proportion in post menopausal patients as compared to premenopausal cancer patients. These findings suggest the presence of specific PRL binding sites in breast tissue. The staining was restricted to epithelial cells and background staining of the stroma was minimally seen in these cases. Positively stained breast carcinoma may represent an apocrine subset of the carcinoma.

  20. The conformational state of the nucleosome entry–exit site modulates TATA box-specific TBP binding

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    Hieb, Aaron R.; Gansen, Alexander; Böhm, Vera; Langowski, Jörg

    2014-01-01

    The TATA binding protein (TBP) is a critical transcription factor used for nucleating assembly of the RNA polymerase II machinery. TBP binds TATA box elements with high affinity and kinetic stability and in vivo is correlated with high levels of transcription activation. However, since most promoters use less stable TATA-less or TATA-like elements, while also competing with nucleosome occupancy, further mechanistic insight into TBP's DNA binding properties and ability to access chromatin is needed. Using bulk and single-molecule FRET, we find that TBP binds a minimal consensus TATA box as a two-state equilibrium process, showing no evidence for intermediate states. However, upon addition of flanking DNA sequence, we observe non-specific cooperative binding to multiple DNA sites that compete for TATA-box specificity. Thus, we conclude that TBP binding is defined by a branched pathway, wherein TBP initially binds with little sequence specificity and is thermodynamically positioned by its kinetic stability to the TATA box. Furthermore, we observed the real-time access of TBP binding to TATA box DNA located within the DNA entry–exit site of the nucleosome. From these data, we determined salt-dependent changes in the nucleosome conformation regulate TBP's access to the TATA box, where access is highly constrained under physiological conditions, but is alleviated by histone acetylation and TFIIA. PMID:24829456

  1. The conformational state of the nucleosome entry-exit site modulates TATA box-specific TBP binding.

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    Hieb, Aaron R; Gansen, Alexander; Böhm, Vera; Langowski, Jörg

    2014-07-01

    The TATA binding protein (TBP) is a critical transcription factor used for nucleating assembly of the RNA polymerase II machinery. TBP binds TATA box elements with high affinity and kinetic stability and in vivo is correlated with high levels of transcription activation. However, since most promoters use less stable TATA-less or TATA-like elements, while also competing with nucleosome occupancy, further mechanistic insight into TBP's DNA binding properties and ability to access chromatin is needed. Using bulk and single-molecule FRET, we find that TBP binds a minimal consensus TATA box as a two-state equilibrium process, showing no evidence for intermediate states. However, upon addition of flanking DNA sequence, we observe non-specific cooperative binding to multiple DNA sites that compete for TATA-box specificity. Thus, we conclude that TBP binding is defined by a branched pathway, wherein TBP initially binds with little sequence specificity and is thermodynamically positioned by its kinetic stability to the TATA box. Furthermore, we observed the real-time access of TBP binding to TATA box DNA located within the DNA entry-exit site of the nucleosome. From these data, we determined salt-dependent changes in the nucleosome conformation regulate TBP's access to the TATA box, where access is highly constrained under physiological conditions, but is alleviated by histone acetylation and TFIIA. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. Identification of specific binding sites for glucagon-like peptide-1 on the posterior lobe of the rat pituitary.

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    Göke, R; Larsen, P J; Mikkelsen, J D; Sheikh, S P

    1995-08-01

    Glucagon-like peptide-1 (GLP-1) immunoreactivity has been found in autonomic and neuroendocrine brain regions, whereas only limited data are available regarding the characterization and localization of brain GLP-1 receptors. In the present study, using quantitative in vitro autoradiography, a high density of specific binding sites for GLP-1 was characterized on sections of the posterior pituitary lobe of the rat. Low specific binding of radiolabeled GLP-1 was found in the anterior lobe and no specific binding in the intermediate lobe. To examine the specificity of GLP-1 binding sites, sections of the posterior lobe were incubated with radiolabeled GLP-1 in the presence of various peptides. Radiolabeled [Tyr39]exendin-4, a specific GLP-1 agonist, bound to these receptor sites with the same affinity as GLP-1, while glucagon and vasoactive intestinal peptide (VIP) were unable to displace 125I-GLP-1. Both unlabeled exendin-4 and GLP-1 inhibited this binding with equally high affinity. Using 125I-[Tyr39]exendin-4 as radiolabel, the concentration of biding sites was found to be 7.8 +/- 0.4 fmol/mg tissue. Further analysis of the binding data from experiments with tissue slices revealed the presence of high and low affinity binding sites. In experiments with unlabeled [Tyr39]exendin-4, the KdS were 6.2 +/- 1.4 x 10(-12) and 9.3 +/- 1.5 x 10(-10) M, respectively, and in experiments with unlabeled GLP-1, 3.4 +/- 1.8 x 10(-12) and 5.9 +/- 1.5 x 10(-10) M, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Specific /sup 3/H-DMCM binding to a non-benzodiazepine binding site after silver ion treatment of rat brain membranes

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    Honore, T.; Nielsen, M.; Braestrup, C.

    1984-11-26

    Specific binding of the BZ-receptor ligand /sup 3/H-DMCM to rat cortical membranes was dramatically enhanced by preincubation of the homogenate with 0.1 mM silver (Ag/sup +/) nitrate. The binding was completely inhibited by midazolam. Nevertheless, the pharmacological specificity of the Ag/sup +/-enhanced /sup 3/H-DMCM binding was different from that of BZ-receptors. Furthermore, the B/sub max/ value, the regional distribution and the molecular target size determined by radiation inactivation analysis of the Ag/sup +/-enhanced binding site were different from those of BZ-receptors. The results indicate that Ag/sup +/-enhanced /sup 3/H-DMCM binding represent a high affinity metal complex formation between /sup 3/H-DMCM and an unknown brain specific protein of approximately 100,000 daltons molecular weight. 11 references, 3 figures, 4 tables.

  4. Comprehensive Identification and Annotation of Cell Type-Specific and Ubiquitous CTCF-Binding Sites in the Human Genome

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    Shu, Wenjie; Bo, Xiaochen; Wang, Shengqi

    2012-01-01

    Chromatin insulators are DNA elements that regulate the level of gene expression either by preventing gene silencing through the maintenance of heterochromatin boundaries or by preventing gene activation by blocking interactions between enhancers and promoters. CCCTC-binding factor (CTCF), a ubiquitously expressed 11-zinc-finger DNA-binding protein, is the only protein implicated in the establishment of insulators in vertebrates. While CTCF has been implicated in diverse regulatory functions, CTCF has only been studied in a limited number of cell types across human genome. Thus, it is not clear whether the identified cell type-specific differences in CTCF-binding sites are functionally significant. Here, we identify and characterize cell type-specific and ubiquitous CTCF-binding sites in the human genome across 38 cell types designated by the Encyclopedia of DNA Elements (ENCODE) consortium. These cell type-specific and ubiquitous CTCF-binding sites show uniquely versatile transcriptional functions and characteristic chromatin features. In addition, we confirm the insulator barrier function of CTCF-binding and explore the novel function of CTCF in DNA replication. These results represent a critical step toward the comprehensive and systematic understanding of CTCF-dependent insulators and their versatile roles in the human genome. PMID:22829947

  5. Tb3+-cleavage assays reveal specific Mg2+ binding sites necessary to pre-fold the btuB riboswitch for AdoCbl binding

    Science.gov (United States)

    Choudhary, Pallavi K.; Gallo, Sofia; Sigel, Roland K. O.

    2017-03-01

    Riboswitches are RNA elements that bind specific metabolites in order to regulate the gene expression involved in controlling the cellular concentration of the respective molecule or ion. Ligand recognition is mostly facilitated by Mg2+ mediated pre-organization of the riboswitch to an active tertiary fold. To predict these specific Mg2+ induced tertiary interactions of the btuB riboswitch from E. coli, we here report Mg2+ binding pockets in its aptameric part in both, the ligand-free and the ligand-bound form. An ensemble of weak and strong metal ion binding sites distributed over the entire aptamer was detected by terbium(III) cleavage assays, Tb3+ being an established Mg2+ mimic. Interestingly many of the Mn+ (n = 2 or 3) binding sites involve conserved bases within the class of coenzyme B12-binding riboswitches. Comparison with the published crystal structure of the coenzyme B12 riboswitch of S. thermophilum aided in identifying a common set of Mn+ binding sites that might be crucial for tertiary interactions involved in the organization of the aptamer. Our results suggest that Mn+ binding at strategic locations of the btuB riboswitch indeed facilitates the assembly of the binding pocket needed for ligand recognition. Binding of the specific ligand, coenzyme B12 (AdoCbl), to the btuB aptamer does however not lead to drastic alterations of these Mn+ binding cores, indicating the lack of a major rearrangement within the three-dimensional structure of the RNA. This finding is strengthened by Tb3+ mediated footprints of the riboswitch's structure in its ligand-free and ligand-bound state indicating that AdoCbl indeed induces local changes rather than a global structural rearrangement.

  6. Role of site-specific binding to plasma albumin in drug availability to brain.

    Science.gov (United States)

    Mandula, Haritha; Parepally, Jagan Mohan R; Feng, Rose; Smith, Quentin R

    2006-05-01

    Many studies have reported greater drug uptake into brain than that predicted based upon existing models using the free fraction (f(u)) of drug in arterial serum. To explain this difference, circulating plasma proteins have been suggested to interact with capillary membrane in vivo to produce a conformational change that favors net drug dissociation and elevation of f(u). Albumin, the principal binding protein in plasma, has two main drug binding sites, Sudlow I and II. We tested this hypothesis using drugs that bind selectively to either site I (warfarin) or site II (ibuprofen), as well as mixed ligands that have affinity for both sites (tolbutamide and valproate). Brain uptake was determined in the presence and absence of albumin using the in situ rat brain perfusion technique. Unidirectional brain uptake transfer constants (K(in)) were measured and compared with those predicted using the modified Kety-Crone-Renkin model: K(in) = F(1-e(-f(u) x PS(u)/F)), where F is perfusion flow and PS(u) is the permeability-surface area product to free drug of brain capillaries. The results demonstrated good agreement between measured and predicted K(in) over a 100-fold range in perfusion fluid albumin concentration using albumin from three different species (i.e., human, bovine, and rat), as well as whole-rat serum. K(in) decreased in the presence of albumin in direct proportion to perfusion fluid f(u) with constant PS(u). The results show that brain uptake of selected Sudlow site I and II ligands matches that predicted by the modified Kety-Crone-Renkin model with no evidence for enhanced dissociation.

  7. New binding site on common molecular scaffold provides HERG channel specificity of scorpion toxin BeKm-1

    DEFF Research Database (Denmark)

    Korolkova, Yuliya V; Bocharov, Eduard V; Angelo, Kamilla

    2002-01-01

    resolved by NMR consists of a short alpha-helix and a triple-stranded antiparallel beta-sheet. By toxin mutagenesis study we identified the residues that are important for the binding of BeKm-1 to the human ERG K+ (HERG) channel. The most critical residues (Tyr-11, Lys-18, Arg-20, Lys-23) are located...... in the alpha-helix and following loop whereas the "traditional" functional site of other short scorpion toxins is formed by residues from the beta-sheet. Thus the unique location of the binding site of BeKm-1 provides its specificity toward the HERG channel....

  8. Primate-specific Melanoma Antigen-A11 Regulates Isoform-specific Human Progesterone Receptor-B Transactivation*

    Science.gov (United States)

    Su, Shifeng; Blackwelder, Amanda J.; Grossman, Gail; Minges, John T.; Yuan, Lingwen; Young, Steven L.; Wilson, Elizabeth M.

    2012-01-01

    Progesterone acting through the progesterone receptor (PR) and its coregulators prepares the human endometrium for receptivity to embryo implantation and maintains pregnancy. The menstrual cycle-dependent expression of melanoma antigen-A11 (MAGE-11) in the mid-secretory human endometrium suggested a novel function in human PR signaling. Here we show that MAGE-11 is an isoform-specific coregulator responsible for the greater transcriptional activity of human PR-B relative to PR-A. PR was recruited to progesterone response regions of progesterone-regulated FK506-binding protein 5 (FKBP5) immunophilin and small Ras family G protein cell growth inhibitor RASD1 genes. Expression of MAGE-11 lentivirus shRNA in human endometrial Ishikawa cells expressing PR-B showed that MAGE-11 is required for isoform-specific PR-B up-regulation of FKBP5. In contrast, MAGE-11 was not required for progesterone up-regulation of RASD1 in endometrial cells expressing the PR-A/B heterodimer. Target gene specificity of PR-B depended on the synergistic actions of MAGE-11 and p300 mediated by the unique PR-B NH2-terminal 110LLXXVLXXLL119 motif that interacts with the MAGE-11 F-box region in a phosphorylation- and ubiquitinylation-dependent manner. A progesterone-dependent mechanism is proposed in which MAGE-11 and p300 increase PR-B up-regulation of the FKBP5 gene. MAGE-11 down-regulates PR-B, similar to the effects of progesterone, and interacts with FKBP5 to stabilize a complex with PR-B. We conclude that the coregulator function of MAGE-11 extends to isoform-specific regulation of PR-B during the cyclic development of the human endometrium. PMID:22891251

  9. Importance of the Sequence-Directed DNA Shape for Specific Binding Site Recognition by the Estrogen-Related Receptor

    Directory of Open Access Journals (Sweden)

    Kareem Mohideen-Abdul

    2017-06-01

    Full Text Available Most nuclear receptors (NRs bind DNA as dimers, either as hetero- or as homodimers on DNA sequences organized as two half-sites with specific orientation and spacing. The dimerization of NRs on their cognate response elements (REs involves specific protein–DNA and protein–protein interactions. The estrogen-related receptor (ERR belongs to the steroid hormone nuclear receptor (SHR family and shares strong similarity in its DNA-binding domain (DBD with that of the estrogen receptor (ER. In vitro, ERR binds with high affinity inverted repeat REs with a 3-bps spacing (IR3, but in vivo, it preferentially binds to single half-site REs extended at the 5′-end by 3 bp [estrogen-related response element (ERREs], thus explaining why ERR was often inferred as a purely monomeric receptor. Since its C-terminal ligand-binding domain is known to homodimerize with a strong dimer interface, we investigated the binding behavior of the isolated DBDs to different REs using electrophoretic migration, multi-angle static laser light scattering (MALLS, non-denaturing mass spectrometry, and nuclear magnetic resonance. In contrast to ER DBD, ERR DBD binds as a monomer to EREs (IR3, such as the tff1 ERE-IR3, but we identified a DNA sequence composed of an extended half-site embedded within an IR3 element (embedded ERRE/IR3, where stable dimer binding is observed. Using a series of chimera and mutant DNA sequences of ERREs and IR3 REs, we have found the key determinants for the binding of ERR DBD as a dimer. Our results suggest that the sequence-directed DNA shape is more important than the exact nucleotide sequence for the binding of ERR DBD to DNA as a dimer. Our work underlines the importance of the shape-driven DNA readout mechanisms based on minor groove recognition and electrostatic potential. These conclusions may apply not only to ERR but also to other members of the SHR family, such as androgen or glucocorticoid, for which a strong well-conserved half-site

  10. Importance of the Sequence-Directed DNA Shape for Specific Binding Site Recognition by the Estrogen-Related Receptor

    Science.gov (United States)

    Mohideen-Abdul, Kareem; Tazibt, Karima; Bourguet, Maxime; Hazemann, Isabelle; Lebars, Isabelle; Takacs, Maria; Cianférani, Sarah; Klaholz, Bruno P.; Moras, Dino; Billas, Isabelle M. L.

    2017-01-01

    Most nuclear receptors (NRs) bind DNA as dimers, either as hetero- or as homodimers on DNA sequences organized as two half-sites with specific orientation and spacing. The dimerization of NRs on their cognate response elements (REs) involves specific protein–DNA and protein–protein interactions. The estrogen-related receptor (ERR) belongs to the steroid hormone nuclear receptor (SHR) family and shares strong similarity in its DNA-binding domain (DBD) with that of the estrogen receptor (ER). In vitro, ERR binds with high affinity inverted repeat REs with a 3-bps spacing (IR3), but in vivo, it preferentially binds to single half-site REs extended at the 5′-end by 3 bp [estrogen-related response element (ERREs)], thus explaining why ERR was often inferred as a purely monomeric receptor. Since its C-terminal ligand-binding domain is known to homodimerize with a strong dimer interface, we investigated the binding behavior of the isolated DBDs to different REs using electrophoretic migration, multi-angle static laser light scattering (MALLS), non-denaturing mass spectrometry, and nuclear magnetic resonance. In contrast to ER DBD, ERR DBD binds as a monomer to EREs (IR3), such as the tff1 ERE-IR3, but we identified a DNA sequence composed of an extended half-site embedded within an IR3 element (embedded ERRE/IR3), where stable dimer binding is observed. Using a series of chimera and mutant DNA sequences of ERREs and IR3 REs, we have found the key determinants for the binding of ERR DBD as a dimer. Our results suggest that the sequence-directed DNA shape is more important than the exact nucleotide sequence for the binding of ERR DBD to DNA as a dimer. Our work underlines the importance of the shape-driven DNA readout mechanisms based on minor groove recognition and electrostatic potential. These conclusions may apply not only to ERR but also to other members of the SHR family, such as androgen or glucocorticoid, for which a strong well-conserved half-site is

  11. Prothrombin requires two sequential metal-dependent conformational transitions to bind phospholipid. Conformation-specific antibodies directed against the phospholipid-binding site on prothrombin

    Energy Technology Data Exchange (ETDEWEB)

    Borowski, M.; Furie, B.C.; Bauminger, S.; Furie, B.

    1986-11-15

    Prothrombin is a gamma-carboxyglutamic acid-containing protein that binds to phospholipid vesicles in the presence of calcium ions after undergoing a metal ion-induced conformational transition. To integrate recent data into a scheme that is compatible with our knowledge of prothrombin-metal interaction, we have proposed a new model of prothrombin structure. In this model prothrombin undergoes two metal-dependent conformational transitions: PT----PT'----PT*. The first transition is not cation-specific, but the second transition is metal-selective for Ca(II), Sr(II), or Ba(II). Only the PT* conformer binds to phospholipid surfaces. To test this model, anti-prothrombin antibodies that only bind to prothrombin in the presence of Ca(II) but not Mg(II) (PT*-specific) were isolated, and termed anti-prothrombin X Ca(II)-specific. Half-maximal binding of antibody to prothrombin was observed at 0.1 mM CaCl/sub 2/ or 1 mM SrCl/sub 2/, but no binding was observed with Mg(II), Mn(II), or Ba(II). However, prothrombin in the presence of both Mg(II)/Ba(II) or Mn(II)/Ba(II) demonstrated significant interaction with the antibody. Prothrombin binding to phospholipid vesicles was inhibited by the anti-prothrombin X Ca(II)-specific antibody or its Fab fragment, but was not inhibited by anti-prothrombin X Mg(II) antibody or its Fab fragment directed at the PT' conformer. These results support this three-state model for prothrombin. The metal specificity characteristic of prothrombin-phospholipid interaction is a property required for the expression of the phospholipid-binding site in the binary prothrombin-metal complex.

  12. Metal Ion Binding at the Catalytic Site Induces Widely Distributed Changes in a Sequence Specific Protein-DNA Complex.

    Science.gov (United States)

    Sinha, Kaustubh; Sangani, Sahil S; Kehr, Andrew D; Rule, Gordon S; Jen-Jacobson, Linda

    2016-11-08

    Metal ion cofactors can alter the energetics and specificity of sequence specific protein-DNA interactions, but it is unknown if the underlying effects on structure and dynamics are local or dispersed throughout the protein-DNA complex. This work uses EcoRV endonuclease as a model, and catalytically inactive lanthanide ions, which replace the Mg 2+ cofactor. Nuclear magnetic resonance (NMR) titrations indicate that four Lu 3+ or two La 3+ cations bind, and two new crystal structures confirm that Lu 3+ binding is confined to the active sites. NMR spectra show that the metal-free EcoRV complex with cognate (GATATC) DNA is structurally distinct from the nonspecific complex, and that metal ion binding sites are not assembled in the nonspecific complex. NMR chemical shift perturbations were determined for 1 H- 15 N amide resonances, for 1 H- 13 C Ile-δ-CH 3 resonances, and for stereospecifically assigned Leu-δ-CH 3 and Val-γ-CH 3 resonances. Many chemical shifts throughout the cognate complex are unperturbed, so metal binding does not induce major conformational changes. However, some large perturbations of amide and side chain methyl resonances occur as far as 34 Å from the metal ions. Concerted changes in specific residues imply that local effects of metal binding are propagated via a β-sheet and an α-helix. Both amide and methyl resonance perturbations indicate changes in the interface between subunits of the EcoRV homodimer. Bound metal ions also affect amide hydrogen exchange rates for distant residues, including a distant subdomain that contacts DNA phosphates and promotes DNA bending, showing that metal ions in the active sites, which relieve electrostatic repulsion between protein and DNA, cause changes in slow dynamics throughout the complex.

  13. Metal Ion Binding at the Catalytic Site Induces Widely Distributed Changes in a Sequence Specific Protein–DNA Complex

    Science.gov (United States)

    2016-01-01

    Metal ion cofactors can alter the energetics and specificity of sequence specific protein–DNA interactions, but it is unknown if the underlying effects on structure and dynamics are local or dispersed throughout the protein–DNA complex. This work uses EcoRV endonuclease as a model, and catalytically inactive lanthanide ions, which replace the Mg2+ cofactor. Nuclear magnetic resonance (NMR) titrations indicate that four Lu3+ or two La3+ cations bind, and two new crystal structures confirm that Lu3+ binding is confined to the active sites. NMR spectra show that the metal-free EcoRV complex with cognate (GATATC) DNA is structurally distinct from the nonspecific complex, and that metal ion binding sites are not assembled in the nonspecific complex. NMR chemical shift perturbations were determined for 1H–15N amide resonances, for 1H–13C Ile-δ-CH3 resonances, and for stereospecifically assigned Leu-δ-CH3 and Val-γ-CH3 resonances. Many chemical shifts throughout the cognate complex are unperturbed, so metal binding does not induce major conformational changes. However, some large perturbations of amide and side chain methyl resonances occur as far as 34 Å from the metal ions. Concerted changes in specific residues imply that local effects of metal binding are propagated via a β-sheet and an α-helix. Both amide and methyl resonance perturbations indicate changes in the interface between subunits of the EcoRV homodimer. Bound metal ions also affect amide hydrogen exchange rates for distant residues, including a distant subdomain that contacts DNA phosphates and promotes DNA bending, showing that metal ions in the active sites, which relieve electrostatic repulsion between protein and DNA, cause changes in slow dynamics throughout the complex. PMID:27786446

  14. Eubacterial SpoVG homologs constitute a new family of site-specific DNA-binding proteins.

    Directory of Open Access Journals (Sweden)

    Brandon L Jutras

    Full Text Available A site-specific DNA-binding protein was purified from Borrelia burgdorferi cytoplasmic extracts, and determined to be a member of the highly conserved SpoVG family. This is the first time a function has been attributed to any of these ubiquitous bacterial proteins. Further investigations into SpoVG orthologues indicated that the Staphylococcus aureus protein also binds DNA, but interacts preferentially with a distinct nucleic acid sequence. Site-directed mutagenesis and domain swapping between the S. aureus and B. burgdorferi proteins identified that a 6-residue stretch of the SpoVG α-helix contributes to DNA sequence specificity. Two additional, highly conserved amino acid residues on an adjacent β-sheet are essential for DNA-binding, apparently by contacts with the DNA phosphate backbone. Results of these studies thus identified a novel family of bacterial DNA-binding proteins, developed a model of SpoVG-DNA interactions, and provide direction for future functional studies on these wide-spread proteins.

  15. SEARCHPATTOOL: a new method for mining the most specific frequent patterns for binding sites with application to prokaryotic DNA sequences

    Directory of Open Access Journals (Sweden)

    Nason Martha

    2007-09-01

    Full Text Available Abstract Background Computational methods to predict transcription factor binding sites (TFBS based on exhaustive algorithms are guaranteed to find the best patterns but are often limited to short ones or impose some constraints on the pattern type. Many patterns for binding sites in prokaryotic species are not well characterized but are known to be large, between 16–30 base pairs (bp and contain at least 2 conserved bases. The length of prokaryotic species promoters (about 400 bp and our interest in studying a small set of genes that could be a cluster of co-regulated genes from microarray experiments led to the development of a new exhaustive algorithm targeting these large patterns. Results We present Searchpattool, a new method to search for and select the most specific (conservative frequent patterns. This method does not impose restrictions on the density or the structure of the pattern. The best patterns (motifs are selected using several statistics, including a new application of a z-score based on the number of matching sequences. We compared Searchpattool against other well known algorithms on a Bacillus subtilis group of 14 input sequences and found that in our experiments Searchpattool always performed the best based on performance scores. Conclusion Searchpattool is a new method for pattern discovery relative to transcription factor binding sites for species or genes with short promoters. It outputs the most specific significant patterns and helps the biologist to choose the best candidates.

  16. Binding of the mannose-specific lectin, Griffithsin, to HIV-1 gp120 exposes the CD4-binding site

    CSIR Research Space (South Africa)

    Alexandre, Kabamba B

    2011-09-01

    Full Text Available The glycans on HIV-1 gp120 play an important role in shielding neutralization-sensitive epitopes from antibody recognition. They also serve as targets for lectins that bind mannose-rich glycans. In this study, the authors investigated...

  17. Polyhydroxylated [60]fullerene binds specifically to functional recognition sites on a monomeric and a dimeric ubiquitin

    Science.gov (United States)

    Zanzoni, Serena; Ceccon, Alberto; Assfalg, Michael; Singh, Rajesh K.; Fushman, David; D'Onofrio, Mariapina

    2015-04-01

    The use of nanoparticles (NPs) in biomedical applications requires an in-depth understanding of the mechanisms by which NPs interact with biomolecules. NPs associating with proteins may interfere with protein-protein interactions and affect cellular communication pathways, however the impact of NPs on biomolecular recognition remains poorly characterized. In this respect, particularly relevant is the study of NP-induced functional perturbations of proteins implicated in the regulation of key biochemical pathways. Ubiquitin (Ub) is a prototypical protein post-translational modifier playing a central role in numerous essential biological processes. To contribute to the understanding of the interactions between this universally distributed biomacromolecule and NPs, we investigated the adsorption of polyhydroxylated [60]fullerene on monomeric Ub and on a minimal polyubiquitin chain in vitro at atomic resolution. Site-resolved chemical shift and intensity perturbations of Ub's NMR signals, together with 15N spin relaxation rate changes, exchange saturation transfer effects, and fluorescence quenching data were consistent with the reversible formation of soluble aggregates incorporating fullerenol clusters. The specific interaction epitopes were identified, coincident with functional recognition sites in a monomeric and lysine48-linked dimeric Ub. Fullerenol appeared to target the open state of the dynamic structure of a dimeric Ub according to a conformational selection mechanism. Importantly, the protein-NP association prevented the enzyme-catalyzed synthesis of polyubiquitin chains. Our findings provide an experiment-based insight into protein/fullerenol recognition, with implications in functional biomolecular communication, including regulatory protein turnover, and for the opportunity of therapeutic intervention in Ub-dependent cellular pathways.The use of nanoparticles (NPs) in biomedical applications requires an in-depth understanding of the mechanisms by which

  18. Characterisation of a GII-4 norovirus variant-specific surface-exposed site involved in antibody binding

    Directory of Open Access Journals (Sweden)

    Gray Jim J

    2009-09-01

    Full Text Available Abstract Background The human noroviruses are a highly diverse group of viruses with a single-stranded RNA genome encoding a single major structural protein (VP1, which has a hypervariable domain (P2 domain as the most exposed part of the virion. The noroviruses are classified on the basis of nucleotide sequence diversity in the VP1-encoding ORF2 gene, which divides the majority of human noroviruses into two genogroups (GI and GII. GII-4 noroviruses are the major aetiological agent of outbreaks of gastroenteritis around the world. During a winter season the diversity among the GII-4 noroviruses has been shown to fluctuate, driving the appearance of new virus variants in the population. We have previously shown that sequence data and in silico modelling experiments suggest there are two surface-exposed sites (site A and site B in the hypervariable P2 domain. We predict these sites may form a functional variant-specific epitope that evolves under selective pressure from the host immune response and gives rise to antibody escape mutants. Results In this paper, we describe the construction of recombinant baculoviruses to express VLPs representing one pre-epidemic and one epidemic variant of GII-4 noroviruses, and the production of monoclonal antibodies against them. We use these novel reagents to provide evidence that site A and site B form a conformational, variant-specific, surface-exposed site on the GII-4 norovirus capsid that is involved in antibody binding. Conclusion As predicted by our earlier study, significant amino acid changes at site A and site B give rise to GII-4 norovirus epidemic variants that are antibody escape mutants.

  19. Progesterone as an immunomodulatory molecule.

    Science.gov (United States)

    Szekeres-Bartho, J; Barakonyi, A; Par, G; Polgar, B; Palkovics, T; Szereday, L

    2001-06-01

    Increased progesterone sensitivity of pregnancy lymphocytes is due to activation-induced appearance of progesterone binding sites in the lymphocytes. Following recognition of fetally derived antigens gamma/delta TCR+ cells develop progesterone receptors. Progesterone binding results in the synthesis of a mediator protein named the progesterone-induced blocking factor (PIBF). PIBF by acting on the phospholipase A2 enzyme interferes with arachidonic acid metabolism, induces a Th2 biased immune response, and by controlling NK activity exerts an anti-abortive effect.

  20. Binding of the mannose-specific lectin, griffithsin, to HIV-1 gp120 exposes the CD4-binding site

    CSIR Research Space (South Africa)

    Alexandre, Kabamba B

    2011-09-01

    Full Text Available :359-69. 37 8. Burrer, R., S. Haessig-Einius, A. M. Aubertin, and C. Moog. 2005. 38 Neutralizing as well as non-neutralizing polyclonal immunoglobulin (Ig)G 39 from infected patients capture HIV-1 via antibodies directed against the 40 principal.... Moore, G. D. Tomaras, C. K. 5 Wibmer, A. Puren, A. DeCamp, P. B. Gilbert, B. Wood, D. C. Montefiori, 6 J. M. Binley, G. M. Shaw, B. F. Haynes, J. R. Mascola, and L. Morris. 7 2009. Antibody specificities associated with neutralization breadth...

  1. Cardiac myosin binding protein C phosphorylation affects cross-bridge cycle's elementary steps in a site-specific manner.

    Directory of Open Access Journals (Sweden)

    Li Wang

    Full Text Available Based on our recent finding that cardiac myosin binding protein C (cMyBP-C phosphorylation affects muscle contractility in a site-specific manner, we further studied the force per cross-bridge and the kinetic constants of the elementary steps in the six-state cross-bridge model in cMyBP-C mutated transgenic mice for better understanding of the influence of cMyBP-C phosphorylation on contractile functions. Papillary muscle fibres were dissected from cMyBP-C mutated mice of ADA (Ala273-Asp282-Ala302, DAD (Asp273-Ala282-Asp302, SAS (Ser273-Ala282-Ser302, and t/t (cMyBP-C null genotypes, and the results were compared to transgenic mice expressing wide-type (WT cMyBP-C. Sinusoidal analyses were performed with serial concentrations of ATP, phosphate (Pi, and ADP. Both t/t and DAD mutants significantly reduced active tension, force per cross-bridge, apparent rate constant (2πc, and the rate constant of cross-bridge detachment. In contrast to the weakened ATP binding and enhanced Pi and ADP release steps in t/t mice, DAD mice showed a decreased ADP release without affecting the ATP binding and the Pi release. ADA showed decreased ADP release, and slightly increased ATP binding and cross-bridge detachment steps, whereas SAS diminished the ATP binding step and accelerated the ADP release step. t/t has the broadest effects with changes in most elementary steps of the cross-bridge cycle, DAD mimics t/t to a large extent, and ADA and SAS predominantly affect the nucleotide binding steps. We conclude that the reduced tension production in DAD and t/t is the result of reduced force per cross-bridge, instead of the less number of strongly attached cross-bridges. We further conclude that cMyBP-C is an allosteric activator of myosin to increase cross-bridge force, and its phosphorylation status modulates the force, which is regulated by variety of protein kinases.

  2. Replicative senescence is associated with nuclear reorganization and with DNA methylation at specific transcription factor binding sites.

    Science.gov (United States)

    Hänzelmann, Sonja; Beier, Fabian; Gusmao, Eduardo G; Koch, Carmen M; Hummel, Sebastian; Charapitsa, Iryna; Joussen, Sylvia; Benes, Vladimir; Brümmendorf, Tim H; Reid, George; Costa, Ivan G; Wagner, Wolfgang

    2015-01-01

    Primary cells enter replicative senescence after a limited number of cell divisions. This process needs to be considered in cell culture experiments, and it is particularly important for regenerative medicine. Replicative senescence is associated with reproducible changes in DNA methylation (DNAm) at specific sites in the genome. The mechanism that drives senescence-associated DNAm changes remains unknown - it may involve stochastic DNAm drift due to imperfect maintenance of epigenetic marks or it is directly regulated at specific sites in the genome. In this study, we analyzed the reorganization of nuclear architecture and DNAm changes during long-term culture of human fibroblasts and mesenchymal stromal cells (MSCs). We demonstrate that telomeres shorten and shift towards the nuclear center at later passages. In addition, DNAm profiles, either analyzed by MethylCap-seq or by 450k IlluminaBeadChip technology, revealed consistent senescence-associated hypermethylation in regions associated with H3K27me3, H3K4me3, and H3K4me1 histone marks, whereas hypomethylation was associated with chromatin containing H3K9me3 and lamina-associated domains (LADs). DNA hypermethylation was significantly enriched in the vicinity of genes that are either up- or downregulated at later passages. Furthermore, specific transcription factor binding motifs (e.g. EGR1, TFAP2A, and ETS1) were significantly enriched in differentially methylated regions and in the promoters of differentially expressed genes. Senescence-associated DNA hypermethylation occurs at specific sites in the genome and reflects functional changes in the course of replicative senescence. These results indicate that tightly regulated epigenetic modifications during long-term culture contribute to changes in nuclear organization and gene expression.

  3. Perspectives for on-site monitoring of progesterone

    NARCIS (Netherlands)

    Posthuma-Trumpie, G.A.; Amerongen, van A.; Korf, J.; Berkel, van W.J.H.

    2009-01-01

    The steroid hormone progesterone is the primary biomarker of the reproductive status of female mammals. Current techniques of monitoring progesterone are based predominantly on (enzyme) immunoassays, but these are too expensive to be affordable in daily screening programmes because of their

  4. Progesterone stimulates adipocyte determination and differentiation 1/sterol regulatory element-binding protein 1c gene expression. potential mechanism for the lipogenic effect of progesterone in adipose tissue.

    Science.gov (United States)

    Lacasa, D; Le Liepvre, X; Ferre, P; Dugail, I

    2001-04-13

    Fatty acid synthase (FAS), a nutritionally regulated lipogenic enzyme, is transcriptionally controlled by ADD1/SREBP1c (adipocyte determination and differentiation 1/sterol regulatory element-binding protein 1c), through insulin-mediated stimulation of ADD1/SREBP1c expression. Progesterone exerts lipogenic effects on adipocytes, and FAS is highly induced in breast tumor cell lines upon progesterone treatment. We show here that progesterone up-regulates ADD1/SREBP1c expression in the MCF7 breast cancer cell line and the primary cultured preadipocyte from rat parametrial adipose tissue. In MCF7, progesterone induced ADD1/SREBP1c and Metallothionein II (a well known progesterone-regulated gene) mRNAs, with comparable potency. In preadipocytes, progesterone increased ADD1/SREBP1c mRNA dose-dependently, but not SREBP1a or SREBP2. Run-on experiments demonstrated that progesterone action on ADD1/SREBP1c was primarily at the transcriptional level. The membrane-bound and mature nuclear forms of ADD1/SREBP1 protein accumulated in preadipocytes cultured with progesterone, and FAS induction could be abolished by adenovirus-mediated overexpression of a dominant negative form of ADD1/SREBP1 in these cells. Finally, in the presence of insulin, progesterone was unable to up-regulate ADD1/SREBP1c mRNA in preadipocytes, whereas its effect was restored after 24 h of insulin deprivation. Together these results demonstrate that ADD1/SREBP1c is controlled by progesterone, which, like insulin, acts by increasing ADD1/SREBP1c gene transcription. This provides a potential mechanism for the lipogenic actions of progesterone on adipose tissue.

  5. DOF-binding sites additively contribute to guard cell-specificity of AtMYB60 promoter

    Directory of Open Access Journals (Sweden)

    Cominelli Eleonora

    2011-11-01

    Full Text Available Abstract Background We previously demonstrated that the Arabidopsis thaliana AtMYB60 protein is an R2R3MYB transcription factor required for stomatal opening. AtMYB60 is specifically expressed in guard cells and down-regulated at the transcriptional levels by the phytohormone ABA. Results To investigate the molecular mechanisms governing AtMYB60 expression, its promoter was dissected through deletion and mutagenesis analyses. By studying different versions of AtMYB60 promoter::GUS reporter fusions in transgenic plants we were able to demonstrate a modular organization for the AtMYB60 promoter. Particularly we defined: a minimal promoter sufficient to confer guard cell-specific activity to the reporter gene; the distinct roles of different DOF-binding sites organised in a cluster in the minimal promoter in determining guard cell-specific expression; the promoter regions responsible for the enhancement of activity in guard cells; a promoter region responsible for the negative transcriptional regulation by ABA. Moreover from the analysis of single and multiple mutants we could rule out the involvement of a group of DOF proteins, known as CDFs, already characterised for their involvement in flowering time, in the regulation of AtMYB60 expression. Conclusions These findings shed light on the regulation of gene expression in guard cells and provide new promoter modules as useful tools for manipulating gene expression in guard cells, both for physiological studies and future biotechnological applications.

  6. Specificity and versatility of substrate binding sites in four catalytic domains of human N-terminal acetyltransferases.

    Directory of Open Access Journals (Sweden)

    Cédric Grauffel

    Full Text Available Nt-acetylation is among the most common protein modifications in eukaryotes. Although thought for a long time to protect proteins from degradation, the role of Nt-acetylation is still debated. It is catalyzed by enzymes called N-terminal acetyltransferases (NATs. In eukaryotes, several NATs, composed of at least one catalytic domain, target different substrates based on their N-terminal sequences. In order to better understand the substrate specificity of human NATs, we investigated in silico the enzyme-substrate interactions in four catalytic subunits of human NATs (Naa10p, Naa20p, Naa30p and Naa50p. To date hNaa50p is the only human subunit for which X-ray structures are available. We used the structure of the ternary hNaa50p/AcCoA/MLG complex and a structural model of hNaa10p as a starting point for multiple molecular dynamics simulations of hNaa50p/AcCoA/substrate (substrate=MLG, EEE, MKG, hNaa10p/AcCoA/substrate (substrate=MLG, EEE. Nine alanine point-mutants of the hNaa50p/AcCoA/MLG complex were also simulated. Homology models of hNaa20p and hNaa30p were built and compared to hNaa50p and hNaa10p. The simulations of hNaa50p/AcCoA/MLG reproduce the interactions revealed by the X-ray data. We observed strong hydrogen bonds between MLG and tyrosines 31, 138 and 139. Yet the tyrosines interacting with the substrate's backbone suggest that their role in specificity is limited. This is confirmed by the simulations of hNaa50p/AcCoA/EEE and hNaa10p/AcCoA/MLG, where these hydrogen bonds are still observed. Moreover these tyrosines are all conserved in hNaa20p and hNaa30p. Other amino acids tune the specificity of the S1' sites that is different for hNaa10p (acidic, hNaa20p (hydrophobic/basic, hNaa30p (basic and hNaa50p (hydrophobic. We also observe dynamic correlation between the ligand binding site and helix [Formula: see text] that tightens under substrate binding. Finally, by comparing the four structures we propose maps of the peptide

  7. Selectivity in progesterone and androgen receptor binding of progestagens used in oral contraceptives

    Energy Technology Data Exchange (ETDEWEB)

    Kloosterboer, H.J.; Vonk-Noordegraaf, C.A.; Turpijn, E.W.

    1988-09-01

    The relative binding affinities (RBAs) of four progestational compounds (norethisterone, levonorgestrel, 3-keto-desogestrel and gestodene) for the human progesterone and androgen receptors were measured in MCF-7 cytosol and intact MCF-7 cells. For the binding to the progesterone receptor, both Org 2058 and Org 3236 (or 3-keto-desogestrel) were used as labelled ligands. The following ranking (low to high) for the RBA of the nuclear (intact cells) progesterone receptor irrespective of the ligand used is found: norethisterone much less than levonorgestrel less than 3-keto-destogestrel less than gestodene. The difference between the various progestagens is significant with the exception of that between 3-keto-desogestrel and gestodene, when Org 2058 is used as ligand. For the cytosolic progesterone receptor, the same order is found with the exception that similar RBAs are found for gestodene and 3-keto-desogestrel. The four progestagens clearly differ with respect to binding to the androgen receptor using dihydrotestosterone as labelled ligand in intact cells; the ranking (low to high) is: norethisterone less than 3 keto-desogestrel less than levonorgestrel and gestodene. The difference between 3-keto-desogestrel and levonorgestrel or gestodene is significant. The selectivity indices (ratio of the mean RBA for the progesterone receptor to that of androgen receptor) in intact cells are significantly higher for 3-keto-desogestrel and gestodene than for levonorgestrel and norethisterone. From these results we conclude that the introduction of the 18-methyl in norethisterone (levonorgestel) increases both the binding to the progesterone and androgen receptors.

  8. Mutations in the substrate binding site of human heat-shock protein 70 indicate specific interaction with HLA-DR outside the peptide binding groove.

    Science.gov (United States)

    Rohrer, Karin M; Haug, Markus; Schwörer, Daniela; Kalbacher, Hubert; Holzer, Ursula

    2014-06-01

    Heat-shock protein 70 (Hsp70)-peptide complexes are involved in MHC class I- and II-restricted antigen presentation, enabling enhanced activation of T cells. As shown previously, mammalian cytosolic Hsp70 (Hsc70) molecules interact specifically with HLA-DR molecules. This interaction might be of significance as Hsp70 molecules could transfer bound antigenic peptides in a ternary complex into the binding groove of HLA-DR molecules. The present study provides new insights into the distinct interaction of Hsp70 with HLA-DR molecules. Using a quantitative binding assay, it could be demonstrated that a point mutation of amino acids alanine 406 and valine 438 in the substrate binding pocket led to reduced peptide binding compared with the wild-type Hsp70 whereas HLA-DR binding remains unaffected. The removal of the C-terminal lid neither altered the substrate binding capacity nor the Hsp70 binding characteristics to HLA-DR. A truncated variant lacking the nucleotide binding domain showed no binding interactions with HLA-DR. Furthermore, the truncated ATPase subunit of constitutively expressed Hsc70 revealed similar binding affinities to HLA-DR compared with the complete Hsc70. Hence, it can be assumed that the Hsp70-HLA-DR interaction takes place outside the peptide binding groove and is attributed to the ATPase domain of HSP70 molecules. The Hsp70-chaperoned peptides might thereby be directly transferred into the binding groove of HLA-DR, so enabling enhanced presentation of the peptide on antigen-presenting cells and leading to an improved proliferation of responding T cells as shown previously. © 2014 John Wiley & Sons Ltd.

  9. Solubilization and characterization of haloperidol-sensitive (+)-( sup 3 H)SKF-10,047 binding sites (sigma sites) from rat liver membranes

    Energy Technology Data Exchange (ETDEWEB)

    McCann, D.J.; Su, T.P. (National Institute on Drug Abuse, Baltimore, MD (USA))

    1991-05-01

    The zwitterionic detergent 3-((3-cholamidopropyl)dimethylamino)-1-propanesulfonate (CHAPS) produced optimal solubilization of (+)-({sup 3}H)SKF-10,047 binding sites from rat liver membranes at a concentration of 0.2%, well below the critical micellular concentration of the detergent. The pharmacological selectivity of the liver (+)-({sup 3}H)SKF-10,047 binding sites corresponds to that of sigma sites from rat and guinea pig brain. When the affinities of 18 different drugs at (+)-({sup 3}H)SKF-10,047 binding sites in membranes and solubilized preparations were compared, a correlation coefficient of 0.99 and a slope of 1.03 were obtained, indicating that the pharmacological selectivity of rat liver sigma sites is retained after solubilization. In addition, the binding of 20 nM ({sup 3}H)progesterone to solubilized rat liver preparations was found to exhibit a pharmacological selectivity appropriate for sigma sites. A stimulatory effect of phenytoin on (+)-({sup 3}H)SKF-10,047 binding to sigma sites persisted after solubilization. When the solubilized preparation was subjected to molecular sizing chromatography, a single peak exhibiting specific (+)-({sup 3}H)SKF-10,047 binding was obtained. The binding activity of this peak was stimulated symmetrically when assays were performed in the presence of 300 microM phenytoin. The molecular weight of the CHAPS-solubilized sigma site complex was estimated to be 450,000 daltons. After solubilization with CHAPS, rat liver sigma sites were enriched to 12 pmol/mg of protein. The present results demonstrate a successful solubilization of sigma sites from rat liver membranes and provide direct evidence that the gonadal steroid progesterone binds to sigma sites. The results also suggest that the anticonvulsant phenytoin binds to an associated allosteric site on the sigma site complex.

  10. Phenylacetic acids and the structurally related non-steroidal anti-inflammatory drug diclofenac bind to specific gamma-hydroxybutyric acid sites in rat brain

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Høg, Signe; Skonberg, Christian

    2009-01-01

    Gamma-Hydroxybutyric acid (GHB) is a proposed neurotransmitter or neuromodulator with a yet unresolved mechanism of action. GHB binds to both specific high-affinity GHB binding sites and to gamma-aminobutyric acid subtype B (GABA(B)) receptors in the brain. To separate specific GHB effects from...... GABA(B) receptor effects, it is imperative to develop GHB selective and potent compounds. We generated the compound, 4-(biphen-4-yl)-4-hydroxybutyric acid, which is the 4-hydroxyl analogue of the non-steroidal anti-inflammatory drug (NSAID) fenbufen (referred to as gamma-hydroxyfenbufen). When measured...... in a rat brain homogenate [(3)H]NCS-382 binding assay, gamma-hydroxyfenbufen inhibited [(3)H]NCS-382 binding with a 10-fold higher affinity than GHB (K(i) 0.44 microM), thus establishing it as a novel lead structure. The active metabolite of fenbufen, 4-biphenylacetic acid inhibited [(3)H]NCS-382 binding...

  11. Site-specific binding of a water molecule to the sulfa drugs sulfamethoxazole and sulfisoxazole: a laser-desorption isomer-specific UV and IR study.

    Science.gov (United States)

    Uhlemann, Thomas; Seidel, Sebastian; Müller, Christian W

    2018-02-20

    To determine the preferred water molecule binding sites of the polybasic sulfa drugs sulfamethoxazole (SMX) and sulfisoxazole (SIX), we have studied their monomers and monohydrated complexes through laser-desorption conformer-specific UV and IR spectroscopy. Both the SMX and SIX monomer adopt a single conformer in the molecular beam. On the basis of their conformer-specific IR spectra in the NH stretch region, these conformers were assigned to the SMX and SIX global minimum structures, both exhibiting a staggered sulfonamide group and an intramolecular C-HO[double bond, length as m-dash]S hydrogen bond. The SMX-H 2 O and SIX-H 2 O complexes each adopt a single isomer in the molecular beam. Their isomeric structures were determined based on their isomer-specific IR spectra in the NH/OH stretch region. Quantum Theory of Atoms in Molecules analysis of the calculated electron densities revealed that in the SMX-H 2 O complex the water molecule donates an O-HN hydrogen bond to the heterocycle nitrogen atom and accepts an N-HO hydrogen bond from the sulfonamide NH group. In the SIX-H 2 O complex, however, the water molecule does not bind to the heterocycle but instead donates an O-HO[double bond, length as m-dash]S hydrogen bond to the sulfonamide group and accepts an N-HO hydrogen bond from the sulfonamide NH group. Both water complexes are additionally stabilized by a C ph -HOH 2 hydrogen bond. Interacting Quantum Atoms analysis suggests that all intermolecular hydrogen bonds are dominated by the short-range exchange-correlation contribution.

  12. pocketZebra: a web-server for automated selection and classification of subfamily-specific binding sites by bioinformatic analysis of diverse protein families.

    Science.gov (United States)

    Suplatov, Dmitry; Kirilin, Eugeny; Arbatsky, Mikhail; Takhaveev, Vakil; Svedas, Vytas

    2014-07-01

    The new web-server pocketZebra implements the power of bioinformatics and geometry-based structural approaches to identify and rank subfamily-specific binding sites in proteins by functional significance, and select particular positions in the structure that determine selective accommodation of ligands. A new scoring function has been developed to annotate binding sites by the presence of the subfamily-specific positions in diverse protein families. pocketZebra web-server has multiple input modes to meet the needs of users with different experience in bioinformatics. The server provides on-site visualization of the results as well as off-line version of the output in annotated text format and as PyMol sessions ready for structural analysis. pocketZebra can be used to study structure-function relationship and regulation in large protein superfamilies, classify functionally important binding sites and annotate proteins with unknown function. The server can be used to engineer ligand-binding sites and allosteric regulation of enzymes, or implemented in a drug discovery process to search for potential molecular targets and novel selective inhibitors/effectors. The server, documentation and examples are freely available at http://biokinet.belozersky.msu.ru/pocketzebra and there are no login requirements. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. H19-DMR allele-specific methylation analysis reveals epigenetic heterogeneity of CTCF binding site 6 but not of site 5 in head-and-neck carcinomas

    DEFF Research Database (Denmark)

    De Castro Valente Esteves, Leda Isabel; De Karla Cervigne, Nilva; Do Carmo Javaroni, Afonso

    2006-01-01

    of CTCF binding sites 5 and 6 using methylation-sensitive restriction enzyme PCR followed by RFLP analysis in matched tumoral and lymphocyte DNA from head-and-neck squamous cell carcinoma (HNSCC) patients, as well as in lymphocyte DNA from control individuals who were cancer-free. The monoallelic...

  14. Alpha-human atrial natriuretic polypeptide (. cap alpha. -hANP) specific binding sites in bovine adrenal gland

    Energy Technology Data Exchange (ETDEWEB)

    Higuchi, K.; Nawata, H.; Kato, K.I.; Ibayashi, H.; Matsuo, H.

    1986-06-13

    The effects of synthetic ..cap alpha..-human atrial natriuretic polypeptide (..cap alpha..-hANP) on steroidogenesis in bovine adrenocortical cells in primary monolayer culture were investigated. ..cap alpha..-hANP did not inhibit basal aldosterone secretion. ..cap alpha..-hANP induced a significant dose-dependent inhibition of basal levels of cortisol and dehydroepiandrosterone (DHEA) secretion and also of aCTH (10/sup -8/M)-stimulated increases in aldosterone, cortisol and DHEA secretion. Visualization of (/sup 125/I) ..cap alpha..-hANP binding sites in bovine adrenal gland by an in vitro autoradiographic technique demonstrated that these sites were highly localized in the adrenal cortex, especially the zona glomerulosa. These results suggest that the adrenal cortex may be a target organ for direct receptor-mediated actions of ..cap alpha..-hANP.

  15. Multidrug ABC transporter Cdr1 of Candida albicans harbors specific and overlapping binding sites for human steroid hormones transport.

    Science.gov (United States)

    Baghel, Pratima; Rawal, Manpreet Kaur; Khan, Mohammad Firoz; Sen, Sobhan; Siddiqui, Mohammed Haris; Chaptal, Vincent; Falson, Pierre; Prasad, Rajendra

    2017-10-01

    The present study examines the kinetics of steroids efflux mediated by the Candida drug resistance protein 1 (Cdr1p) and evaluates their interaction with the protein. We exploited our in-house mutant library for targeting the 252 residues forming the twelve transmembrane helices (TMHs) of Cdr1p. The screening revealed 65 and 58 residues critical for β-estradiol and corticosterone transport, respectively. Notably, up to 83% critical residues for corticosterone face the lipid interface compared to 54% for β-estradiol. Molecular docking identified a possible peripheral corticosterone-binding site made of 8/14 critical/non-critical residues between TMHs 3, 4 and 6. β-estradiol transport was severely hampered by alanine replacements of Cdr1p core residues involving TMHs 2, 5 and 8, in a binding site made of 10/14 critical residues mainly shared with rhodamine 6G with which it competes. By contrast, TMH11 was poorly impacted, although being part of the core domain. Finally, we observed the presence of several contiguous stretches of 3-5 critical residues in TMHs 2, 5 and 10 that points to a rotation motion of these helices during the substrate transport cycle. The selective structural arrangement of the steroid-binding pockets in the core region and at the lipid-TMD interface, which was never reported before, together with the possible rotation of some TMHs may be the structural basis of the drug-transport mechanism achieved by these type II ABC transporters. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Oxidative stress effect on progesterone-induced blocking factor (PIBF) binding to PIBF-receptor in lymphocytes.

    Science.gov (United States)

    de la Haba, Carlos; Palacio, José R; Palkovics, Tamas; Szekeres-Barthó, Júlia; Morros, Antoni; Martínez, Paz

    2014-01-01

    Receptor-ligand binding is an essential interaction for biological function. Oxidative stress can modify receptors and/or membrane lipid dynamics, thus altering cell physiological functions. The aim of this study is to analyze how oxidative stress may alter receptor-ligand binding and lipid domain distribution in the case of progesterone-induced blocking factor/progesterone-induced blocking factor-receptor. For membrane fluidity regionalization analysis of MEC-1 lymphocytes, two-photon microscopy was used in individual living cells. Lymphocytes were also double stained with AlexaFluor647/progesterone-induced blocking factor and Laurdan to evaluate -induced blocking factor/progesterone-induced blocking factor-receptor distribution in the different membrane domains, under oxidative stress. A new procedure has been developed which quantitatively analyzes the regionalization of a membrane receptor among the lipid domains of different fluidity in the plasma membrane. We have been able to establish a new tool which detects and evaluates lipid raft clustering from two-photon microscopy images of individual living cells. We show that binding of progesterone-induced blocking factor to progesterone-induced blocking factor-receptor causes a rigidification of plasma membrane which is related to an increase of lipid raft clustering. However, this clustering is inhibited under oxidative stress conditions. In conclusion, oxidative stress decreases membrane fluidity, impairs receptor-ligand binding and reduces lipid raft clustering. © 2013.

  17. Progesterone and non-specific immunologic mechanisms in pregnancy.

    Science.gov (United States)

    Szekeres-Bartho, J; Par, G; Szereday, L; Smart, C Y; Achatz, I

    1997-09-01

    Progesterone-dependent immunomodulation is one of the mechanisms that enables pregnancy to proceed to term. Immunologic effects of progesterone are mediated by a protein named the progesterone-induced blocking factor (PIBF). Among other effects this protein inhibits natural killer (NK) activity and displays an anti-abortive effect in mice. Recently, we have shown that PIBF induces a Th2 shift in vitro. The present study was aimed at investigating the in vivo effect of PIBF on cytokine production, as well as the relationship between cytokine production, NK activity, and pregnancy loss. Balb-c mice on day 8.5 of pregnancy were injected intraperitoneally with 0.5 mg of rabbit anti-PIBF immunoglobulin G (IgG). Another group of mice was simultaneously treated with anti-NK monoclonal antibodies. Mice treated with the same amount of normal rabbit serum or untreated mice of similar gestational age were used as controls. The animals were sacrificed and their uteri were inspected. The ratio of living and resorbed embryos was determined. NK activity as well as cytokine expression on the spleen cells were determined by immunocytochemistry and enzyme-linked immunoadsorbent assay (ELISA). Mitogen-activated spleen cells from anti-PIBF-treated mice produced significantly (P < 0.001) less IL-10 than those of pregnant control mice. A significantly higher percentage (P < 0.001) of spleen cells from anti-PIBF-treated mice expressed interferon-gamma (IFN gamma) as determined by immunocytochemistry, than those of untreated pregnant mice. There was a positive relationship between the percentage of IFN gamma-positive spleen cells and resorption rates, and an inverse relationship between the latter and interleukin-10 (IL-10) production. All these effects were corrected by treatment with anti-NK antibodies. Our data suggest that PIBF contributes to the success of gestation via cytokine-mediated inhibition of NK activity.

  18. Site-specific copper-catalyzed oxidation of α-synuclein: tightening the link between metal binding and protein oxidative damage in Parkinson's disease.

    Science.gov (United States)

    Miotto, Marco C; Rodriguez, Esaú E; Valiente-Gabioud, Ariel A; Torres-Monserrat, Valentina; Binolfi, Andrés; Quintanar, Liliana; Zweckstetter, Markus; Griesinger, Christian; Fernández, Claudio O

    2014-05-05

    Amyloid aggregation of α-synuclein (AS) has been linked to the pathological effects associated with Parkinson's disease (PD). Cu(II) binds specifically at the N-terminus of AS and triggers its aggregation. Site-specific Cu(I)-catalyzed oxidation of AS has been proposed as a plausible mechanism for metal-enhanced AS amyloid formation. In this study, Cu(I) binding to AS was probed by NMR spectroscopy, in combination with synthetic peptide models, site-directed mutagenesis, and C-terminal-truncated protein variants. Our results demonstrate that both Met residues in the motif (1)MDVFM(5) constitute key structural determinants for the high-affinity binding of Cu(I) to the N-terminal region of AS. The replacement of one Met residue by Ile causes a dramatic decrease in the binding affinity for Cu(I), whereas the removal of both Met residues results in a complete lack of binding. Moreover, these Met residues can be oxidized rapidly after air exposure of the AS-Cu(I) complex, whereas Met-116 and Met-127 in the C-terminal region remain unaffected. Met-1 displays higher susceptibility to oxidative damage compared to Met-5 because it is directly involved in both Cu(II) and Cu(I) coordination, resulting in closer exposure to the reactive oxygen species that may be generated by the redox cycling of copper. Our findings support a mechanism where the interaction of AS with copper ions leads to site-specific metal-catalyzed oxidation in the protein under physiologically relevant conditions. In light of recent biological findings, these results support a role for AS-copper interactions in neurodegeneration in PD.

  19. Hydrogen storage in a potassium-ion-bound metal-organic framework incorporating crown ether struts as specific cation binding sites.

    Science.gov (United States)

    Lim, Dae-Woon; Chyun, Seung An; Suh, Myunghyun Paik

    2014-07-21

    To develop a metal-organic framework (MOF) for hydrogen storage, SNU-200 incorporating a 18-crown-6 ether moiety as a specific binding site for selected cations has been synthesized. SNU-200 binds K(+), NH4(+), and methyl viologen (MV(2+)) through single-crystal to single-crystal transformations. It exhibits characteristic gas-sorption properties depending on the bound cation. SNU-200 activated with supercritical CO2 shows a higher isosteric heat (Qst) of H2 adsorption (7.70 kJ mol(-1)) than other zinc-based MOFs. Among the cation inclusions, K(+) is the best for enhancing the isosteric heat of the H2 adsorption (9.92 kJ mol(-1)) as a result of the accessible open metal sites on the K(+) ion. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. The human β-globin gene 3' enhancer contains multiple binding sites for an erythroid specific protein.

    NARCIS (Netherlands)

    L. Wall (Lee); E. de Boer (Ernie); F.G. Grosveld (Frank)

    1988-01-01

    textabstractWe have shown that the minimal enhancer fragment present in the 3'-flanking region of the human beta-globin gene contains four regions that bind nuclear proteins in vitro. By using gel mobility shift and DNase I footprinting assays, we were able to show that each of these regions binds

  1. Site-Specific Innovation

    DEFF Research Database (Denmark)

    Reeh, Henrik; Hemmersam, Peter

    2015-01-01

    Currently, cities across the Northern European region are actively redeveloping their former industrial harbours. Indeed, harbours areas are essential in the long-term transition from industrial to information and experience societies; harbours are becoming sites for new businesses and residences...... question is how innovation may contribute to urban life and site-specific qualities....

  2. MicroRNAs Form Triplexes with Double Stranded DNA at Sequence-Specific Binding Sites; a Eukaryotic Mechanism via which microRNAs Could Directly Alter Gene Expression.

    Directory of Open Access Journals (Sweden)

    Steven W Paugh

    2016-02-01

    Full Text Available MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA and typically down-regulating their stability or translation. Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence (i.e., NMR, FRET, SPR that purine or pyrimidine-rich microRNAs of appropriate length and sequence form triple-helical structures with purine-rich sequences of duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident to search genome-wide for potential triplex-forming sites and show that several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, p<2.2 × 10(-16 for genes whose expression is positively correlated with expression of microRNAs targeting triplex binding sequences. This work has thus revealed a new mechanism by which microRNAs could interact with gene promoter regions to modify gene transcription.

  3. Possible evidence that dehydroepiandrosterone sulfate (DHA-S) stimulates cervical ripening by a membrane-mediated process: Specific binding-sites in plasma membrane from human uterine cervix

    Energy Technology Data Exchange (ETDEWEB)

    Ohno, T.; Imai, A.; Tamaya, T. (Department of Obstetrics and Gynecology, Gifu University School of Medicine (Japan))

    1991-04-01

    Fetal adrenal steroid, dehydroepiandrosterone sulfate (DHA-S) is well known to promote cervical ripening in late pregnancy. The presence of sites specifically binding the DHA-S in plasma membrane was studied in human cervical fibroblasts prepared from pregnant uterus. The fibroblasts were incubated with {sup 3}H DHA-S and then fractionated into plasma membranes, cytosol, nuclei, and other organella debris. The specific activity of 3H-count in the plasma membrane fraction was enriched {approximately} 7-fold compared with the whole homogenate. When the isolated plasma membrane preparations from the fibroblasts were exposed to {sup 3}H DHA-S, the binding showed saturation kinetics; an apparent equilibrium dissociation constant (Kd) of 12 nM, and the binding capacity (Bmax) of 1.25 pmol/mg protein. The present results suggest that DHA is bound to and recognized by components in plasma membrane, and may exert its action on cervical ripening through the membrane-mediated processes.

  4. Loss of the Otx2-Binding Site in the Nanog Promoter Affects the Integrity of Embryonic Stem Cell Subtypes and Specification of Inner Cell Mass-Derived Epiblast

    Directory of Open Access Journals (Sweden)

    Dario Acampora

    2016-06-01

    Full Text Available Mouse embryonic stem cells (ESCs and the inner cell mass (ICM-derived epiblast exhibit naive pluripotency. ESC-derived epiblast stem cells (EpiSCs and the postimplantation epiblast exhibit primed pluripotency. Although core pluripotency factors are well-characterized, additional regulators, including Otx2, recently have been shown to function during the transition from naive to primed pluripotency. Here we uncover a role for Otx2 in the control of the naive pluripotent state. We analyzed Otx2-binding activity in ESCs and EpiSCs and identified Nanog, Oct4, and Sox2 as direct targets. To unravel the Otx2 transcriptional network, we targeted the strongest Otx2-binding site in the Nanog promoter, finding that this site modulates the size of specific ESC-subtype compartments in cultured cells and promotes Nanog expression in vivo, predisposing ICM differentiation to epiblast. Otx2-mediated Nanog regulation thus contributes to the integrity of the ESC state and cell lineage specification in preimplantation development.

  5. Non-specificity and synergy at the binding site of the carboplatin-induced DNA adduct via molecular dynamics simulations of the MutSα-DNA recognition complex

    Science.gov (United States)

    Negureanu, Lacramioara; Salsbury, Freddie R

    2013-01-01

    MutSα is the most abundant mismatch binding factor of human DNA mismatch repair (MMR) proteins. MMR maintain genetic stability by recognizing and repairing DNA defects. Failure to accomplish their function may lead to cancer. In addition, MutSα recognizes at least some types of DNA damage making it a target for anticancer agents. Here, complementing scarce experimental data, we report unique hydrogen bonding motifs associated with the recognition of the carboplatin induced DNA damage by MutSα. These data predict that carboplatin and cisplatin induced damaging DNA adducts are recognized by MutSα in a similar manner. Our simulations also indicate that loss of base pairing at the damage site results in (1) non-specific binding and (2) changes in the atomic flexibility at the lesion site and beyond. To further quantify alterations at MutSα-DNA interface in response to damage recognition non-bonding interactions and salt bridges were investigated. These data indicate (1) possible different packing and (2) disruption of the salt bridges at the MutSα-DNA interface in the damaged complex. These findings (1) underscore the general observation of disruptions at the MutSα-DNA interface and (2) highlight the nature of the anticancer effect of the carboplatin agent. The analysis was carried out from atomistic simulations. PMID:23799640

  6. Progesterone binding nano-carriers based on hydrophobically modified hyperbranched polyglycerols

    Science.gov (United States)

    Alizadeh Noghani, M.; Brooks, D. E.

    2016-02-01

    Progesterone (Pro) is a potent neurosteroid and promotes recovery from moderate Traumatic Brain Injury but its clinical application is severely impeded by its poor water solubility. Here we demonstrate that reversibly binding Pro within hydrophobically modified hyperbranched polyglycerol (HPG-Cn-MPEG) enhances its solubility, stability and bioavailability. Synthesis, characterization and Pro loading into HPG-Cn-MPEG is described. The release kinetics are correlated with structural properties and the results of Differential Scanning Calorimetry studies of a family of HPG-Cn-MPEGs of varying molecular weight and alkylation. While the maximum amount of Pro bound correlates well with the amount of alkyl carbon per molecule contributing to its hydrophobicity, the dominant first order rate constant for Pro release correlates strongly with the amount of structured or bound water in the dendritic domain of the polymer. The results provide evidence to justify more detailed studies of interactions with biological systems, both single cells and in animal models.Progesterone (Pro) is a potent neurosteroid and promotes recovery from moderate Traumatic Brain Injury but its clinical application is severely impeded by its poor water solubility. Here we demonstrate that reversibly binding Pro within hydrophobically modified hyperbranched polyglycerol (HPG-Cn-MPEG) enhances its solubility, stability and bioavailability. Synthesis, characterization and Pro loading into HPG-Cn-MPEG is described. The release kinetics are correlated with structural properties and the results of Differential Scanning Calorimetry studies of a family of HPG-Cn-MPEGs of varying molecular weight and alkylation. While the maximum amount of Pro bound correlates well with the amount of alkyl carbon per molecule contributing to its hydrophobicity, the dominant first order rate constant for Pro release correlates strongly with the amount of structured or bound water in the dendritic domain of the polymer. The

  7. Antibodies to the CD4-binding site of HIV-1 gp120 suppress gp120-specific CD4 T cell response while enhancing antibody response

    Directory of Open Access Journals (Sweden)

    Hioe Catarina E

    2008-07-01

    Full Text Available Abstract Background The binding of Abs to the CD4-binding site (CD4bs of HIV-1 envelope gp120 has been shown to obstruct the processing and generation of helper epitopes from this antigen, resulting in poor presentation of various gp120 epitopes by MHC class II to CD4 T cells. However, the physiologic significance of these inhibitory anti-CD4bs Abs in vivo has remained unclear. In this study, we evaluated the immunologic effects of anti-CD4bs Abs in vivo using a murine model. Results Animals were immunized with recombinant envelope proteins with or without CD4-binding activity (designated CD4bs+ Env and CD4bs– Env, respectively. As expected, anti-CD4bs Abs were generated only after immunization with CD4bs+ Env and not with CD4bs– Env. The presence of anti-CD4bs Abs was associated with lower levels of envelope-specific lymphoproliferation in animals immunized with CD4bs+ Env. To further determine the specific role of the anti-CD4bs Abs, we immunized mice with gp120 in the presence of an inhibitory anti-CD4bs mAb or a non-inhibitory anti-gp120 mAb. The data show that the presence of anti-CD4bs mAb reduced CD4 T cell responses to gp120. However, we also detected significantly higher titers of anti-gp120 Abs following immunization with gp120 and the anti-CD4bs mAb. Conclusion Anti-CD4bs Abs can exert discordant effects on the gp120-specific CD4 T cell and Ab responses in vivo, indicating the importance of these particular Abs in influencing both the cellular and the humoral immune responses against HIV-1.

  8. Bitopic Ligands and Metastable Binding Sites

    DEFF Research Database (Denmark)

    Fronik, Philipp; Gaiser, Birgit I; Sejer Pedersen, Daniel

    2017-01-01

    G protein-coupled receptors (GPCRs) belong to a large superfamily of membrane receptors mediating a variety of physiological functions. As such they are attractive targets for drug therapy. However, it remains a challenge to develop subtype selective GPCR ligands due to the high conservation...... sites. Computational studies on ligand binding to GPCRs have revealed transient, low-affinity binding sites, termed metastable binding sites. Metastable binding sites may provide a new source of allosteric binding sites that could be exploited in the design of bitopic ligands. Unlike the bitopic ligands...

  9. Pharmacological characterization of intracellular, membrane, and plasma binding sites for corticosterone in house sparrows.

    Science.gov (United States)

    Breuner, Creagh W; Orchinik, Miles

    2009-09-01

    The diversity and specificity of glucocorticoid effects are dependent on cell-specific receptor mechanisms. Three known corticosteroid receptors mediate tissue effects of glucocorticoids in vertebrates: two intracellular receptors that act primarily as ligand-activated transcription factors, and a membrane-associated receptor. The intracellular receptor sub-types have been well characterized in mammals, however relatively little is known about them across non-mammalian vertebrates. The membrane-associated receptors are poorly characterized in most vertebrate taxa. To explore the basis for glucocorticoid action in birds, we pharmacologically characterized the three putative corticosteroid receptors in the brain, as well as a plasma corticosterone binding globulin, in the house sparrow (Passer domesticus). We found that house sparrow brain cytosol contained two distinguishable binding sites for corticosterone. A high affinity, mineralocorticoid-like receptor had subnanomolar affinity for corticosterone (K(d) approximately 0.2 nM). However, this 'MR-like' high-affinity receptor did not bind RU28318 or canrenoic acid, two compounds that bind mammalian MR with high affinity. A lower-affinity, glucocorticoid-like receptor in brain cytosol bound corticosterone with an average K(d)=5.61 nM. This GR-like receptor showed subnanomolar affinity for RU 486. MR- and GR-like receptors were found in equal numbers in whole brain assays (average B(max)=69 and 62 fmol/mg protein, respectively). House sparrow brain membranes contain a single binding site specific for glucocorticoids, with characteristics consistent with a steroid/receptor interaction. Corticosterone affinity for this putative membrane receptor was approximately 24 nM, with apparent B(max)=177 fmol/mg protein. House sparrow plasma contained a single binding site for [(3)H]corticosterone. Specific binding to plasma sites was inhibited by glucocorticoids, progesterone, and testosterone. Testosterone binding to this

  10. The Molecular Switch of Telomere Phages: High Binding Specificity of the PY54 Cro Lytic Repressor to a Single Operator Site

    Directory of Open Access Journals (Sweden)

    Jens Andre Hammerl

    2015-06-01

    Full Text Available Temperate bacteriophages possess a molecular switch, which regulates the lytic and lysogenic growth. The genomes of the temperate telomere phages N15, PY54 and ɸKO2 harbor a primary immunity region (immB comprising genes for the prophage repressor, the lytic repressor and a putative antiterminator. The roles of these products are thought to be similar to those of the lambda proteins CI, Cro and Q, respectively. Moreover, the gene order and the location of several operator sites in the prototype telomere phage N15 and in ɸKO2 are also reminiscent of lambda-like phages. By contrast, in silico analyses revealed the presence of only one operator (O\\(_{\\rm{R}}\\3 in PY54. The purified PY54 Cro protein was used for EMSA studies demonstrating that it exclusively binds to a 16-bp palindromic site (O\\(_{\\rm{R}}\\3 upstream of the prophage repressor gene. The O\\(_{\\rm{R}}\\3 operator sequences of PY54 and ɸKO2/N15 only differ by their peripheral base pairs, which are responsible for Cro specificity. PY54 cI and cro transcription is regulated by highly active promoters initiating the synthesis of a homogenious species of leaderless mRNA. The location of the PY54 Cro binding site and of the identified promoters suggests that the lytic repressor suppresses cI transcription but not its own synthesis. The results indicate an unexpected diversity of the growth regulation mechanisms in lambda-related phages.

  11. 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.

  12. Active site studies of cytochrome P=450CAM. I. Specific cysteine labeling with the affinity reagent isobornyl bromoacetate as a model for substrate binding.

    Science.gov (United States)

    Murray, R I; Gunsalus, I C; Dus, K M

    1982-11-10

    A model is presented suggesting a function of specific cysteine residue of cytochrome P-450CAM in binding the substrate camphor, via a thiohemiketal bond, for its correct orientation to the heme iron and for the subsequent transfer of nascent product to facilitate its release. This model was developed to explain the results of affinity labeling with isobornyl bromoacetate. This reagent couples to the proteins via a thioether bond to cysteine, eliciting a type I transition in the difference spectrum. Formation of this covalent complex, which is strongly inhibited by the substrate, can be monitored by quantitation of S-carboxymethylcysteine in acid hydrolyzates. While addition of one equivalent of label yields 0.3 equivalents of the cysteine derivative after 5 min, increasing to 0.8 equivalents after 24 h, the spectral shift decays with time. Kinetic analysis of the spectral decay and of covalent coupling strongly suggests that thioether bond formation occurs at the substrate binding-site, in a reaction step prior to, and distinct from, the step associated with the spectral decay. The P-450CAM derivative, when titrated with camphor, produced again a type I spectrum virtually identical with the spectrum of the native P-450CAM-substrate complex. While the model presented here is not the only possible interpretation of the results, it is fully consistent with them and provides an excellent framework for further study of the catalytic mechanism of P-450CAM.

  13. Binding specificity of Escherichia coli trigger factor

    Science.gov (United States)

    Patzelt, Holger; Rüdiger, Stefan; Brehmer, Dirk; Kramer, Günter; Vorderwülbecke, Sonja; Schaffitzel, Elke; Waitz, Andreas; Hesterkamp, Thomas; Dong, Liying; Schneider-Mergener, Jens; Bukau, Bernd; Deuerling, Elke

    2001-01-01

    The ribosome-associated chaperone trigger factor (TF) assists the folding of newly synthesized cytosolic proteins in Escherichia coli. Here, we determined the substrate specificity of TF by examining its binding to 2842 membrane-coupled 13meric peptides. The binding motif of TF was identified as a stretch of eight amino acids, enriched in basic and aromatic residues and with a positive net charge. Fluorescence spectroscopy verified that TF exhibited a comparable substrate specificity for peptides in solution. The affinity to peptides in solution was low, indicating that TF requires ribosome association to create high local concentrations of nascent polypeptide substrates for productive interaction in vivo. Binding to membrane-coupled peptides occurred through the central peptidyl-prolyl-cis/trans isomerase (PPIase) domain of TF, however, independently of prolyl residues. Crosslinking experiments showed that a TF fragment containing the PPIase domain linked to the ribosome via the N-terminal domain is sufficient for interaction with nascent polypeptide substrates. Homology modeling of the PPIase domain revealed a conserved FKBP(FK506-binding protein)-like binding pocket composed of exposed aromatic residues embedded in a groove with negative surface charge. The features of this groove complement well the determined substrate specificity of TF. Moreover, a mutation (E178V) in this putative substrate binding groove known to enhance PPIase activity also enhanced TF's association with a prolyl-free model peptide in solution and with nascent polypeptides. This result suggests that both prolyl-independent binding of peptide substrates and peptidyl-prolyl isomerization involve the same binding site. PMID:11724963

  14. Site-specific

    Directory of Open Access Journals (Sweden)

    Adel M.E. Mohamed

    2013-06-01

    Full Text Available In order to quantify the near-surface seismic properties (P- and S-wave velocities, and the dynamic elastic properties with respect to the depth at a specific area (6th of October club, we conducted a non-invasive and low cost active seismic survey. The primary wave velocity is determined by conducting a P-wave shallow seismic refraction survey. The dispersive characteristics of Rayleigh type surface waves were utilized for imaging the shallow subsurface layers by estimating the 1D (depth and 2D (depth and surface location shear wave velocities. The reliability of the Multi-channel Analysis of Surface Waves (MASW depends on the accurate determination of phase velocities for horizontally traveling fundamental mode Rayleigh waves. Consequently, the elastic properties are evaluated empirically. The Vs30 (average shear wave velocity down to 30 m depth, which is obtained from the MASW technique, plays a critical role in evaluating the site response of the upper 30 m depth. The distribution of the obtained values of Vs30 through the studied area demonstrates site classes of C and D, according to the NEHRP (National Earthquake Hazard Reduction Program and IBC (International Building Code standards.

  15. Recent advances in structure of progestins and their binding to progesterone receptors.

    Science.gov (United States)

    Cabeza, Marisa; Heuze, Yvonne; Sánchez, Araceli; Garrido, Mariana; Bratoeff, Eugene

    2015-02-01

    The role of progesterone in women's cancers as well as the knowledge of the progesterone receptor (PR) structure has prompted the design of different therapies. The aim of this review is to describe the basic structure of PR agonists and antagonists as well as the recent treatments for illness associated with the progesterone receptor. The rational design for potent and effective drugs for the treatment of female cancer must consider the structural changes of the androgen and progestogen skeleton which are an indicator of their activity as progestins or antiprogestins. The presence of a hydroxyl group at C-17 in the progesterone skeleton brings about a loss of progestational activity whereas acetylation induces a progestational effect. The incorporation of an ethynyl functional group to the testosterone framework results in a loss of androgenic activity with a concomitant enhancement of the progestational effect. On the other hand, an ester function at C-3 of dehydroepiandrosterone skeleton induces partial antagonism to the PR.

  16. Phosphorylation of a specific cdk site in E2F-1 affects its electrophoretic mobility and promotes pRB-binding in vitro

    DEFF Research Database (Denmark)

    Peeper, D S; Keblusek, P; Helin, K

    1995-01-01

    of the retinoblastoma gene (pRB). We find that E2F-1 proteins are heterogeneously phosphorylated in insect cells, as a result of which they migrate as a doublet on SDS-polyacrylamide gels. This electrophoretic shift is shown to be dependent upon specific phosphorylation of E2F-1 on serine-375 (S375), near the pRB......-binding site. Phosphorylation on S375 also occurs in human cells. E2F-1 was most efficiently phosphorylated on this residue by cyclin A/cdk2 kinase, and to a lesser extent by cyclin A/cdk2, irrespective of the presence of the pRB-related p107 protein. Phosphorylation of E2F-1 on S375 greatly enhanced its......The E2F transcription factor family participates in growth control presumably through transcriptional activation of genes that promote entry into S phase. E2F activity is believed to be controlled across the cell cycle by association with various cellular proteins, including the product...

  17. Site-specific immobilization of recombinant antibody fragments through material-binding peptides for the sensitive detection of antigens in enzyme immunoassays.

    Science.gov (United States)

    Kumada, Yoichi

    2014-11-01

    The immobilization of an antibody is one of the key technologies that are used to enhance the sensitivity and efficiency of the detection of target molecules in immunodiagnosis and immunoseparation. Recombinant antibody fragments such as VHH, scFv and Fabs produced by microorganisms are the next generation of ligand antibodies as an alternative to conventional whole Abs due to a smaller size and the possibility of site-directed immobilization with uniform orientation and higher antigen-binding activity in the adsorptive state. For the achievement of site-directed immobilization, affinity peptides for a certain ligand molecule or solid support must be introduced to the recombinant antibody fragments. In this mini-review, immobilization technologies for the whole antibodies (whole Abs) and recombinant antibody fragments onto the surfaces of plastics are introduced. In particular, the focus here is on immobilization technologies of recombinant antibody fragments utilizing affinity peptide tags, which possesses strong binding affinity towards the ligand molecules. Furthermore, I introduced the material-binding peptides that are capable of direct recognition of the target materials. Preparation and immobilization strategies for recombinant antibody fragments linked to material-binding peptides (polystyrene-binding peptides (PS-tags) and poly (methyl methacrylate)-binding peptide (PMMA-tag)) are the focus here, and are based on the enhancement of sensitivity and a reduction in the production costs of ligand antibodies. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Ethylene binding site affinity in ripening apples

    Energy Technology Data Exchange (ETDEWEB)

    Blankenship, S.M. (North Carolina State Univ., Raleigh, NC (United States). Dept. of Horticultural Science); Sisler, E.C. (North Carolina State Univ., Raleigh, NC (United States))

    1993-09-01

    Scatchard plots for ethylene binding in apples (Malus domestica Borkh.), which were harvested weekly for 5 weeks to include the ethylene climacteric rise, showed C[sub 50] values (concentration of ethylene needed to occupy 50% of the ethylene binding sites) of 0.10, 0.11, 0.34, 0.40, and 0.57 [mu]l ethylene/liter[sup [minus]1], respectively, for each of the 5 weeks. Higher ethylene concentrations were required to saturate the binding sites during the climacteric rise than at other times. Diffusion of [sup 14]C-ethylene from the binding sites was curvilinear and did not show any indication of multiple binding sites. Ethylene was not metabolized by apple tissue.

  19. Specific gamma-hydroxybutyrate-binding sites but loss of pharmacological effects of gamma-hydroxybutyrate in GABA-B1-deficient mice

    DEFF Research Database (Denmark)

    Kaupmann, Klemens; Cryan, John F; Wellendorph, Petrine

    2003-01-01

    functional GABAB receptors. Autoradiography reveals a similar spatial distribution of [3H]GHB-binding sites in brains of GABAB(1)-/- and wild-type mice. The maximal number of binding sites and the KD values for the putative GHB antagonist [3H]6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene acetic...... in brain membrane preparations from wild-type but not GABAB(1)-/- mice. The GTPgamma[35S] responses in wild-type mice were blocked by the GABAB antagonist [3-[[1-(S)-(3,4dichlorophenyl)ethyl]amino]-2-(S)-hydroxy-propyl]-cyclohexylmethyl phosphinic acid hydrochloride (CGP54626) but not by NCS-382....... Altogether, these findings suggest that the GHB-induced GTPgamma[35S] responses are mediated by GABAB receptors. Following GHB or GBL application, GABAB(1)-/- mice showed neither the hypolocomotion, hypothermia, increase in striatal dopamine synthesis nor electroencephalogram delta-wave induction seen...

  20. Fluorescent protein-imprinted polymers capable of signal transduction of specific binding events prepared by a site-directed two-step post-imprinting modification.

    Science.gov (United States)

    Sunayama, Hirobumi; Ooya, Tooru; Takeuchi, Toshifumi

    2014-02-09

    Protein recognition polymers capable of highly specific transduction of protein binding events into fluorescence change were prepared by molecular imprinting in conjunction with a newly developed two-step post-imprinting chemical modification of functional groups located within the protein recognition cavity.

  1. Low affinity glucocorticoid binding site ligands as potential anti-fibrogenics

    OpenAIRE

    Marek, Carylyn J; Wallace, Karen; Durward, Elaine; Koruth, Matthew; Leel, Val; Leiper, Lucy J.; Wright, Matthew C.

    2009-01-01

    Background Pregnane X receptor (PXR) agonists inhibit liver fibrosis. However, the rodent PXR activator pregnenolone 16? carbonitrile (PCN) blocks, in vitro, hepatic stellate cell-to-myofibroblast trans-differentiation and proliferation in cells from mice with a disrupted PXR gene, suggesting there is an additional anti-fibrogenic drug target for PCN. The role of the low affinity glucocorticoid binding site (LAGS) ? which may be identical or associated with the progesterone receptor membrane ...

  2. Plasmodium vivax Duffy binding protein peptides specifically bind to reticulocytes.

    Science.gov (United States)

    Ocampo, Marisol; Vera, Ricardo; Eduardo Rodriguez, Luis; Curtidor, Hernando; Urquiza, Mauricio; Suarez, Jorge; Garcia, Javier; Puentes, Alvaro; Lopez, Ramsés; Trujillo, Mary; Torres, Elizabeth; Patarroyo, Manuel Elkin

    2002-01-01

    Plasmodium vivax Duffy Binding Protein (Pv-DBP) is essential during merozoite invasion of reticulocytes. Reticulocyte binding region identification is important for understanding Pv-DBP reticulocyte recognition. Fifty 20 mer non-overlapping peptides, spanning Pv-DBP sequences, were tested in erythrocyte and reticulocyte binding assays. Ten HARBPs, mainly located in region II (Kd 50-130 nM), were High Activity Reticulocyte Binding Peptides (HARBPs); one bound to erythrocytes. Reticulocyte trypsin-, chymotrypsin- or neuraminidase- treatment affects HARBP binding differently, suggesting that these peptides have different reticulocyte-binding-sites. Some peptides bound to a Coomasie non-stainable 40 Kda band. Some HARBPs were able to block recombinant PvRII binding (Pv-DBP region II) to Duffy positive reticulocytes.

  3. 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.

  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

    2013-01-01

    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. PMID:23641004

  5. Cell differentiation by interaction of two HMG-box proteins: Mat1-Mc activates M cell-specific genes in S.pombe by recruiting the ubiquitous transcription factor Ste11 to weak binding sites

    DEFF Research Database (Denmark)

    Kjaerulff, S; Dooijes, D; Clevers, H

    1997-01-01

    The Schizosaccharomyces pombe mfm1 gene is expressed in an M cell-specific fashion. This regulation requires two HMG-box proteins: the ubiquitous Ste11 transcription factor and the M cell-controlling protein Mat1-Mc. Here we report that the mfm1 promoter contains a single, weak Stell-binding site...

  6. A membrane-associated progesterone-binding protein, 25-Dx, is regulated by progesterone in brain regions involved in female reproductive behaviors

    Science.gov (United States)

    Krebs, Christopher J.; Jarvis, Erich D.; Chan, Johnny; Lydon, John P.; Ogawa, Sonoko; Pfaff, Donald W.

    2000-01-01

    The ventromedial hypothalamus (VMH) plays a central role in the regulation of the female reproductive behavior lordosis, a behavior dependent upon the sequential activation of receptors for the ovarian steroid hormones estradiol (E) and progesterone (P). These receptors function as transcription factors to alter the expression of target genes. To discover behaviorally relevant genes targeted by E and P in the VMH, we used the differential display PCR to identify messenger RNAs that are differentially expressed in the hypothalamus of ovariectomized (ovx) rats treated with E alone compared with ovariectomized rats treated with E and P. We show here that one interesting mRNA within the hypothalamus that is repressed by P after E priming encodes the protein 25-Dx, the rat homolog of the human membrane-associated P-binding protein Hpr6.6. Neurons in the brain containing the highest levels of 25-Dx are located in several nuclei of the basal forebrain, including the VMH. 25-Dx expression is also higher in the hypothalamus of female P receptor “knockout” mice than in their wild-type littermates. These findings suggest a mechanism in which the activation of nuclear P receptor represses expression of a membrane P receptor, 25-Dx, during lordosis facilitation. PMID:11070092

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

    Directory of Open Access Journals (Sweden)

    Evangelia Petsalaki

    2009-03-01

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

  8. Progesterone impairs antigen-non-specific immune protection by CD8 T memory cells via interferon-γ gene hypermethylation.

    Directory of Open Access Journals (Sweden)

    Yushi Yao

    2017-11-01

    Full Text Available Pregnant women and animals have increased susceptibility to a variety of intracellular pathogens including Listeria monocytogenes (LM, which has been associated with significantly increased level of sex hormones such as progesterone. CD8 T memory(Tm cell-mediated antigen-non-specific IFN-γ responses are critically required in the host defense against LM. However, whether and how increased progesterone during pregnancy modulates CD8 Tm cell-mediated antigen-non-specific IFN-γ production and immune protection against LM remain poorly understood. Here we show in pregnant women that increased serum progesterone levels are associated with DNA hypermethylation of IFN-γ gene promoter region and decreased IFN-γ production in CD8 Tm cells upon antigen-non-specific stimulation ex vivo. Moreover, IFN-γ gene hypermethylation and significantly reduced IFN-γ production post LM infection in antigen-non-specific CD8 Tm cells are also observed in pregnant mice or progesterone treated non-pregnant female mice, which is a reversible phenotype following demethylation treatment. Importantly, antigen-non-specific CD8 Tm cells from progesterone treated mice have impaired anti-LM protection when adoptive transferred in either pregnant wild type mice or IFN-γ-deficient mice, and demethylation treatment rescues the adoptive protection of such CD8 Tm cells. These data demonstrate that increased progesterone impairs immune protective functions of antigen-non-specific CD8 Tm cells via inducing IFN-γ gene hypermethylation. Our findings thus provide insights into a new mechanism through which increased female sex hormone regulate CD8 Tm cell functions during pregnancy.

  9. Peptide binding specificity of the chaperone calreticulin

    DEFF Research Database (Denmark)

    Sandhu, N.; Duus, K.; Jorgensen, C.S.

    2007-01-01

    Calreticulin is a molecular chaperone with specificity for polypeptides and N-linked monoglucosylated glycans. In order to determine the specificity of polypeptide binding, the interaction of calreticulin with polypeptides was investigated using synthetic peptides of different length and composit...

  10. Monoclonal Antibodies Specific for the V2, V3, CD4-Binding Site, and gp41 of HIV-1 Mediate Phagocytosis in a Dose-Dependent Manner.

    Science.gov (United States)

    Musich, Thomas; Li, Liuzhe; Liu, Lily; Zolla-Pazner, Susan; Robert-Guroff, Marjorie; Gorny, Miroslaw K

    2017-04-15

    In light of the weak or absent neutralizing activity mediated by anti-V2 monoclonal antibodies (MAbs), we tested whether they can mediate Ab-dependent cellular phagocytosis (ADCP), which is an important element of anti-HIV-1 immunity. We tested six anti-V2 MAbs and compared them with 21 MAbs specific for V3, the CD4-binding site (CD4bs), and gp41 derived from chronically HIV-1-infected individuals and produced by hybridoma cells. ADCP activity was measured by flow cytometry using uptake by THP-1 monocytic cells of fluorescent beads coated with gp120, gp41, BG505 SOSIP.664, or BG505 DS-SOSIP.664 complexed with MAbs. The measurement of ADCP activity by the area under the curve showed significantly higher activity of anti-gp41 MAbs than of the members of the three other groups of MAbs tested using beads coated with monomeric gp41 or gp120; anti-V2 MAbs were dominant compared to anti-V3 and anti-CD4bs MAbs against clade C gp120 ZM109 ADCP activity mediated by V2 and V3 MAbs was positive against stabilized DS-SOSIP.664 trimer but negligible against SOSIP.664 targets, suggesting that a closed envelope conformation better exposes the variable loops. Two IgG3 MAbs against the V2 and V3 regions displayed dominant ADCP activity compared to a panel of IgG1 MAbs. This superior ADCP activity was confirmed when two of three recombinant IgG3 anti-V2 MAbs were compared to their IgG1 counterparts. The study demonstrated dominant ADCP activity of anti-gp41 against monomers but not trimers, with some higher activity of anti-V2 MAbs than of anti-V3 and anti-CD4bs MAbs. The ability to mediate ADCP suggests a mechanism by which anti-HIV-1 envelope Abs can contribute to protective efficacy. IMPORTANCE Anti-V2 antibodies (Abs) correlated with reduced risk of HIV-1 infection in recipients of the RV144 vaccine, suggesting that they play a protective role, but a mechanism providing such protection remains to be determined. The rare and weak neutralizing activities of anti-V2 MAbs prompted us

  11. A single, specific thymine mutation in the ComK-Binding site severely decreases binding and transcription activation by the competence transcription factor ComK of Bacillus subtilis

    NARCIS (Netherlands)

    Susanna, Kim A.; Mironczuk, Aleksandra M.; Smits, Wiep Klaas; Hamoen, Leendert W.; Kuipers, Oscar P.

    The competence transcription factor ComK plays a central role in competence development in Bacillus subtilis by activating the transcription of the K regulon. ComK-activated genes are characterized by the presence of a specific sequence to which ComK binds, a K-box, in their upstream DNA region.

  12. Enhanced binding of antibodies to the DTR motif of MUC1 tandem repeat peptide is mediated by site-specific glycosylation.

    Science.gov (United States)

    Karsten, U; Diotel, C; Klich, G; Paulsen, H; Goletz, S; Müller, S; Hanisch, F G

    1998-06-15

    The epithelial mucin MUC1 is an important tumor marker of breast cancer and other carcinomas. Its immunodominant DTR motif, which is the principal target for immunotherapeutic approaches, has been assumed until recently not to be glycosylated in both normal and tumor MUC1 and to acquire its immunogenic conformation by virtue of a certain number of tandem repeats. We present evidence that the antigenicity of the single repeat toward a considerable number of antibodies to the DTR motif is greatly enhanced if it is glycosylated within this motif, and only in this position. Twenty-eight monoclonal anti-MUC1 antibodies with DTR specificity were tested for binding to synthetic 21-mer (AHG21) or 20-mer (HGV20) tandem repeat peptides O-glycosylated with galactose beta1-3N-acetylgalactosamine alpha or N-acetylgalactosamine alpha at defined Thr or Ser positions. Binding was measured in ELISA experiments using the glycopeptides as plate-immobilized antigens or as inhibitors in solution. At least 12 antibodies revealed significantly enhanced binding to the peptides glycosylated at the DTR motif (Thr-10) as compared to positional isomers glycosylated at Thr-5, Ser-6, Ser-16, or Thr-17 and to the nonglycosylated peptides. Six antibodies (VU-3-C6, A76-A/C7, Ma552, VU-11-D1, VU-12-E1, and VU-11-E2) that were unreactive with the monomeric repeat peptide did bind to the DTR-glycosylated peptide. Several lines of evidence suggest that glycosylation with N-acetylgalactosamine is sufficient for the observed enhancement effect. Our results are of special interest in conjunction with the recent observation that the DTR motif of lactation-associated MUC1 is O-glycosylated in vivo (Müller et al., J. Biol. Chem., 272: 24780-24793, 1997). They may have consequences for the design of efficient tumor vaccines.

  13. Specific insulin binding in bovine chromaffin cells; demonstration of preferential binding to adrenalin-storing cells

    Energy Technology Data Exchange (ETDEWEB)

    Serck-Hanssen, G.; Soevik, O.

    1987-12-28

    Insulin binding was studied in subpopulations of bovine chromaffin cells enriched in adrenalin-producing cells (A-cells) or noradrenalin-producing cells (NA-cells). Binding of /sup 125/I-insulin was carried out at 15/sup 0/C for 3 hrs in the absence or presence of excess unlabeled hormone. Four fractions of cells were obtained by centrifugation on a stepwise bovine serum albumin gradient. The four fractions were all shown to bind insulin in a specific manner and the highest binding was measured in the cell layers of higher densities, containing mainly A-cells. The difference in binding of insulin to the four subpopulations of chromaffin cells seemed to be related to differences in numbers of receptors as opposed to receptor affinities. The authors conclude that bovine chromaffin cells possess high affinity binding sites for insulin and that these binding sites are mainly confined to A-cells. 24 references, 2 figures, 1 table.

  14. Multiple Copies of a Simple MYB-Binding Site Confers Trans-regulation by Specific Flavonoid-Related R2R3 MYBs in Diverse Species

    Directory of Open Access Journals (Sweden)

    Cyril Brendolise

    2017-10-01

    Full Text Available In apple, the MYB transcription factor MYB10 controls the accumulation of anthocyanins. MYB10 is able to auto-activate its expression by binding its own promoter at a specific motif, the R1 motif. In some apple accessions a natural mutation, termed R6, has more copies of this motif within the MYB10 promoter resulting in stronger auto-activation and elevated anthocyanins. Here we show that other anthocyanin-related MYBs selected from apple, pear, strawberry, petunia, kiwifruit and Arabidopsis are able to activate promoters containing the R6 motif. To examine the specificity of this motif, members of the R2R3 MYB family were screened against a promoter harboring the R6 mutation. Only MYBs from subgroups 5 and 6 activate expression by binding the R6 motif, with these MYBs sharing conserved residues in their R2R3 DNA binding domains. Insertion of the apple R6 motif into orthologous promoters of MYB10 in pear (PcMYB10 and Arabidopsis (AtMY75 elevated anthocyanin levels. Introduction of the R6 motif into the promoter region of an anthocyanin biosynthetic enzyme F3′5′H of kiwifruit imparts regulation by MYB10. This results in elevated levels of delphinidin in both tobacco and kiwifruit. Finally, an R6 motif inserted into the promoter the vitamin C biosynthesis gene GDP-L-Gal phosphorylase increases vitamin C content in a MYB10-dependent manner. This motif therefore provides a tool to re-engineer novel MYB-regulated responses in plants.

  15. DNA binding site analysis of Burkholderia thailandensis response regulators.

    Science.gov (United States)

    Nowak-Lovato, Kristy L; Hickmott, Alexana J; Maity, Tuhin S; Bulyk, Martha L; Dunbar, John; Hong-Geller, Elizabeth

    2012-07-01

    Bacterial response regulators (RR) that function as transcription factors in two component signaling pathways are crucial for ensuring tight regulation and coordinated expression of the genome. Currently, consensus DNA binding sites in the promoter for very few bacterial RRs have been identified. A systematic method to characterize these DNA binding sites for RRs would enable prediction of specific gene expression patterns in response to extracellular stimuli. To identify RR DNA binding sites, we functionally activated RRs using beryllofluoride and applied them to a protein-binding microarray (PBM) to discover DNA binding motifs for RRs expressed in Burkholderia, a Gram-negative bacterial genus. We identified DNA binding motifs for conserved RRs in Burkholderia thailandensis, including KdpE, RisA, and NarL, as well as for a previously uncharacterized RR at locus BTH_II2335 and its ortholog in the human pathogen Burkholderia pseudomallei at locus BPSS2315. We further demonstrate RR binding of predicted genomic targets for the two orthologs using gel shift assays and reveal a pattern of RR regulation of expression of self and other two component systems. Our studies illustrate the use of PBMs to identify DNA binding specificities for bacterial RRs and enable prediction of gene regulatory networks in response to two component signaling. Published by Elsevier B.V.

  16. Hanford Site environmental management specification

    Energy Technology Data Exchange (ETDEWEB)

    Grygiel, M.L.

    1998-06-10

    The US Department of Energy, Richland Operations Office (RL) uses this Hanford Site Environmental Management Specification (Specification) to document top-level mission requirements and planning assumptions for the prime contractors involved in Hanford Site cleanup and infrastructure activities under the responsibility of the US Department of Energy, Office of Environmental Management. This Specification describes at a top level the activities, facilities, and infrastructure necessary to accomplish the cleanup of the Hanford Site and assigns this scope to Site contractors and their respective projects. This Specification also references the key National Environmental Policy Act of 1969 (NEPA), Comprehensive Environmental Response, Compensation, and Liability Act of 1980 (CERCLA), and safety documentation necessary to accurately describe the cleanup at a summary level. The information contained in this document reflects RL`s application of values, priorities, and critical success factors expressed by those involved with and affected by the Hanford Site project. The prime contractors and their projects develop complete baselines and work plans to implement this Specification. These lower-level documents and the data that support them, together with this Specification, represent the full set of requirements applicable to the contractors and their projects. Figure 1-1 shows the relationship of this Specification to the other basic Site documents. Similarly, the documents, orders, and laws referenced in this specification represent only the most salient sources of requirements. Current and contractual reference data contain a complete set of source documents.

  17. Molt-inhibiting hormone stimulates vitellogenesis at advanced ovarian developmental stages in the female blue crab, Callinectes sapidus 2: novel specific binding sites in hepatopancreas and cAMP as a second messenger

    Science.gov (United States)

    Zmora, Nilli; Sagi, Amir; Zohar, Yonathan; Chung, J Sook

    2009-01-01

    The finding that molt-inhibiting hormone (MIH) regulates vitellogenesis in the hepatopancreas of mature Callinectes sapidus females, raised the need for the characterization of its mode of action. Using classical radioligand binding assays, we located specific, saturable, and non-cooperative binding sites for MIH in the Y-organs of juveniles (J-YO) and in the hepatopancreas of vitellogenic adult females. MIH binding to the hepatopancreas membranes had an affinity 77 times lower than that of juvenile YO membranes (KD values: 3.22 × 10-8 and 4.19 × 10-10 M/mg protein, respectively). The number of maximum binding sites (BMAX) was approximately two times higher in the hepatopancreas than in the YO (BMAX values: 9.24 × 10-9 and 4.8 × 10-9 M/mg protein, respectively). Furthermore, MIH binding site number in the hepatopancreas was dependent on ovarian stage and was twice as high at stage 3 than at stages 2 and 1. SDS-PAGE separation of [125I] MIH or [125I] crustacean hyperglycemic hormone (CHH) crosslinked to the specific binding sites in the membranes of the J-YO and hepatopancreas suggests a molecular weight of ~51 kDa for a MIH receptor in both tissues and a molecular weight of ~61 kDa for a CHH receptor in the hepatopancreas. The use of an in vitro incubation of hepatopancreas fragments suggests that MIH probably utilizes cAMP as a second messenger in this tissue, as cAMP levels increased in response to MIH. Additionally, 8-Bromo-cAMP mimicked the effects of MIH on vitellogenin (VtG) mRNA and heterogeneous nuclear (hn) VtG RNA levels. The results imply that the functions of MIH in the regulation of molt and vitellogenesis are mediated through tissue specific receptors with different kinetics and signal transduction. MIH ability to regulate vitellogenesis is associated with the appearance of MIH specific membrane binding sites in the hepatopancreas upon pubertal/final molt. PMID:19583849

  18. Improved mapping of protein binding sites

    Science.gov (United States)

    Kortvelyesi, Tamas; Silberstein, Michael; Dennis, Sheldon; Vajda, Sandor

    2003-02-01

    Computational mapping methods place molecular probes - small molecules or functional groups - on a protein surface in order to identify the most favorable binding positions by calculating an interaction potential. Mapping is an important step in a number of flexible docking and drug design algorithms. We have developed improved algorithms for mapping protein surfaces using small organic molecules as molecular probes. The calculations reproduce the binding of eight organic solvents to lysozyme as observed by NMR, as well as the binding of four solvents to thermolysin, in good agreement with x-ray data. Application to protein tyrosine phosphatase 1B shows that the information provided by the mapping can be very useful for drug design. We also studied why the organic solvents bind in the active site of proteins, in spite of the availability of alternative pockets that can very tightly accommodate some of the probes. A possible explanation is that the binding in the relatively large active site retains a number of rotational states, and hence leads to smaller entropy loss than the binding elsewhere else. Indeed, the mapping reveals that the clusters of the ligand molecules in the protein's active site contain different rotational-translational conformers, which represent different local minima of the free energy surface. In order to study the transitions between different conformers, reaction path and molecular dynamics calculations were performed. Results show that most of the rotational states are separated by low free energy barriers at the experimental temperature, and hence the entropy of binding in the active site is expected to be high.

  19. Site-specific conjugation of a lanthanide chelator and its effects on the chemical synthesis and receptor binding affinity of human relaxin-2 hormone

    Energy Technology Data Exchange (ETDEWEB)

    Shabanpoor, Fazel [Florey Neuroscience Institutes, The University of Melbourne, Parkville, VIC 3010 (Australia); School of Chemistry, The University of Melbourne, Parkville, VIC 3010 (Australia); Bathgate, Ross A.D.; Belgi, Alessia [Florey Neuroscience Institutes, The University of Melbourne, Parkville, VIC 3010 (Australia); Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, VIC 3010 (Australia); Chan, Linda J.; Nair, Vinojini B.; Wade, John D. [Florey Neuroscience Institutes, The University of Melbourne, Parkville, VIC 3010 (Australia); School of Chemistry, The University of Melbourne, Parkville, VIC 3010 (Australia); Hossain, Mohammed Akhter, E-mail: akhter.hossain@florey.edu.au [Florey Neuroscience Institutes, The University of Melbourne, Parkville, VIC 3010 (Australia); School of Chemistry, The University of Melbourne, Parkville, VIC 3010 (Australia)

    2012-04-06

    Highlights: Black-Right-Pointing-Pointer A mono-Eu-DTPA conjugated peptide ligand, Eu-DTPA-(A)-H2, has been developed. Black-Right-Pointing-Pointer The choice of a site for incorporation of a chelator is critical. Black-Right-Pointing-Pointer The labeled peptide retains full activity at the RXFP1 receptor. Black-Right-Pointing-Pointer It is markedly cheaper to produce and easier to use than radioactive probes. -- Abstract: Diethylenetriamine pentaacetic acid (DTPA) is a popular chelator agent for enabling the labeling of peptides for their use in structure-activity relationship study and biodistribution analysis. Solid phase peptide synthesis was employed to couple this commercially available chelator at the N-terminus of either the A-chain or B-chain of H2 relaxin. The coupling of the DTPA chelator at the N-terminus of the B-chain and subsequent loading of a lanthanide (europium) ion into the chelator led to a labeled peptide (Eu-DTPA-(B)-H2) in low yield and having very poor water solubility. On the other hand, coupling of the DTPA and loading of Eu at the N-terminus of the A-chain led to a water-soluble peptide (Eu-DTPA-(A)-H2) with a significantly improved final yield. The conjugation of the DTPA chelator at the N-terminus of the A-chain did not have any impact on the secondary structure of the peptide determined by circular dichroism spectroscopy (CD). On the other hand, it was not possible to determine the secondary structure of Eu-DTPA-(B)-H2 because of its insolubility in phosphate buffer. The B-chain labeled peptide Eu-DTPA-(B)-H2 required solubilization in DMSO prior to carrying out binding assays, and showed lower affinity for binding to H2 relaxin receptor, RXFP1, compared to the water-soluble A-chain labeled peptide Eu-DTPA-(A)-H2. The mono-Eu-DTPA labeled A-chain peptide, Eu-DTPA-(A)-H2, thus can be used as a valuable probe to study ligand-receptor interactions of therapeutically important H2 relaxin analogs. Our results show that it is critical to

  20. 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.

  1. Binding sites for gonadotropins in human postmenopausal ovaries

    Energy Technology Data Exchange (ETDEWEB)

    Nakano, R.; Shima, K.; Yamoto, M.; Kobayashi, M.; Nishimori, K.; Hiraoka, J.

    1989-02-01

    The binding of human LH and human FSH to postmenopausal ovarian tissue from 21 patients with cervical carcinoma was analyzed. The binding sites for FSH and LH were demonstrated in postmenopausal ovarian tissue. The surface-binding sites for gonadotropins were localized in the cells of cortical stroma of the postmenopausal ovary. In addition, diffuse cytoplasmic staining of endogenous estrogen and 3 beta-hydroxysteroid dehydrogenase activity were detected immunohistochemically and histochemically in the cells of the cortical stroma. Electron microscopic study also suggested steroidogenic function in the cells of the cortical stroma. The results of the present study suggest that postmenopausal ovaries contain specific binding sites for pituitary gonadotropins and play a role in ovarian steroidogenesis.

  2. An HIV-1 envelope immunogen with W427S mutation in CD4 binding site induced more T follicular helper memory cells and reduced non-specific antibody responses.

    Directory of Open Access Journals (Sweden)

    Hao-Tong Yu

    Full Text Available The CD4 binding site (CD4BS of the HIV-1 envelope glycoprotein (Env contains epitopes for broadly neutralizing antibody (nAb and is the target for the vaccine development. However, the CD4BS core including residues 425-430 overlaps the B cell superantigen site and may be related to B cell exhaustion in HIV-1 infection. Furthermore, production of nAb and high-affinity plasma cells needs germinal center reaction and the help of T follicular helper (Tfh cells. We believe that strengthening the ability of Env CD4BS in inducing Tfh response and decreasing the effects of the superantigen are the strategies for eliciting nAb and development of HIV-1 vaccine. We constructed a gp120 mutant W427S of an HIV-1 primary R5 strain and examined its ability in the elicitation of Ab and the production of Tfh by immunization of BALB/c mice. We found that the trimeric wild-type gp120 can induce more non-specific antibody-secreting plasma cells, higher serum IgG secretion, and more Tfh cells by splenocyte. The modified W427S gp120 elicits higher levels of specific binding antibodies as well as nAbs though it produces less Tfh cells. Furthermore, higher Tfh cell frequency does not correlate to the specific binding Abs or nAbs indicating that the wild-type gp120 induced some non-specific Tfh that did not contribute to the production of specific Abs. This gp120 mutant led to more memory Tfh production, especially, the effector memory Tfh cells. Taken together, W427S gp120 could induce higher level of specific binding and neutralizing Ab production that may be associated with the reduction of non-specific Tfh but strengthening of the memory Tfh.

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

    Directory of Open Access Journals (Sweden)

    Bitter István

    2010-10-01

    , except for few specific cases, the shapes of the binding pockets have relatively low weights in the determination of the affinity profiles of proteins. Since the MAF profile is closely related to the target specificity of ligand binding sites we can conclude that the shape of the binding site is not a pivotal factor in selecting drug targets. Nonetheless, based on strong specific associations between certain MAF profiles and specific geometric descriptors we identified, the shapes of the binding sites do have a crucial role in virtual drug design for certain drug categories, including morphine derivatives, benzodiazepines, barbiturates and antihistamines.

  4. Druggability of methyl-lysine binding sites

    Science.gov (United States)

    Santiago, C.; Nguyen, K.; Schapira, M.

    2011-12-01

    Structural modules that specifically recognize—or read—methylated or acetylated lysine residues on histone peptides are important components of chromatin-mediated signaling and epigenetic regulation of gene expression. Deregulation of epigenetic mechanisms is associated with disease conditions, and antagonists of acetyl-lysine binding bromodomains are efficacious in animal models of cancer and inflammation, but little is known regarding the druggability of methyl-lysine binding modules. We conducted a systematic structural analysis of readers of methyl marks and derived a predictive druggability landscape of methyl-lysine binding modules. We show that these target classes are generally less druggable than bromodomains, but that some proteins stand as notable exceptions.

  5. 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.

  6. Probing the phalloidin binding site of actin.

    Science.gov (United States)

    Faulstich, H; Zobeley, S; Heintz, D; Drewes, G

    1993-03-08

    Phallotoxins form tight complexes with filamentous actin and stabilize the polymer against shearing stress. In the present study a phalloidin derivative containing a thiol-capturing moiety was prepared and reacted with single thiol groups of monomeric muscle actin. Sites of attachment in the protein were Cys-374 next to the C-terminus and Cys-10, close to the N-terminus; the latter was recently shown to be uncovered during a slow but reversible conformational transition occurring in ADP-G-actin. Phalloidin bound to Cys-374 stabilizes filaments against shearing stress almost as effectively as free phalloidin, indicating that the phalloidin binding site cannot be far from the C-terminus of actin. Stabilization was also achieved when the phalloidin reagent was added to F-actin, however, the subsequent formation of a covalent linkage with Cys-374 was not observed, most likely due to a restricted mobility of the reactants. In contrast to the efficient stabilization of filaments by phalloidin linked to Cys-374 a destabilizing effect was observed when phalloidin was attached to Cys-10. It appears that phalloidin located close to the N-terminus is unable to bind to the normal binding site in its own filament. Pronounced gelification of this actin derivative suggests that the toxin is able to mediate crosslinking with neighbouring filaments. From these results we conclude that the phalloidin binding site of actin is distant from the N-terminus, but close to the C-terminus. Furthermore, the data provide evidence that binding of phalloidin reduces the mobility of the C-terminus.

  7. Quantitative modeling of transcription factor binding specificities using DNA shape.

    Science.gov (United States)

    Zhou, Tianyin; Shen, Ning; Yang, Lin; Abe, Namiko; Horton, John; Mann, Richard S; Bussemaker, Harmen J; Gordân, Raluca; Rohs, Remo

    2015-04-14

    DNA binding specificities of transcription factors (TFs) are a key component of gene regulatory processes. Underlying mechanisms that explain the highly specific binding of TFs to their genomic target sites are poorly understood. A better understanding of TF-DNA binding requires the ability to quantitatively model TF binding to accessible DNA as its basic step, before additional in vivo components can be considered. Traditionally, these models were built based on nucleotide sequence. Here, we integrated 3D DNA shape information derived with a high-throughput approach into the modeling of TF binding specificities. Using support vector regression, we trained quantitative models of TF binding specificity based on protein binding microarray (PBM) data for 68 mammalian TFs. The evaluation of our models included cross-validation on specific PBM array designs, testing across different PBM array designs, and using PBM-trained models to predict relative binding affinities derived from in vitro selection combined with deep sequencing (SELEX-seq). Our results showed that shape-augmented models compared favorably to sequence-based models. Although both k-mer and DNA shape features can encode interdependencies between nucleotide positions of the binding site, using DNA shape features reduced the dimensionality of the feature space. In addition, analyzing the feature weights of DNA shape-augmented models uncovered TF family-specific structural readout mechanisms that were not revealed by the DNA sequence. As such, this work combines knowledge from structural biology and genomics, and suggests a new path toward understanding TF binding and genome function.

  8. Penicillin-binding site on the Escherichia coli cell envelope

    Energy Technology Data Exchange (ETDEWEB)

    Amaral, L.; Lee, Y.; Schwarz, U.; Lorian, V.

    1986-08-01

    The binding of /sup 35/S-labeled penicillin to distinct penicillin-binding proteins (PBPs) of the cell envelope obtained from the sonication of Escherichia coli was studied at different pHs ranging from 4 to 11. Experiments distinguishing the effect of pH on penicillin binding by PBP 5/6 from its effect on beta-lactamase activity indicated that although substantial binding occurred at the lowest pH, the amount of binding increased with pH, reaching a maximum at pH 10. Based on earlier studies, it is proposed that the binding at high pH involves the formation of a covalent bond between the C-7 of penicillin and free epsilon amino groups of the PBPs. At pHs ranging from 4 to 8, position 1 of penicillin, occupied by sulfur, is considered to be the site that establishes a covalent bond with the sulfhydryl groups of PBP 5. The use of specific blockers of free epsilon amino groups or sulfhydryl groups indicated that wherever the presence of each had little or no effect on the binding of penicillin by PBP 5, the presence of both completely prevented binding. The specific blocker of the hydroxyl group of serine did not affect the binding of penicillin.

  9. Biophysical fitness landscapes for transcription factor binding sites.

    Directory of Open Access Journals (Sweden)

    Allan Haldane

    2014-07-01

    Full Text Available Phenotypic states and evolutionary trajectories available to cell populations are ultimately dictated by complex interactions among DNA, RNA, proteins, and other molecular species. Here we study how evolution of gene regulation in a single-cell eukaryote S. cerevisiae is affected by interactions between transcription factors (TFs and their cognate DNA sites. Our study is informed by a comprehensive collection of genomic binding sites and high-throughput in vitro measurements of TF-DNA binding interactions. Using an evolutionary model for monomorphic populations evolving on a fitness landscape, we infer fitness as a function of TF-DNA binding to show that the shape of the inferred fitness functions is in broad agreement with a simple functional form inspired by a thermodynamic model of two-state TF-DNA binding. However, the effective parameters of the model are not always consistent with physical values, indicating selection pressures beyond the biophysical constraints imposed by TF-DNA interactions. We find little statistical support for the fitness landscape in which each position in the binding site evolves independently, indicating that epistasis is common in the evolution of gene regulation. Finally, by correlating TF-DNA binding energies with biological properties of the sites or the genes they regulate, we are able to rule out several scenarios of site-specific selection, under which binding sites of the same TF would experience different selection pressures depending on their position in the genome. These findings support the existence of universal fitness landscapes which shape evolution of all sites for a given TF, and whose properties are determined in part by the physics of protein-DNA interactions.

  10. Biophysical fitness landscapes for transcription factor binding sites.

    Science.gov (United States)

    Haldane, Allan; Manhart, Michael; Morozov, Alexandre V

    2014-07-01

    Phenotypic states and evolutionary trajectories available to cell populations are ultimately dictated by complex interactions among DNA, RNA, proteins, and other molecular species. Here we study how evolution of gene regulation in a single-cell eukaryote S. cerevisiae is affected by interactions between transcription factors (TFs) and their cognate DNA sites. Our study is informed by a comprehensive collection of genomic binding sites and high-throughput in vitro measurements of TF-DNA binding interactions. Using an evolutionary model for monomorphic populations evolving on a fitness landscape, we infer fitness as a function of TF-DNA binding to show that the shape of the inferred fitness functions is in broad agreement with a simple functional form inspired by a thermodynamic model of two-state TF-DNA binding. However, the effective parameters of the model are not always consistent with physical values, indicating selection pressures beyond the biophysical constraints imposed by TF-DNA interactions. We find little statistical support for the fitness landscape in which each position in the binding site evolves independently, indicating that epistasis is common in the evolution of gene regulation. Finally, by correlating TF-DNA binding energies with biological properties of the sites or the genes they regulate, we are able to rule out several scenarios of site-specific selection, under which binding sites of the same TF would experience different selection pressures depending on their position in the genome. These findings support the existence of universal fitness landscapes which shape evolution of all sites for a given TF, and whose properties are determined in part by the physics of protein-DNA interactions.

  11. Computational identification of uncharacterized cruzain binding sites.

    Directory of Open Access Journals (Sweden)

    Jacob D Durrant

    2010-05-01

    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.

  12. GlnR negatively regulates the transcription of the alanine dehydrogenase encoding gene ald in Amycolatopsis mediterranei U32 under nitrogen limited conditions via specific binding to its major transcription initiation site.

    Directory of Open Access Journals (Sweden)

    Ying Wang

    Full Text Available Ammonium assimilation is catalyzed by two enzymatic pathways, i.e., glutamine synthetase/glutamate synthase (GS/GOGAT and alanine dehydrogenase (AlaDH in Amycolatopsis mediterranei U32. Under nitrogen-rich conditions, the AlaDH pathway is the major route for ammonium assimilation, while the GS/GOGAT pathway takes over when the extracellular nitrogen supply is limited. The global nitrogen regulator GlnR was previously characterized to activate the transcription of the GS encoding gene glnA in response to nitrogen limitation and is demonstrated in this study as a repressor for the transcription of the AlaDH encoding gene ald, whose regulation is consistent with the switch of the ammonium assimilation pathways from AlaDH to GS/GOGAT responding to nitrogen limitation. Three transcription initiation sites (TISs of ald were determined with primer extension assay, among which transcription from aldP2 contributed the major transcripts under nitrogen-rich conditions but was repressed to an undetectable level in response to nitrogen limitation. Through DNase I footprinting assay, two separate regions were found to be protected by GlnR within ald promoter, within which three GlnR binding sites (a1, b1 sites in region I and a2 site in region II were defined. Interestingly, the major TIS aldP2 is located in the middle of a2 site within region II. Therefore, one may easily conclude that GlnR represses the transcription of ald via specific binding to the GlnR binding sites, which obviously blocks the transcription initiation from aldP2 and therefore reduces ald transcripts.

  13. Localization of binding sites of Ulex europaeus I, Helix pomatia and Griffonia simplicifolia I-B4 lectins and analysis of their backbone structures by several glycosidases and poly-N-acetyllactosamine-specific lectins in human breast carcinomas.

    Science.gov (United States)

    Ito, N; Imai, S; Haga, S; Nagaike, C; Morimura, Y; Hatake, K

    1996-09-01

    Several studies have shown the deletion of blood group A or B antigens and the accumulation of H antigens in human breast carcinomas. Other studies have independently demonstrated that the binding sites of lectins such as Helix pomatia agglutinin (HPA) and Griffonia simplicifolia agglutinin I-B4 (GSAI-B4) are highly expressed in these cells. In order to clarify the molecular mechanisms of malignant transformation and metastasis of carcinoma cells, it is important to understand the relationship between such phenotypically distinct events. For this purpose, we examined whether the binding sites of these lectins and Ulex europaeus agglutinin I (UEA-I) are expressed concomitantly in the same carcinoma cells and analyzed their backbone structures. The expression of the binding sites of these lectins was observed independently of the blood group (ABO) of the patients and was not affected by the histological type of the carcinomas. Observation of serial sections stained with these lectins revealed that the distribution of HPA binding sites was almost identical to that of GSAI-B4 in most cases. Furthermore, in some cases, UEA-I binding patterns were similar to those of HPA and GSAI-B4 but in other cases, mosaic staining patterns with these lectins were also observed, i.e., some cell clusters were stained with both HPA and GSAI-B4 but not with UEA-I and adjacent cell clusters were stained only with UEA-I. Digestion with endo-beta-galactosidase or N-glycosidase F markedly reduced the staining intensity of these lectins. Together with the reduction of staining by these lectins, reactivity with Griffonia simplicifolia agglutinin II appeared in carcinoma cells following endo-beta-galactosidase digestion. Among the lectins specific to poly-N-acetyllactosamine, Lycopersicon esculentum agglutinin (LEA) most vividly and consistently stained the cancer cells. Next to LEA, pokeweed mitogen agglutinin was also effective in staining these cells. Carcinoma cells reactive with these

  14. 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.

  15. LIGAND-BINDING SITES ON THE MYCOBACTERIUM TUBERCULOSIS UREASE

    Directory of Open Access Journals (Sweden)

    Lisnyak Yu. V.

    2017-10-01

    Full Text Available Introduction. Mycobacterium tuberculosis is the causative agent of tuberculosis that remains a serious medical and social health problem. Despite intensive efforts have been made in the past decade, there are no new efficient anti-tuberculosis drugs today, and that need is growing due to the spread of drug-resistant strains of M.tuberculosis. M. tuberculosis urease (MTU, being an important factor of the bacterium viability and virulence, is an attractive target for anti-tuberculosis drugs acting by inhibition of urease activity. However, the commercially available urease inhibitors are toxic and unstable, that prevent their clinical use. Therefore, new more potent anti-tuberculosis drugs inhibiting new targets are urgently needed. A useful tool for the search of novel inhibitors is a computational drug design. The inhibitor design is significantly easier if binding sites on the enzyme are identified in advance. This paper aimed to determine the probable ligand binding sites on the surface of M. tuberculosis urease. Methods. To identify ligand binding sites on MTU surface, сomputational solvent mapping method FTSite was applied by the use of MTU homology model we have built earlier. The method places molecular probes (small organic molecules containing various functional groups on a dense grid defined around the enzyme, and for each probe finds favorable positions. The selected poses are refined by free energy minimization, the low energy conformations are clustered, and the clusters are ranked on the basis of the average free energy. FTSite server outputs the protein residues delineating a binding sites and the probe molecules representing each cluster. To predict allosteric pockets on MTU, AlloPred and AlloSite servers were applied. AlloPred uses the normal mode analysis (NMA and models how the dynamics of a protein would be altered in the presence of a modulator at a specific pocket. Pockets on the enzyme are predicted using the Fpocket

  16. Sex-Dependent, Osteoblast Stage-Specific Effects of Progesterone Receptor on Bone Acquisition.

    Science.gov (United States)

    Zhong, Zhendong A; Kot, Alexander; Lay, Yu-An E; Zhang, Hongliang; Jia, Junjing; Lane, Nancy E; Yao, Wei

    2017-09-01

    The role of the progesterone receptor (PR) in the regulation of sexual dimorphism in bone has yet to be determined. Here we utilized genetic fate mapping and Western blotting to demonstrate age-dependent PR expression in the mouse femoral metaphysis and diaphysis. To define sex-dependent and osteoblast stage-specific effects of PR on bone acquisition, we selectively deleted PR at different stages of osteoblast differentiation. We found that when Prx1-Cre mice were crossed with PR floxed mice to generate a mesenchymal stem cell (MSC) conditional KO model (Prx1; PRcKO), the mutant mice developed greater trabecular bone volume with higher mineral apposition rate and bone formation. This may be explained by increased number of MSCs and greater osteogenic potential, particularly in males. Age-related trabecular bone loss was similar between the Prx1; PRcKO mice and their WT littermates in both sexes. Hormone deficiency during the period of rapid bone growth induced rapid trabecular bone loss in both the WT and the Prx1; PRcKO mice in both sexes. No differences in trabecular bone mass was observed when PR was deleted in mature osteoblasts using osteocalcin-Cre (Bglap-Cre). Also, there were no differences in cortical bone mass in all three PRcKO mice. In conclusion, PR inactivation in early osteoprogenitor cells but not in mature osteoblasts influenced trabecular bone accrual in a sex-dependent manner. PR deletion in osteoblast lineage cells did not affect cortical bone mass. © 2017 American Society for Bone and Mineral Research. © 2017 American Society for Bone and Mineral Research.

  17. Peripheral benzodiazepine binding sites on striated muscles of the rat: Properties and effect of denervation

    Energy Technology Data Exchange (ETDEWEB)

    Mueller, W.E.; Ickstadt, A. (Mainz Univ. (Germany, F.R.). Pharmakologisches Inst.); Hopf, H.Ch. (Mainz Univ. (Germany, F.R.))

    1985-01-01

    In order to test the hypothesis that peripheral benzodiazepine binding sites mediate some direct effects of benzodiazepines on striated muscles, the properties of specific /sup 3/H-Ro 5-4864 binding to rat biceps and rat diaphragm homogenates were investigated. In both tissues a single population of sites was found with a Ksub(D) value of 3 nmol/l. The density of these sites in both muscles was higher than the density in rat brain, but was considerably lower than in rat kidney. Competition experiments indicate a substrate specificity of specific /sup 3/H-Ro 5-4864 binding similar to the properties already demonstrated for the specific binding of this ligand to peripheral benzodiazepine binding sites in many other tissues. The properties of these sites in the rat diaphragm are not changed after motoric denervation by phrenicectomy. It is concluded that peripheral benzodiazepine binding sites are not involved in direct effects of benzodiazepines on striated muscles.

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

    OpenAIRE

    Hofmann, C; Vanderbruggen, H; Höfte, H.; Van Rie, J.; Jansens, S.; Van Mellaert, H

    1988-01-01

    Binding studies were performed with two 125I-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 ...

  19. Binding of two desmin derivatives to the plasma membrane and the nuclear envelope of avian erythrocytes: evidence for a conserved site-specificity in intermediate filament-membrane interactions

    Energy Technology Data Exchange (ETDEWEB)

    Georgatos, S.D.; Weber, K.; Geisler, N.; Blobel, G.

    1987-10-01

    Using solution binding assays, the authors found that a 45-kDa fragment of /sup 125/I-labelled desmin, lacking 67 residues from the N terminus, could specifically associate with avian erythrocyte nuclear envelopes but not with plasma membranes from the same cells. It was also observed that a 50-kDa desmin peptide, missing 27 C-terminal residues, retained the ability to bind to both membrane preparations. Displacement experiments with an excess of purified vimentin suggested that the two desmin derivatives were interacting with a previously identified vimentin receptor at the nuclear envelope, the protein lamin B. Additional analysis by affinity chromatography confirmed this conclusion. Employing an overlay assay, they demonstrated that the 50-kDa fragment, but not the 45-kDa desmin peptide, was capable of interacting with the plasma membrane polypeptide ankyrin (a known vimentin attachment site), as was intact vimentin. Conversely, the nuclear envelope protein lamin B was recognized by both fragments but not by a chymotryptic peptide composed solely of the helical rod domain of desmin. These data imply that the lamin B-binding site on desmin resides within the 21 residues following its helical rod domain, whereas the ankyrin-associating region is localized within its N-terminal head domain, exactly as in the case of vimentin.

  20. Progesterone receptor membrane component 1 and its role in ovarian follicle growth

    Directory of Open Access Journals (Sweden)

    John J Peluso

    2013-06-01

    Full Text Available Progesterone (P4 is synthesized in the ovary and acts directly on granulosa cells of developing ovarian follicles to suppress their rate of mitosis and apoptosis. Granulosa cells do not express nuclear progesterone receptor (PGR but rather progesterone receptor membrane component-1 (PGRMC1. PGRMC1 binds P4 and mediates P4’s actions, as evidenced by PGRMC1 siRNA studies. PGRMC1 acts by binding plasminogen activator inhibitor 1 RNA binding protein and regulating gene expression. Specifically, PGRMC1 suppresses some genes that promote cell death (i.e. Bad, Caspase-3, Caspase-4. P4 regulates gene expression in part by inhibiting PGRMC1 binding to Tcf/Lef transcription sites, thereby reducing Tcf/Lef transcriptional activity. Since Tcf/Lef transcription sites are located within the promoters of genes that initiate mitosis and/or apoptosis (i.e. c-jun and c-myc, P4-PGRMC1 mediated suppression of these Tcf/Lef regulated genes could account for P4’s actions. PGRMC1 expression is also altered in women with polycystic ovarian syndrome, premature ovarian failure and infertility. Collectively, these observations support a role for PGRMC1 in regulating human ovarian follicle development.

  1. A single glycine-alanine exchange directs ligand specificity of the elephant progestin receptor.

    Directory of Open Access Journals (Sweden)

    Michael Wierer

    Full Text Available The primary gestagen of elephants is 5α-dihydroprogesterone (DHP, which is unlike all other mammals studied until now. The level of DHP in elephants equals that of progesterone in other mammals, and elephants are able to bind DHP with similar affinity to progesterone indicating a unique ligand-binding specificity of the elephant progestin receptor (PR. Using site-directed mutagenesis in combination with in vitro binding studies we here report that this change in specificity is due to a single glycine to alanine exchange at position 722 (G722A of PR, which specifically increases DHP affinity while not affecting binding of progesterone. By conducting molecular dynamics simulations comparing human and elephant PR ligand-binding domains (LBD, we observed that the alanine methyl group at position 722 is able to push the DHP A-ring into a position similar to progesterone. In the human PR, the DHP A-ring position is twisted towards helix 3 of PR thereby disturbing the hydrogen bond pattern around the C3-keto group, resulting in a lower binding affinity. Furthermore, we observed that the elephant PR ligand-binding pocket is more rigid than the human analogue, which probably explains the higher affinity towards both progesterone and DHP. Interestingly, the G722A substitution is not elephant-specific, rather it is also present in five independent lineages of mammalian evolution, suggesting a special role of the substitution for the development of distinct mammalian gestagen systems.

  2. Binding sites analyser (BiSA: software for genomic binding sites archiving and overlap analysis.

    Directory of Open Access Journals (Sweden)

    Matloob Khushi

    Full Text Available Genome-wide mapping of transcription factor binding and histone modification reveals complex patterns of interactions. Identifying overlaps in binding patterns by different factors is a major objective of genomic studies, but existing methods to archive large numbers of datasets in a personalised database lack sophistication and utility. Therefore we have developed transcription factor DNA binding site analyser software (BiSA, for archiving of binding regions and easy identification of overlap with or proximity to other regions of interest. Analysis results can be restricted by chromosome or base pair overlap between regions or maximum distance between binding peaks. BiSA is capable of reporting overlapping regions that share common base pairs; regions that are nearby; regions that are not overlapping; and average region sizes. BiSA can identify genes located near binding regions of interest, genomic features near a gene or locus of interest and statistical significance of overlapping regions can also be reported. Overlapping results can be visualized as Venn diagrams. A major strength of BiSA is that it is supported by a comprehensive database of publicly available transcription factor binding sites and histone modifications, which can be directly compared to user data. The documentation and source code are available on http://bisa.sourceforge.net.

  3. Effects of mutagenesis of aspartic acid residues in the putative phosphoribosyl diphosphate binding site of Escherichia coli phosphoribosyl diphosphate synthetase on metal ion specificity and ribose-5-phosphate binding

    DEFF Research Database (Denmark)

    Willemoës, Martin; Nilsson, Dan; Hove-Jensen, Bjarne

    1996-01-01

    The three conserved aspartic acid residues of the 5-phospho-d-ribosyl a-1-diphosphate binding site (213-GRDCVLVDDMIDTGGT-228) of Escherichia coli phosphoribosyl diphosphate synthetase were studied by analysis of the mutant enzymes D220E, D220F, D221A, D224A, and D224S. The mutant enzymes showed...... an increase in KM for ribose 5-phosphate in the presence of at least one of the divalent metal ions Mg2+, Mn2+, Co2+, or Cd2+, with the most dramatic changes revealed by the D220E and D220F enzymes in the presence of Co2+ and the D221A enzyme in the presence of Mn2+ or Co2+. The D220F and D221A enzymes both...... showed large decreases in Vapp in the presence of the various divalent metal ions, except for the D221A enzyme in the presence of Co2+. Vapp of the D220E enzyme was similar to that of the wild-type enzyme in the presence of Mg2+, Mn2+, or Cd2+, whereas the Vapp was increased in the presence of Co2+. Vapp...

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

    Science.gov (United States)

    Khorshid, Mohsen; Rodak, Christoph; Zavolan, Mihaela

    2011-01-01

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

  5. Cell-specific localization of progesterone receptors in the bovine ovary at different stages of the oestrous cycle.

    Science.gov (United States)

    D'Haeseleer, M; Simoens, P; Van den Broeck, W

    2007-04-01

    This immunohistochemical study describes the localization of progesterone receptors (PR) in the bovine ovary of 23 cows at different stages of the oestrous cycle. In primordial, primary and secondary follicles the score for PR in the follicle cells increased progressively with the maturation of the follicle. In vital tertiary follicles and cystic atretic follicles a moderate score for PR was found, while in obliterative atretic follicles the score was much lower. Scores were high in corpora hemorrhagica, low in corpora lutea and still lower in corpora albicantia. Low PR scores were also found in the tunica albuginea and surface epithelium. Cyclic variations of PR immunoreactivity were manifest in most ovarian tissues. Follicular scores for PR were high in oestrus and decreased during the following stages, whereas scores in corpora lutea cells varied according to a characteristic pattern with high levels during oestrus and metoestrus. The variations in the scores for PR in the different ovarian cell types suggest a cell-specific and cycle-dependent influence of progesterone. A negative correlation was found between the PR scores and the plasma progesterone concentration.

  6. Binding site number variation and high-affinity binding consensus of Myb-SANT-like transcription factor Adf-1 in Drosophilidae

    OpenAIRE

    Lang, Michael; Juan, Elvira

    2010-01-01

    There is a growing interest in the evolution of transcription factor binding sites and corresponding functional change of transcriptional regulation. In this context, we have examined the structural changes of the ADF-1 binding sites at the Adh promoters of Drosophila funebris and D. virilis. We detected an expanded footprinted region in D. funebris that contains various adjacent binding sites with different binding affinities. ADF-1 was described to direct sequence-specific DNA binding to si...

  7. Design of sequence-specific DNA-binding molecules.

    Science.gov (United States)

    Dervan, P B

    1986-04-25

    Base sequence information can be stored in the local structure of right-handed double-helical DNA (B-DNA). The question arises as to whether a set of rules for the three-dimensional readout of the B-DNA helix can be developed. This would allow the design of synthetic molecules that bind DNA of any specific sequence and site size. There are four stages of development for each new synthetic sequence-specific DNA-binding molecule: design, synthesis, testing for sequence specificity, and reevaluation of the design. This approach has produced bis(distamycin)fumaramide, a synthetic, crescent-shaped oligopeptide that binds nine contiguous adenine-thymine base pairs in the minor groove of double-helical DNA.

  8. Structural Fingerprints of Transcription Factor Binding Site Regions

    Directory of Open Access Journals (Sweden)

    Peter Willett

    2009-03-01

    Full Text Available Fourier transforms are a powerful tool in the prediction of DNA sequence properties, such as the presence/absence of codons. We have previously compiled a database of the structural properties of all 32,896 unique DNA octamers. In this work we apply Fourier techniques to the analysis of the structural properties of human chromosomes 21 and 22 and also to three sets of transcription factor binding sites within these chromosomes. We find that, for a given structural property, the structural property power spectra of chromosomes 21 and 22 are strikingly similar. We find common peaks in their power spectra for both Sp1 and p53 transcription factor binding sites. We use the power spectra as a structural fingerprint and perform similarity searching in order to find transcription factor binding site regions. This approach provides a new strategy for searching the genome data for information. Although it is difficult to understand the relationship between specific functional properties and the set of structural parameters in our database, our structural fingerprints nevertheless provide a useful tool for searching for function information in sequence data. The power spectrum fingerprints provide a simple, fast method for comparing a set of functional sequences, in this case transcription factor binding site regions, with the sequences of whole chromosomes. On its own, the power spectrum fingerprint does not find all transcription factor binding sites in a chromosome, but the results presented here show that in combination with other approaches, this technique will improve the chances of identifying functional sequences hidden in genomic data.

  9. LASAGNA: a novel algorithm for transcription factor binding site alignment.

    Science.gov (United States)

    Lee, Chih; Huang, Chun-Hsi

    2013-03-24

    Scientists routinely scan DNA sequences for transcription factor (TF) binding sites (TFBSs). Most of the available tools rely on position-specific scoring matrices (PSSMs) constructed from aligned binding sites. Because of the resolutions of assays used to obtain TFBSs, databases such as TRANSFAC, ORegAnno and PAZAR store unaligned variable-length DNA segments containing binding sites of a TF. These DNA segments need to be aligned to build a PSSM. While the TRANSFAC database provides scoring matrices for TFs, nearly 78% of the TFs in the public release do not have matrices available. As work on TFBS alignment algorithms has been limited, it is highly desirable to have an alignment algorithm tailored to TFBSs. We designed a novel algorithm named LASAGNA, which is aware of the lengths of input TFBSs and utilizes position dependence. Results on 189 TFs of 5 species in the TRANSFAC database showed that our method significantly outperformed ClustalW2 and MEME. We further compared a PSSM method dependent on LASAGNA to an alignment-free TFBS search method. Results on 89 TFs whose binding sites can be located in genomes showed that our method is significantly more precise at fixed recall rates. Finally, we described LASAGNA-ChIP, a more sophisticated version for ChIP (Chromatin immunoprecipitation) experiments. Under the one-per-sequence model, it showed comparable performance with MEME in discovering motifs in ChIP-seq peak sequences. We conclude that the LASAGNA algorithm is simple and effective in aligning variable-length binding sites. It has been integrated into a user-friendly webtool for TFBS search and visualization called LASAGNA-Search. The tool currently stores precomputed PSSM models for 189 TFs and 133 TFs built from TFBSs in the TRANSFAC Public database (release 7.0) and the ORegAnno database (08Nov10 dump), respectively. The webtool is available at http://biogrid.engr.uconn.edu/lasagna_search/.

  10. Low affinity glucocorticoid binding site ligands as potential anti-fibrogenics.

    Science.gov (United States)

    Marek, Carylyn J; Wallace, Karen; Durward, Elaine; Koruth, Matthew; Leel, Val; Leiper, Lucy J; Wright, Matthew C

    2009-05-11

    Pregnane X receptor (PXR) agonists inhibit liver fibrosis. However, the rodent PXR activator pregnenolone 16alpha carbonitrile (PCN) blocks, in vitro, hepatic stellate cell-to-myofibroblast trans-differentiation and proliferation in cells from mice with a disrupted PXR gene, suggesting there is an additional anti-fibrogenic drug target for PCN. The role of the low affinity glucocorticoid binding site (LAGS) - which may be identical or associated with the progesterone receptor membrane component 1 (PGRMC1) - in mediating this anti-fibrogenic effect has been examined, since binding of dexamethasone to the LAGS in liver microsomal membranes has previously been shown to be inhibited by PCN. Quiescent rat and human hepatic stellate cells (HSC) were isolated from livers and cultured to generate liver myofibroblasts. HSC and myofibroblasts expressed PGRMC1 as determined by RT-PCR and Western blotting. Quiescent rat HSC also expressed the truncated HC5 variant of rPGRMC1. Rat PGRMC1 was cloned and expression in COS-7 cells gave rise to specific binding of radiolabelled dexamethasone in cell extracts that was inhibited by PCN, suggesting that PGRMC1 may be identical to LAGS or activates LAGS binding activity. Liver microsomes were used to screen a range of structurally related compounds for their ability to inhibit radiolabelled dexamethasone binding to rat LAGS. These compounds were also screened for their ability to activate rat and human PXR and to inhibit rat HSC-to-myofibroblast trans-differentiation/proliferation. A compound (4 androstene-3-one 17beta-carboxylic acid methyl ester) was identified which bound rat LAGS with high affinity and inhibited both rat and human HSC trans-differentiation/proliferation to fibrogenic myofibroblasts without showing evidence of rat or human PXR agonism. However, despite potent anti-fibrogenic effects in vitro, this compound did not modulate liver fibrosis severity in a rat model of liver fibrosis. Immunohistochemical analysis showed

  11. The in Silico Map-Based Cloning of Pi36, a Rice Coiled-Coil Nucleotide-Binding Site Leucine-Rich Repeat Gene That Confers Race-Specific Resistance to the Blast Fungus

    National Research Council Canada - National Science Library

    Liu, Xinqiong; Lin, Fei; Wang, Ling; Pan, Qinghua

    2007-01-01

    ...) gene content of the interval and hence for the identification of candidate gene(s) for Pi36. Three such sequences, which all had both a nucleotide-binding site and a leucine-rich repeat motif, were present...

  12. Tuning genetic clocks employing DNA binding sites.

    Directory of Open Access Journals (Sweden)

    Shridhar Jayanthi

    Full Text Available Periodic oscillations play a key role in cell physiology from the cell cycle to circadian clocks. The interplay of positive and negative feedback loops among genes and proteins is ubiquitous in these networks. Often, delays in a negative feedback loop and/or degradation rates are a crucial mechanism to obtain sustained oscillations. How does nature control delays and kinetic rates in feedback networks? Known mechanisms include proper selection of the number of steps composing a feedback loop and alteration of protease activity, respectively. Here, we show that a remarkably simple means to control both delays and effective kinetic rates is the employment of DNA binding sites. We illustrate this design principle on a widely studied activator-repressor clock motif, which is ubiquitous in natural systems. By suitably employing DNA target sites for the activator and/or the repressor, one can switch the clock "on" and "off" and precisely tune its period to a desired value. Our study reveals a design principle to engineer dynamic behavior in biomolecular networks, which may be largely exploited by natural systems and employed for the rational design of synthetic circuits.

  13. Ceruloplasmin has two nearly identical sites that bind myeloperoxidase

    Science.gov (United States)

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

    2014-01-01

    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 30 nM 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 120 degrees, which, in turn, exerts tension on MPO and results in conformational change. PMID:25301560

  14. 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 was effici......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...... peptide-MHC complexes may have broad significance in the biology of T cell responses, including generation of the T cell repertoire, the specificity of mixed lymphocyte responses, and the immune surveillance of self and nonself antigens in peripheral lymphoid tissues....

  15. Divergent activity of the gonadotropin-releasing hormone receptor gene promoter among genetic lines of pigs is partially conferred by nuclear factor (NF)-B, specificity protein (SP)1-like and GATA-4 binding sites.

    Science.gov (United States)

    McDonald, Emily A; Smith, Jacqueline E; Cederberg, Rebecca A; White, Brett R

    2016-06-29

    Binding of gonadotropin-releasing hormone (GnRH) to its receptor (GnRHR) on gonadotropes within the anterior pituitary gland is essential to reproduction. In pigs, the GnRHR gene is also located near a genetic marker for ovulation rate, a primary determinant of prolificacy. We hypothesized that pituitary expression of the GnRHR gene is alternatively regulated in genetic strains with elevated ovulation rates (Chinese Meishan and Nebraska Index) vs. standard white crossbred swine (Control). Luciferase reporter vectors containing 5118 bp of GnRHR gene promoter from either the Control, Index or Meishan swine lines were generated. Transient transfection of line-specific, full length, deletion and mutation constructs into gonadotrope-derived αT3-1 cells were performed to compare promoter activity and identify regions necessary for divergent regulation of the porcine GnRHR gene. Additionally, transcription factors that bind the GnRHR promoter from each line were identified with electrophoretic mobility shift assays (EMSA). Dramatic differences in luciferase activity among Control, Index and Meishan promoters (19-, 27- and 49-fold over promoterless control, respectively; P Index promoters. Transient transfection of vectors containing block replacement mutations of either the GATA-4 or NF-κB binding sites within the context of their native promoters resulted in a 50 and 60 % reduction of luciferase activity, respectively (P Index promoters resulted in binding of the p52 and p65 subunits of NF-κB and a specificity protein 1 (SP1)-like factor (-1235) as well as GATA-4 (-845). Vectors containing the full-length Meishan promoter harboring individual mutations spanning these regions reduced luciferase activity by 25 and 20 %, respectively, compared to native sequence (P Index promoters in αT3-1 cells is partially due to three single nucleotide polymorphisms resulting in the unique binding of GATA-4 (-1690), the p52/p65 subunits of NF-kB in combination with a SP1

  16. Domain specific association of small fluorescent probe trans-3-(4-monomethylaminophenyl)-acrylonitrile (MMAPA) with bovine serum albumin (BSA) and its dissociation from protein binding sites by Ag nanoparticles: spectroscopic and molecular docking study.

    Science.gov (United States)

    Ghosh, Shalini; Jana, Sankar; Guchhait, Nikhil

    2012-01-26

    Photoinduced intramolecular charge transfer produced a polar excited state in trans-3-(4-monomethylaminophenyl)acrylonitrile (MMAPA), rendering the resulting emission sensitive to the medium polarity. Strong binding interaction of silver nanoparticles with the probe was observed, causing fluorescence quenching through the static quenching process. The probe MMAPA was found to bind to the less polar hydrophobic, restricted proteinous environment of bovine serum albumin (BSA) resulting in the blue shift of the emission maximum with an increase in emission intensity and fluorescence anisotropy. Studies using site markers of flufenamic acid and phenylbutazone coupled with molecular docking results predicted that the binding site of the probe is in between subdomains IIIA and IB of BSA and is different from the conventional Sudlow sites. The denaturation of the probe-bound BSA by urea or heat released the probe from this proteinous environment to water marked by exactly reverse spectral changes. On the interaction of silver nanoparticles with the probe bound protein, the probe was observed to move from its binding site in the protein to the Ag(0) nanoparticle surface involving conformational changes of the protein near the probe binding site.

  17. A systems biology approach to transcription factor binding site prediction.

    Directory of Open Access Journals (Sweden)

    Xiang Zhou

    2010-03-01

    Full Text Available The elucidation of mammalian transcriptional regulatory networks holds great promise for both basic and translational research and remains one the greatest challenges to systems biology. Recent reverse engineering methods deduce regulatory interactions from large-scale mRNA expression profiles and cross-species conserved regulatory regions in DNA. Technical challenges faced by these methods include distinguishing between direct and indirect interactions, associating transcription regulators with predicted transcription factor binding sites (TFBSs, identifying non-linearly conserved binding sites across species, and providing realistic accuracy estimates.We address these challenges by closely integrating proven methods for regulatory network reverse engineering from mRNA expression data, linearly and non-linearly conserved regulatory region discovery, and TFBS evaluation and discovery. Using an extensive test set of high-likelihood interactions, which we collected in order to provide realistic prediction-accuracy estimates, we show that a careful integration of these methods leads to significant improvements in prediction accuracy. To verify our methods, we biochemically validated TFBS predictions made for both transcription factors (TFs and co-factors; we validated binding site predictions made using a known E2F1 DNA-binding motif on E2F1 predicted promoter targets, known E2F1 and JUND motifs on JUND predicted promoter targets, and a de novo discovered motif for BCL6 on BCL6 predicted promoter targets. Finally, to demonstrate accuracy of prediction using an external dataset, we showed that sites matching predicted motifs for ZNF263 are significantly enriched in recent ZNF263 ChIP-seq data.Using an integrative framework, we were able to address technical challenges faced by state of the art network reverse engineering methods, leading to significant improvement in direct-interaction detection and TFBS-discovery accuracy. We estimated the accuracy

  18. Binding site prediction for protein-protein interactions and novel motif discovery using re-occurring polypeptide sequences.

    Science.gov (United States)

    Amos-Binks, Adam; Patulea, Catalin; Pitre, Sylvain; Schoenrock, Andrew; Gui, Yuan; Green, James R; Golshani, Ashkan; Dehne, Frank

    2011-06-02

    While there are many methods for predicting protein-protein interaction, very few can determine the specific site of interaction on each protein. Characterization of the specific sequence regions mediating interaction (binding sites) is crucial for an understanding of cellular pathways. Experimental methods often report false binding sites due to experimental limitations, while computational methods tend to require data which is not available at the proteome-scale. Here we present PIPE-Sites, a novel method of protein specific binding site prediction based on pairs of re-occurring polypeptide sequences, which have been previously shown to accurately predict protein-protein interactions. PIPE-Sites operates at high specificity and requires only the sequences of query proteins and a database of known binary interactions with no binding site data, making it applicable to binding site prediction at the proteome-scale. PIPE-Sites was evaluated using a dataset of 265 yeast and 423 human interacting proteins pairs with experimentally-determined binding sites. We found that PIPE-Sites predictions were closer to the confirmed binding site than those of two existing binding site prediction methods based on domain-domain interactions, when applied to the same dataset. Finally, we applied PIPE-Sites to two datasets of 2347 yeast and 14,438 human novel interacting protein pairs predicted to interact with high confidence. An analysis of the predicted interaction sites revealed a number of protein subsequences which are highly re-occurring in binding sites and which may represent novel binding motifs. PIPE-Sites is an accurate method for predicting protein binding sites and is applicable to the proteome-scale. Thus, PIPE-Sites could be useful for exhaustive analysis of protein binding patterns in whole proteomes as well as discovery of novel binding motifs. PIPE-Sites is available online at http://pipe-sites.cgmlab.org/.

  19. In vivo binding of PRDM9 reveals interactions with noncanonical genomic sites

    DEFF Research Database (Denmark)

    Grey, Corinne; Clément, Julie A.J.; Buard, Jérôme

    2017-01-01

    In mouse and human meiosis, DNA double-strand breaks (DSBs) initiate homologous recombination and occur at specific sites called hotspots. The localization of these sites is determined by the sequence-specific DNA binding domain of the PRDM9 histone methyl transferase. Here, we performed an exten......In mouse and human meiosis, DNA double-strand breaks (DSBs) initiate homologous recombination and occur at specific sites called hotspots. The localization of these sites is determined by the sequence-specific DNA binding domain of the PRDM9 histone methyl transferase. Here, we performed...

  20. Bioinformatic analysis of cis-regulatory interactions between progesterone and estrogen receptors in breast cancer

    Directory of Open Access Journals (Sweden)

    Matloob Khushi

    2014-11-01

    Full Text Available Chromatin factors interact with each other in a cell and sequence-specific manner in order to regulate transcription and a wealth of publically available datasets exists describing the genomic locations of these interactions. Our recently published BiSA (Binding Sites Analyser database contains transcription factor binding locations and epigenetic modifications collected from published studies and provides tools to analyse stored and imported data. Using BiSA we investigated the overlapping cis-regulatory role of estrogen receptor alpha (ERα and progesterone receptor (PR in the T-47D breast cancer cell line. We found that ERα binding sites overlap with a subset of PR binding sites. To investigate further, we re-analysed raw data to remove any biases introduced by the use of distinct tools in the original publications. We identified 22,152 PR and 18,560 ERα binding sites (<5% false discovery rate with 4,358 overlapping regions among the two datasets. BiSA statistical analysis revealed a non-significant overall overlap correlation between the two factors, suggesting that ERα and PR are not partner factors and do not require each other for binding to occur. However, Monte Carlo simulation by Binary Interval Search (BITS, Relevant Distance, Absolute Distance, Jaccard and Projection tests by Genometricorr revealed a statistically significant spatial correlation of binding regions on chromosome between the two factors. Motif analysis revealed that the shared binding regions were enriched with binding motifs for ERα, PR and a number of other transcription and pioneer factors. Some of these factors are known to co-locate with ERα and PR binding. Therefore spatially close proximity of ERα binding sites with PR binding sites suggests that ERα and PR, in general function independently at the molecular level, but that their activities converge on a specific subset of transcriptional targets.

  1. Identification of a phosphorylation site in the hinge region of the human progesterone receptor and additional amino-terminal phosphorylation sites.

    Science.gov (United States)

    Knotts, T A; Orkiszewski, R S; Cook, R G; Edwards, D P; Weigel, N L

    2001-03-16

    We have previously reported the identification of seven in vivo phosphorylation sites in the amino-terminal region of the human progesterone receptor (PR). From our previous in vivo studies, it was evident that several phosphopeptides remained unidentified. In particular, we wished to determine whether human PR contains a phosphorylation site in the hinge region, as do other steroid receptors including chicken PR, human androgen receptor, and mouse estrogen receptor. Previously, problematic trypsin cleavage sites hampered our ability to detect phosphorylation sites in large incomplete tryptic peptides. Using a combination of mass spectrometry and in vitro phosphorylation, we have identified six previously unidentified phosphorylation sites in human PR. Using nanoelectrospray ionization mass spectrometry, we have identified two new in vivo phosphorylation sites, Ser(20) and Ser(676), in baculovirus-expressed human PR. Ser(676) is analogous to the hinge site identified in other steroid receptors. Additionally, precursor ion scans identified another phosphopeptide that contains Ser(130)-Pro(131), a likely candidate for phosphorylation. In vitro phosphorylation of PR with Cdk2 has revealed five additional in vitro Cdk2 phosphorylation sites: Ser(25), Ser(213), Thr(430), Ser(554), and Ser(676). At least two of these, Ser(213) and Ser(676), are authentic in vivo sites. We confirmed the presence of the Cdk2-phosphorylated peptide containing Ser(213) in PR from in vivo labeled T47D cells, indicating that this is an in vivo site. Our combined studies indicate that most, if not all, of the Ser-Pro motifs in human PR are sites for phosphorylation. Taken together, these data indicate that the phosphorylation of PR is highly complex, with at least 14 phosphorylation sites.

  2. Identification of clustered YY1 binding sites in Imprinting Control Regions

    Energy Technology Data Exchange (ETDEWEB)

    Kim, J D; Hinz, A; Bergmann, A; Huang, J; Ovcharenko, I; Stubbs, L; Kim, J

    2006-04-19

    Mammalian genomic imprinting is regulated by Imprinting Control Regions (ICRs) that are usually associated with tandem arrays of transcription factor binding sites. In the current study, the sequence features derived from a tandem array of YY1 binding sites of Peg3-DMR (differentially methylated region) led us to identify three additional clustered YY1 binding sites, which are also localized within the DMRs of Xist, Tsix, and Nespas. These regions have been shown to play a critical role as ICRs for the regulation of surrounding genes. These ICRs have maintained a tandem array of YY1 binding sites during mammalian evolution. The in vivo binding of YY1 to these regions is allele-specific and only to the unmethylated active alleles. Promoter/enhancer assays suggest that a tandem array of YY1 binding sites function as a potential orientation-dependent enhancer. Insulator assays revealed that the enhancer-blocking activity is detected only in the YY1 binding sites of Peg3-DMR but not in the YY1 binding sites of other DMRs. Overall, our identification of three additional clustered YY1 binding sites in imprinted domains suggests a significant role for YY1 in mammalian genomic imprinting.

  3. Site-directed in vitro immunization leads to a complete human monoclonal IgG4λ that binds specifically to the CDR2 region of CTLA-4 (CD152 without interfering the engagement of natural ligands

    Directory of Open Access Journals (Sweden)

    Hsu Shu-Ching

    2007-08-01

    Full Text Available Abstract Background The ability to acquire fully human monoclonal antibodies (mAbs with pre-defined specificities is critical to the development of molecular tags for the analysis of receptor function in addition to promising immunotherapeutics. Yet most of the arriving affinity maturated and complete human immunoglobulin G (IgG molecules, which are actually derived from single human B cells, have not widely been used to study the conserved self antigens (Ags such as CD152 (cytotoxic T lymphocyte antigen-4, CTLA-4 because proper hosts are lacking. Results Here we developed an optimized protocol for site-directed in vitro immunizing peripheral blood mononuclear cells (PBMC by using a selected epitope of human CD152, an essential receptor involved in down-regulation of T cell activation. The resultant stable trioma cell lines constantly produce anti-CD152 mAb (γ4λhuCD152, which contains variable (V regions of the heavy chain and the light chain derived from the VH3 and Vλ human germline genes, respectively, and yet displays an unusual IgG4 isotype. Interestingly, γ4λhuCD152 has a basic pI not commonly found in myeloid monoclonal IgG4λs as revealed by the isoelectric focusing (IEF analysis. Furthermore, γ4λhuCD152 binds specifically, with nanomolar affinity, to an extracellular constituency encompassing the putative second complementarity determining region (CDR2 of CD152, whereby it can react to activated CD3+ cells. Conclusion In a context of specific cell depletion and conditioned medium,in vitro induction of human Abs against a conserved self Ag was successfully acquired and a relatively basic mAb, γ4λhuCD152, with high affinity to CDR2 of CD152 was thus obtained. Application of such a human IgG4λ mAb with designated CDR2 specificity may impact upon and prefer for CD152 labeling both in situ and ex situ, as it does not affect the binding of endogenous B7 ligands and can localize into the confined immunological synapse which may

  4. Evolution of Metal(Loid) Binding Sites in Transcriptional Regulators

    Energy Technology Data Exchange (ETDEWEB)

    Ordonez, E.; Thiyagarajan, S.; Cook, J.D.; Stemmler, T.L.; Gil, J.A.; Mateos, L.M.; Rosen, B.P.

    2009-05-22

    Expression of the genes for resistance to heavy metals and metalloids is transcriptionally regulated by the toxic ions themselves. Members of the ArsR/SmtB family of small metalloregulatory proteins respond to transition metals, heavy metals, and metalloids, including As(III), Sb(III), Cd(II), Pb(II), Zn(II), Co(II), and Ni(II). These homodimeric repressors bind to DNA in the absence of inducing metal(loid) ion and dissociate from the DNA when inducer is bound. The regulatory sites are often three- or four-coordinate metal binding sites composed of cysteine thiolates. Surprisingly, in two different As(III)-responsive regulators, the metalloid binding sites were in different locations in the repressor, and the Cd(II) binding sites were in two different locations in two Cd(II)-responsive regulators. We hypothesize that ArsR/SmtB repressors have a common backbone structure, that of a winged helix DNA-binding protein, but have considerable plasticity in the location of inducer binding sites. Here we show that an As(III)-responsive member of the family, CgArsR1 from Corynebacterium glutamicum, binds As(III) to a cysteine triad composed of Cys{sup 15}, Cys{sup 16}, and Cys{sup 55}. This binding site is clearly unrelated to the binding sites of other characterized ArsR/SmtB family members. This is consistent with our hypothesis that metal(loid) binding sites in DNA binding proteins evolve convergently in response to persistent environmental pressures.

  5. Sensitivity, specificity, predictive value and accuracy of ultrasonography in pregnancy rate prediction in Sahelian goats after progesterone impregnated sponge synchronization

    Directory of Open Access Journals (Sweden)

    Justin Kouamo

    2014-09-01

    Full Text Available Aim: This study was aimed to evaluate the sensitivity, specificity, predictive value and accuracy of ultrasonography in pregnancy rate (PR prediction in Sahelian goats after progesterone impregnated sponge synchronization within the framework of caprine artificial insemination (AI program in Fatick (Senegal. Materials and Methods: Of 193 candidate goats in AI program, 167 were selected (day 50 in six villages. Estrus was synchronized by progesterone impregnated sponges installed for 11 days. Two days before the time of sponge removal (day 4, each goat was treated with 500 IU of equine chorionic gonadotropin and 50 μg of dcloprostenol. All goats were inseminated (day 0 with alpine goat semen from France at 45±3 h after sponge removal (day 2. Real-time B-mode ultrasonography was performed at day 50, day 13, day 0, day 40 and day 60 post-AI. Results: Selection rate, estrus response rate, AI rate, PR at days 40 and days 60 were 86.53%; 71.85%; 83.34%; 51% and 68% (p<0.05 respectively. Value of sensitivity, specificity, positive and negative predictive value, accuracy, total conformity, conformity of correct positive, conformity of correct negative and discordance of pregnancy diagnosis by trans-abdominal ultrasonography (TU were 98.03%; 63.26%; 73.52%; 3.12%; 81%; 81%; 50%; 31% and 19%, respectively. Conclusion: These results indicate that the TU can be performed in goats under traditional condition and emphasized the importance of re-examination of goats with negative or doubtful TU diagnoses performed at day 40 post-AI.

  6. 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. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  7. A third Na+-binding site in the sodium pump.

    Science.gov (United States)

    Li, Ciming; Capendeguy, Oihana; Geering, Käthi; Horisberger, Jean-Daniel

    2005-09-06

    The sodium pump, or Na,K-ATPase, exports three intracellular sodium ions in exchange for two extracellular potassium ions. In the high resolution structure of the related calcium pump, two cation-binding sites have been identified. The two corresponding sites in the sodium pump are expected to be alternatively occupied by sodium and potassium. The position of a third sodium-specific site is still hypothetical. Here, we report the large effects of single residue substitutions on the voltage-dependent kinetics of the release of sodium to the extracellular side of the membrane. These mutations also alter the apparent affinity for intracellular sodium while one of them does not affect the intrinsic affinity for potassium. These results enable us to locate the third sodium-specific site of the sodium pump in a space between the fifth, sixth, and ninth transmembrane helices of the alpha-subunit and provide an experimental validation of the model proposed by Ogawa and Toyoshima [Ogawa, H. & Toyoshima, C. (2002) Proc. Natl. Acad. Sci. USA 99, 15977-15982].

  8. Binding site number variation and high-affinity binding consensus of Myb-SANT-like transcription factor Adf-1 in Drosophilidae.

    Science.gov (United States)

    Lang, Michael; Juan, Elvira

    2010-10-01

    There is a growing interest in the evolution of transcription factor binding sites and corresponding functional change of transcriptional regulation. In this context, we have examined the structural changes of the ADF-1 binding sites at the Adh promoters of Drosophila funebris and D. virilis. We detected an expanded footprinted region in D. funebris that contains various adjacent binding sites with different binding affinities. ADF-1 was described to direct sequence-specific DNA binding to sites consisting of the multiple trinucleotide repeat . The ADF-1 recognition sites with high binding affinity differ from this trinucleotide repeat consensus sequence and a new consensus sequence is proposed for the high-affinity ADF-1 binding sites. In vitro transcription experiments with the D. funebris and D. virilis ADF-1 binding regions revealed that stronger ADF-1 binding to the expanded D. funebris ADF-1 binding region only moderately lead to increased transcriptional activity of the Adh gene. The potential of this regional expansion is discussed in the context of different ADF-1 cellular concentrations and maintenance of the ADF-1 stimulus. Altogether, evolutionary change of ADF-1 binding regions involves both, rearrangements of complex binding site cluster and also nucleotide substitutions within sites that lead to different binding affinities.

  9. Blocking the QB-binding site of photosystem II by tenuazonic acid, a non-host-specific toxin of Alternaria alternata, activates singlet oxygen-mediated and EXECUTER-dependent signalling in Arabidopsis.

    Science.gov (United States)

    Chen, Shiguo; Kim, Chanhong; Lee, Je Min; Lee, Hyun-Ah; Fei, Zhangjun; Wang, Liangsheng; Apel, Klaus

    2015-06-01

    Necrotrophic fungal pathogens produce toxic compounds that induce cell death in infected plants. Often, the primary targets of these toxins and the way a plant responds to them are not known. In the present work, the effect of tenuazonic acid (TeA), a non-host-specific toxin of Alternaria alternata, on Arabidopsis thaliana has been analysed. TeA blocks the QB -binding site at the acceptor side of photosystem II (PSII). As a result, charge recombination at the reaction centre (RC) of PSII is expected to enhance the formation of the excited triplet state of the RC chlorophyll that promotes generation of singlet oxygen ((1)O₂). (1)O₂ activates a signalling pathway that depends on the two EXECUTER (EX) proteins EX1 and EX2 and triggers a programmed cell death response. In seedlings treated with TeA at half-inhibition concentration (1)O₂-mediated and EX-dependent signalling is activated as indicated by the rapid and transient up-regulation of (1)O₂-responsive genes in wild type, and its suppression in ex1/ex2 mutants. Lesion formation occurs when seedlings are exposed to higher concentrations of TeA for a longer period of time. Under these conditions, the programmed cell death response triggered by (1)O₂-mediated and EX-dependent signalling is superimposed by other events that also contribute to lesion formation. © 2014 John Wiley & Sons Ltd.

  10. Evolution of transcription factor binding sites in mammalian gene regulatory regions: handling counterintuitive results.

    Science.gov (United States)

    Balmer, James E; Blomhoff, Rune

    2009-06-01

    The remodeling of transcription factor binding sites is one of the major engines of evolutionary change, yet almost all available examples of this involve sites from regulatory modules brought online during development. Developmental modules are known to enjoy some type of canalization, which allows considerably more cryptic change than would be assumed for nondevelopmental modules. It remains an open question, then, how frequently binding site remodeling occurs in general. There are strong intuitive reasons to expect that regulatory constancy, and hence binding site conservation in general, is the rule, yet little systematic work has been done to verify this. In the present article, we show that the most obvious way of approaching this problem--which is simply to collect experimentally verified binding sites from the literature without further analysis, create multispecies alignments, and apply conservation algorithms--leads to counterintuitive and ultimately inaccurate results. This is because of the low complexity of typical binding sites and, consequently, because of the frequency with which strings resembling legitimate sites occur throughout the genome. In vitro results can easily be confounded by this. Applying one traditional conservation algorithm and two novel algorithms to a data set that ought to be representative of binding sites in general, but which is taken from the literature directly, we find that only 58% of sites appear to be conserved. However, after the data set is carefully vetted against binding site overloading, and after the likelihood of a specific type of compensatory evolution is evaluated, conservation rates as high as 94% appear reasonable.

  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 acids...

  12. Site-specific ground response analysis

    National Research Council Canada - National Science Library

    L. GovindaRaju; G. V. Ramana; C. HanumanthaRao; T. G. Sitharam

    2004-01-01

    ... modifications to the underlying motion. We highlight the engineering importance of site-specific ground response analysis and difficulties faced in conducting a complete ground response analysis...

  13. Uncoupling metallonuclease metal ion binding sites via nudge mutagenesis.

    Science.gov (United States)

    Papadakos, Grigorios A; Nastri, Horacio; Riggs, Paul; Dupureur, Cynthia M

    2007-05-01

    The hydrolysis of phosphodiester bonds by nucleases is critical to nucleic acid processing. Many nucleases utilize metal ion cofactors, and for a number of these enzymes two active-site metal ions have been detected. Testing proposed mechanistic roles for individual bound metal ions has been hampered by the similarity between the sites and cooperative behavior. In the homodimeric PvuII restriction endonuclease, the metal ion dependence of DNA binding is sigmoidal and consistent with two classes of coupled metal ion binding sites. We reasoned that a conservative active-site mutation would perturb the ligand field sufficiently to observe the titration of individual metal ion binding sites without significantly disturbing enzyme function. Indeed, mutation of a Tyr residue 5.5 A from both metal ions in the enzyme-substrate crystal structure (Y94F) renders the metal ion dependence of DNA binding biphasic: two classes of metal ion binding sites become distinct in the presence of DNA. The perturbation in metal ion coordination is supported by 1H-15N heteronuclear single quantum coherence spectra of enzyme-Ca(II) and enzyme-Ca(II)-DNA complexes. Metal ion binding by free Y94F is basically unperturbed: through multiple experiments with different metal ions, the data are consistent with two alkaline earth metal ion binding sites per subunit of low millimolar affinity, behavior which is very similar to that of the wild type. The results presented here indicate a role for the hydroxyl group of Tyr94 in the coupling of metal ion binding sites in the presence of DNA. Its removal causes the affinities for the two metal ion binding sites to be resolved in the presence of substrate. Such tuning of metal ion affinities will be invaluable to efforts to ascertain the contributions of individual bound metal ions to metallonuclease function.

  14. Lectin-binding sites in the epithelium of normal human appendix vermiformis and in acute appendicitis.

    Science.gov (United States)

    Brinck, U; Bosbach, R; Korabiowska, M; Schauer, A; Gabius, H J

    1995-01-01

    By using histochemical methods, the binding pattern of various lectins in the epithelium of normal human appendix vermiformis was assessed. In addition to plant and invertebrate sugar receptors with nominal monosaccharide specificity for alpha-L-Fuc (UEA-I), alpha-D-Man and alpha-D-Gluc (Con A), alpha-D-GalNAc (DBA), D-GalNAc (SBA, HPA) beta-D-Gal (RCA-I) and D-Gal (VAA), a mammalian beta-galactoside-specific lectin (MW, 14 kDa) was included in the applied panel. The apical surface of enterocytes presented binding sites for RCA-I on all cells, binding sites of UEA-I, DBA, SBA, HPA and VAA heterogeneously and no binding sites of Con A and 14 kDa. Binding sites of DBA, SBA, HPA, VAA and RCA-I within enterocytes were located primarily focally in a supranuclear position, whereas Con A and 14 kDa bound to the cytoplasm both in apical and basal cell parts. In the follicle-associated epithelium more enterocytes expressed SBA- and VAA-binding sites than in the crypt epithelium. No differences between the lectin-binding pattern of M-cells and enterocytes were found in the follicle-associated epithelium. Intraepithelial macrophages were heterogeneously positive for the full panel of applied lectins. In contrast, intraepithelial lymphatic cells expressed binding sites only for RCA-I and less prominently for Con A, VAA and 14 kDa. Goblet cell mucus contained lectin-binding sites in a heterogeneous manner: binding sites for Con A were not detected in goblet cells for DBA, SBA, VAA and 14 kDa in less than 20%, for UEA-I in 20-40%, for HPA in 40-60% and for RCA-I in 60-100% of the goblet cells. Secreted mucus differed in its lectin-binding capacity from intracellular goblet cell mucus selectively by an increase of UEA-I, SBA- and RCA-I-binding sites and a lack of 14 kDa-binding sites. Comparative study of lectin binding to goblet cell mucin in another region of the large intestine, namely the rectosigmoid, demonstrated that DBA, SBA and 14 kDa bound mainly to the distal colon

  15. Specific binding of nerve growth factor to normal and denervated canine heart.

    Science.gov (United States)

    Wells, D J; Strehlo, B L; Kaye, M P

    1981-11-01

    Preparations of membranes from normal and surgically denervated canine heart were tested for binding of radiolabeled nerve growth factor. Specific binding was detected in both normal and denervated hearts. Binding was nonsaturable and complex for normal atria and ventricles and denervated atria but appeared to be saturable for denervated ventricles. Nerve growth factor bound per microgram of protein was lower in denervated ventricles than normal ventricles, whereas slightly higher binding to denervated atria than normal atria was observed. This binding could not be displaced by cytochrome c, insulin, or epidermal growth factor. The data indicate that a large part of binding to normal ventricles could be due to nerve terminals attached to the heart, but specific binding detected in the denervated ventricles may be an intrinsic property of the tissue itself. These sites may serve as storage or uptake sites to direct sympathetic innervation in the developing or reinnervating myocardium.

  16. Localization of gonadotropin binding sites in human ovarian neoplasms

    Energy Technology Data Exchange (ETDEWEB)

    Nakano, R.; Kitayama, S.; Yamoto, M.; Shima, K.; Ooshima, A. (Wakayama Medical College (Japan))

    1989-10-01

    The binding of human luteinizing hormone and human follicle-stimulating hormone to ovarian tumor biopsy specimens from 29 patients was analyzed. The binding sites for human luteinizing hormone were demonstrated in one tumor of epithelial origin (mucinous cystadenoma) and in one of sex cord-stromal origin (theca cell tumor). The binding sites for human follicle-stimulating hormone were found in three tumors of epithelial origin (serous cystadenoma and mucinous cystadenoma) and in two of sex cord-stromal origin (theca cell tumor and theca-granulosa cell tumor). The surface-binding autoradiographic study revealed that the binding sites for gonadotropins were localized in the stromal tissue. The results suggest that gonadotropic hormones may play a role in the growth and differentiation of a certain type of human ovarian neoplasms.

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

    Science.gov (United States)

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

    2017-09-01

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

  18. SITE-94. Site specific base data for the performance assessment

    Energy Technology Data Exchange (ETDEWEB)

    Geier, J. [ed.] [Clearwater Hardrock Consulting, Monmouth, OR (United States); Tiren, S. [Geosigma AB, Uppsala (Sweden); Dverstorp, B. [Swedish Nuclear Power Inspectorate, Stockholm (Sweden); Glynn, P. [U.S. Geological Survey, Reston, VA (United States)

    1996-06-01

    This report documents the site specific base data that were available, and the utilization of these data within SITE-94. A brief summary is given of SKB`s preliminary site investigations for the Aespoe Hard Rock Laboratory (HRL), which were the main source of site-specific data for SITE-94, and an overview is given of the field methods and instrumentation for the preliminary investigations. A compilation is given of comments concerning the availability and quality of the data for Aespoe, and specific recommendations are given for future site investigations. It was found that the HRL pre-investigations produced a large quantity of data which were, for the most part, of sufficient quality to be valuable for a performance assessment. However, some problems were encountered regarding documentation, procedural consistency, positional information, and storage of the data from the measurements. 77 refs, 4 tabs.

  19. Microbes bind complement inhibitor factor H via a common site.

    Directory of Open Access Journals (Sweden)

    T Meri

    Full Text Available To cause infections microbes need to evade host defense systems, one of these being the evolutionarily old and important arm of innate immunity, the alternative pathway of complement. It can attack all kinds of targets and is tightly controlled in plasma and on host cells by plasma complement regulator factor H (FH. FH binds simultaneously to host cell surface structures such as heparin or glycosaminoglycans via domain 20 and to the main complement opsonin C3b via domain 19. Many pathogenic microbes protect themselves from complement by recruiting host FH. We analyzed how and why different microbes bind FH via domains 19-20 (FH19-20. We used a selection of FH19-20 point mutants to reveal the binding sites of several microbial proteins and whole microbes (Haemophilus influenzae, Bordetella pertussis, Pseudomonas aeruginosa, Streptococcus pneumonia, Candida albicans, Borrelia burgdorferi, and Borrelia hermsii. We show that all studied microbes use the same binding region located on one side of domain 20. Binding of FH to the microbial proteins was inhibited with heparin showing that the common microbial binding site overlaps with the heparin site needed for efficient binding of FH to host cells. Surprisingly, the microbial proteins enhanced binding of FH19-20 to C3b and down-regulation of complement activation. We show that this is caused by formation of a tripartite complex between the microbial protein, FH, and C3b. In this study we reveal that seven microbes representing different phyla utilize a common binding site on the domain 20 of FH for complement evasion. Binding via this site not only mimics the glycosaminoglycans of the host cells, but also enhances function of FH on the microbial surfaces via the novel mechanism of tripartite complex formation. This is a unique example of convergent evolution resulting in enhanced immune evasion of important pathogens via utilization of a "superevasion site."

  20. Solubilization of serotonin-1a and serotonin-1b binding sites from bovine brain

    Energy Technology Data Exchange (ETDEWEB)

    Asarch, K.B.; Shih, J.C.

    1987-05-01

    Serotonin1 (5-hydroxytryptamine1, 5-HT1) binding sites have been solubilized from bovine brain cortex using a mixture of 0.1% Nonidet P-40 and 0.3% digitonin in a low-salt buffer containing 0.1% ascorbic acid. The affinity of (/sup 3/H)5-HT for the soluble cortical binding sites (2.1 nM) is identical to its affinity at membrane-bound binding sites (2.1 nM). (/sup 3/H)8-Hydroxy-2-(di-n-propylamino)tetralin ((/sup 3/H)DPAT), a selective 5-HT1a radioligand, also binds to soluble cortical binding sites with high affinity (1.8 nM) comparable with its affinity in the crude membranes (1.7 nM). A significant correlation exists in the rank order potency of serotonergic agents for (/sup 3/H)5-HT binding and for (/sup 3/H)DPAT binding to crude and soluble membranes. The density of (/sup 3/H)DPAT binding sites relative to the (/sup 3/H)5-HT sites in the solubilized cortical membranes (35%) corresponds well with the proportion of 5-HT1a sites in the crude membranes determined by spiperone displacement (33%), suggesting that both the 5-HT1a and 5-HT1b binding sites have been cosolubilized. (/sup 3/H)5-HT binding in the soluble preparations was inhibited by GTP, suggesting that a receptor complex may have been solubilized. (/sup 3/H)Spiperone-specific binding was not detectable in this preparation, suggesting that 5-HT2 sites were not cosolubilized.

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

    Science.gov (United States)

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

    2013-01-01

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

  2. MODELING LANTHANIDE BINDING SITES ON HUMIC ACID

    OpenAIRE

    Pourret, Olivier; Martinez, Raul,

    2009-01-01

    International audience; Lanthanide (Ln) binding to humic acid (HA) has been investigated by combining ultrafiltration and ICP-MS techniques. A Langmuir sorption isotherm metal complexation model was used in conjunction with a linear programming method (LPM) to fit experimental data representing a wide range of experimental conditions both in HA/Ln ratio (varying between 5 and 20) and in pH range (from 2 to 10) with a ionic strength of 10-3 mol L-1. The LPM approach, not requiring prior knowle...

  3. Chloride binding site of neurotransmitter sodium symporters

    DEFF Research Database (Denmark)

    Kantcheva, Adriana Krassimirova; Quick, Matthias; Shi, Lei

    2013-01-01

    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 ...

  4. 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.

  5. 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.

  6. NMR mapping of copper binding sites in alpha-synuclein.

    Science.gov (United States)

    Sung, Yoon-Hui; Rospigliosi, Carla; Eliezer, David

    2006-01-01

    Copper binding to the Parkinson disease-linked protein alpha-synuclein (aS) has been shown to accelerate its oligomerization in vitro and may therefore play a role in aS-mediated pathology in vivo. We use NMR spectroscopy to identify a number of independent copper binding sites in both the lipid-binding N-terminal domain and the highly acidic C-terminal domain of aS. Most of the sites appear to involve negatively charged amino acid side chains, but binding is also observed to the sole histidine residue located at position 50 and to the N-terminal amino group. Both the N-terminal and the histidine sites, as well as the sites in the C-terminal tail, can also bind copper in the more highly structured conformation adopted by aS upon binding to detergent micelles or lipid vesicles. There is no evidence for the formation of any sites requiring long-range order in the protein.

  7. An Overview of the Prediction of Protein DNA-Binding Sites

    Directory of Open Access Journals (Sweden)

    Jingna Si

    2015-03-01

    Full Text Available Interactions between proteins and DNA play an important role in many essential biological processes such as DNA replication, transcription, splicing, and repair. The identification of amino acid residues involved in DNA-binding sites is critical for understanding the mechanism of these biological activities. In the last decade, numerous computational approaches have been developed to predict protein DNA-binding sites based on protein sequence and/or structural information, which play an important role in complementing experimental strategies. At this time, approaches can be divided into three categories: sequence-based DNA-binding site prediction, structure-based DNA-binding site prediction, and homology modeling and threading. In this article, we review existing research on computational methods to predict protein DNA-binding sites, which includes data sets, various residue sequence/structural features, machine learning methods for comparison and selection, evaluation methods, performance comparison of different tools, and future directions in protein DNA-binding site prediction. In particular, we detail the meta-analysis of protein DNA-binding sites. We also propose specific implications that are likely to result in novel prediction methods, increased performance, or practical applications.

  8. Site specific agreement : Lake Mason Wetland

    Data.gov (United States)

    US Fish and Wildlife Service, Department of the Interior — The site-specific agreement describes purpose and scope of the partnership between Ducks Unlimited, Inc. and U.S. Fish and Wildlife Service to manage wetlands for...

  9. Detection and comparison of specific hemin binding by Porphyromonas gingivalis and Prevotella intermedia.

    OpenAIRE

    Tompkins, G R; Wood, D P; Birchmeier, K R

    1997-01-01

    A radioligand assay was designed to detect and compare specific hemin binding by the periodontal anaerobic black-pigmenting bacteria (BPB) Porphyromonas gingivalis and Prevotella intermedia. The assay included physiological concentrations of the hemin-binding protein rabbit serum albumin (RSA) to prevent self-aggregation and nonspecific interaction of hemin with cellular components. Under these conditions, heme-starved P. intermedia cells (two strains) expressed a single binding site species ...

  10. Characterization of an intracellular hyaluronic acid binding site in isolated rat hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Frost, S.J.; Raja, R.H.; Weigel, P.H. (Univ. of Texas Medical Branch, Galveston (USA))

    1990-11-13

    125I-HA, prepared by chemical modification at the reducing sugar, specifically binds to rat hepatocytes in suspension or culture. Intact hepatocytes have relatively few surface 125I-HA binding sites and show low specific binding. However, permeabilization of hepatocytes with the nonionic detergent digitonin results in increased specific 125I-HA binding (45-65%) and a very large increase in the number of specific 125I-HA binding sites. Scatchard analysis of equilibrium 125I-HA binding to permeabilized hepatocytes in suspension at 4 degrees C indicates a Kd = 1.8 x 10(-7) M and 1.3 x 10(6) molecules of HA (Mr approximately 30,000) bound per cell at saturation. Hepatocytes in primary culture for 24 h show the same affinity but the total number of HA molecules bound per cell at saturation decreases to approximately 6.2 x 10(5). Increasing the ionic strength above physiologic concentrations decreases 125I-HA binding to permeable cells, whereas decreasing the ionic strength above causes an approximately 4-fold increase. The divalent cation chelator EGTA does not prevent binding nor does it release 125I-HA bound in the presence of 2 mM CaCl2, although higher divalent cation concentrations stimulate 125I-HA binding. Ten millimolar CaCl2 or MnCl2 increases HA binding 3-6-fold compared to EGTA-treated cells. Ten millimolar MgCl2, SrCl2, or BaCl2 increased HA binding by 2-fold. The specific binding of 125I-HA to digitonin-treated hepatocytes at 4{degrees}C increased greater than 10-fold at pH 5.0 as compared to pH 7.

  11. OB protein binds specifically to the choroid plexus of mice and rats.

    Science.gov (United States)

    Devos, R; Richards, J G; Campfield, L A; Tartaglia, L A; Guisez, Y; van der Heyden, J; Travernier, J; Plaetinck, G; Burn, P

    1996-05-28

    Binding studies were conducted to identify the anatomical location of brain target sites for OB protein, the ob gene product. 125I-labeled recombinant mouse OB protein or alkaline phosphatase-OB fusion proteins were used for in vitro and in vivo binding studies. Coronal brain sections or fresh tissue from lean, obese ob/ob, and obese db/db mice as well as lean and obese Zucker rats were probed to identify potential central OB protein-binding sites. We report here that recombinant OB protein binds specifically to the choroid plexus. The binding of OB protein (either radiolabeled or the alkaline phosphatase-OB fusion protein) and its displacement by unlabeled OB protein was similar in lean, obese ob/ob, and obese db/db mice as well as lean and obese Zucker rats. These findings suggest that OB protein binds with high affinity to a specific receptor in the choroid plexus. After binding to the choroid plexus receptor, OB protein may then be transported across the blood-brain barrier into the cerebrospinal fluid. Alternatively, binding of OB protein to a specific receptor in the choroid plexus may activate afferent neural inputs to the neural network that regulates feeding behavior and energy balance or may result in the clearance or degradation of OB protein. The identification of the choroid plexus as a brain binding site for OB protein will provide the basis for the construction of expression libraries and facilitate the rapid cloning of the choroid plexus OB receptor.

  12. The Art of Compiling Protein Binding Site Ensembles.

    Science.gov (United States)

    Bietz, Stefan; Fährrolfes, Rainer; Rarey, Matthias

    2016-12-01

    Structure-based drug design starts with the collection, preparation, and initial analysis of protein structures. With more than 115,000 structures publically available in the Protein Data Bank (PDB), fully automated processes reliably performing these important preprocessing steps are needed. Several tools are available for these tasks, however, most of them do not address the special needs of scientists interested in protein-ligand interactions. In this paper, we summarize our research activities towards an automated processing pipeline from raw PDB data towards ready-to-use protein binding site ensembles. Starting from a single protein structure, the pipeline covers the following phases: Extracting structurally related binding sites from the PDB, aligning disconnected binding site sequences, resolving tautomeric forms and protonation, orienting hydrogens and flippable side-chains, structurally aligning the multitude of binding sites, and performing a reasonable reduction of ensemble structures. The pipeline, named SIENA, creates protein-structural ensembles for the analysis of protein flexibility, molecular design efforts like docking or de novo design within seconds. For the first time, we are able to process the whole PDB in order to create a large collection of protein binding site ensembles. SIENA is available as part of the ZBH ProteinsPlus webserver under http://proteinsplus.zbh.uni-hamburg.de. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Divergence of transcription factor binding sites across related yeast species.

    Science.gov (United States)

    Borneman, Anthony R; Gianoulis, Tara A; Zhang, Zhengdong D; Yu, Haiyuan; Rozowsky, Joel; Seringhaus, Michael R; Wang, Lu Yong; Gerstein, Mark; Snyder, Michael

    2007-08-10

    Characterization of interspecies differences in gene regulation is crucial for understanding the molecular basis of both phenotypic diversity and evolution. By means of chromatin immunoprecipitation and DNA microarray analysis, the divergence in the binding sites of the pseudohyphal regulators Ste12 and Tec1 was determined in the yeasts Saccharomyces cerevisiae, S. mikatae, and S. bayanus under pseudohyphal conditions. We have shown that most of these sites have diverged across these species, far exceeding the interspecies variation in orthologous genes. A group of Ste12 targets was shown to be bound only in S. mikatae and S. bayanus under pseudohyphal conditions. Many of these genes are targets of Ste12 during mating in S. cerevisiae, indicating that specialization between the two pathways has occurred in this species. Transcription factor binding sites have therefore diverged substantially faster than ortholog content. Thus, gene regulation resulting from transcription factor binding is likely to be a major cause of divergence between related species.

  14. DNase I footprinting of small molecule binding sites on DNA.

    Science.gov (United States)

    Bailly, Christian; Kluza, Jérôme; Martin, Christopher; Ellis, Thomas; Waring, Michael J

    2005-01-01

    Nuclease footprinting techniques were initially developed to investigate protein-deoxyribonucleic acid (DNA) interactions but these tools of molecular biology have also become instrumental for probing sequence-selective binding of small molecules to DNA. Here, the method is described and technical details are given for performing deoxyribonuclease (DNase) I footprinting with DNA-binding drugs. An example is presented where DNase I is used (as well as DNase II and micrococcal nuclease) to probe the patterns of sequence-selective recognition of DNA by the anticancer antibiotic actinomycin D. DNase I is a convenient endonuclease for detecting and locating the position of actinomycin-binding sites within GC-rich sequences.

  15. Cation specific binding with protein surface charges.

    Science.gov (United States)

    Hess, Berk; van der Vegt, Nico F A

    2009-08-11

    Biological organization depends on a sensitive balance of noncovalent interactions, in particular also those involving interactions between ions. Ion-pairing is qualitatively described by the law of "matching water affinities." This law predicts that cations and anions (with equal valence) form stable contact ion pairs if their sizes match. We show that this simple physical model fails to describe the interaction of cations with (molecular) anions of weak carboxylic acids, which are present on the surfaces of many intra- and extracellular proteins. We performed molecular simulations with quantitatively accurate models and observed that the order K(+) < Na(+) < Li(+) of increasing binding affinity with carboxylate ions is caused by a stronger preference for forming weak solvent-shared ion pairs. The relative insignificance of contact pair interactions with protein surfaces indicates that thermodynamic stability and interactions between proteins in alkali salt solutions is governed by interactions mediated through hydration water molecules.

  16. The binding sites on human heme oxygenase-1 for cytochrome p450 reductase and biliverdin reductase.

    Science.gov (United States)

    Wang, Jinling; de Montellano, Paul R Ortiz

    2003-05-30

    Human heme oxygenase-1 (hHO-1) catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, CO, and free iron. The biliverdin is subsequently reduced to bilirubin by biliverdin reductase. Earlier kinetic studies suggested that biliverdin reductase facilitates the release of biliverdin from hHO-1 (Liu, Y., and Ortiz de Montellano, P. R. (2000) J. Biol. Chem. 275, 5297-5307). We have investigated the binding of P450 reductase and biliverdin reductase to truncated, soluble hHO-1 by fluorescence resonance energy transfer and site-specific mutagenesis. P450 reductase and biliverdin reductase bind to truncated hHO-1 with Kd = 0.4 +/- 0.1 and 0.2 +/- 0.1 microm, respectively. FRET experiments indicate that biliverdin reductase and P450 reductase compete for binding to truncated hHO-1. Mutation of surface ionic residues shows that hHO-1 residues Lys18, Lys22, Lys179, Arg183, Arg198, Glu19, Glu127, and Glu190 contribute to the binding of cytochrome P450 reductase. The mutagenesis results and a computational analysis of the protein surfaces partially define the binding site for P450 reductase. An overlapping binding site including Lys18, Lys22, Lys179, Arg183, and Arg185 is similarly defined for biliverdin reductase. These results confirm the binding of biliverdin reductase to hHO-1 and define binding sites of the two reductases.

  17. Sequence and structural features of binding site residues in protein-protein complexes: comparison with protein-nucleic acid complexes

    Directory of Open Access Journals (Sweden)

    Selvaraj S

    2011-10-01

    Full Text Available Abstract Background Protein-protein interactions are important for several cellular processes. Understanding the mechanism of protein-protein recognition and predicting the binding sites in protein-protein complexes are long standing goals in molecular and computational biology. Methods We have developed an energy based approach for identifying the binding site residues in protein–protein complexes. The binding site residues have been analyzed with sequence and structure based parameters such as binding propensity, neighboring residues in the vicinity of binding sites, conservation score and conformational switching. Results We observed that the binding propensities of amino acid residues are specific for protein-protein complexes. Further, typical dipeptides and tripeptides showed high preference for binding, which is unique to protein-protein complexes. Most of the binding site residues are highly conserved among homologous sequences. Our analysis showed that 7% of residues changed their conformations upon protein-protein complex formation and it is 9.2% and 6.6% in the binding and non-binding sites, respectively. Specifically, the residues Glu, Lys, Leu and Ser changed their conformation from coil to helix/strand and from helix to coil/strand. Leu, Ser, Thr and Val prefer to change their conformation from strand to coil/helix. Conclusions The results obtained in this study will be helpful for understanding and predicting the binding sites in protein-protein complexes.

  18. Quantitative autoradiography of /sup 3/H-nomifensine binding sites in rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Scatton, B.; Dubois, A.; Dubocovich, M.L.; Zahniser, N.R.; Fage, D.

    1985-03-04

    The distribution of /sup 3/H-nomifensine binding sites in the rat brain has been studied by quantitative autoradiography. The binding of /sup 3/H-nomifensine to caudate putamen sections was saturable, specific, of a highly affinity (Kd = 56 nM) and sodium-dependent. The dopamine uptake inhibitors benztropine, nomifensine, cocaine, bupropion and amfonelic acid were the most potent competitors of /sup 3/H-nomifensine binding to striatal sections. The highest levels of (benztropine-displaceable) /sup 3/H-nomifensine binding sites were found in the caudate-putamen, the olfactory tubercle and the nucleus accumbens. 6-Hydroxy-dopamine-induced lesion of the ascending dopaminergic bundle resulted in a marked decrease in the /sup 3/H-ligand binding in these areas. Moderately high concentrations of the /sup 3/H-ligand were observed in the bed nucleus of the stria terminalis, the anteroventral thalamic nucleus, the cingulate cortex, the lateral septum, the hippocampus, the amygdala, the zona incerta and some hypothalamic nuclei. There were low levels of binding sites in the habenula, the dorsolateral geniculate body, the substantia nigra, the ventral tegmental area and the periaqueductal gray matter. These autoradiographic data are consistent with the hypothesis that /sup 3/H-nomifensine binds primarily to the presynaptic uptake site for dopamine but also labels the norepinephrine uptake site. 33 references, 2 figures, 1 table.

  19. Structure-based function prediction of uncharacterized protein using binding sites comparison.

    Science.gov (United States)

    Konc, Janez; Hodošček, Milan; Ogrizek, Mitja; Trykowska Konc, Joanna; Janežič, Dušanka

    2013-01-01

    A challenge in structural genomics is prediction of the function of uncharacterized proteins. When proteins cannot be related to other proteins of known activity, identification of function based on sequence or structural homology is impossible and in such cases it would be useful to assess structurally conserved binding sites in connection with the protein's function. In this paper, we propose the function of a protein of unknown activity, the Tm1631 protein from Thermotoga maritima, by comparing its predicted binding site to a library containing thousands of candidate structures. The comparison revealed numerous similarities with nucleotide binding sites including specifically, a DNA-binding site of endonuclease IV. We constructed a model of this Tm1631 protein with a DNA-ligand from the newly found similar binding site using ProBiS, and validated this model by molecular dynamics. The interactions predicted by the Tm1631-DNA model corresponded to those known to be important in endonuclease IV-DNA complex model and the corresponding binding free energies, calculated from these models were in close agreement. We thus propose that Tm1631 is a DNA binding enzyme with endonuclease activity that recognizes DNA lesions in which at least two consecutive nucleotides are unpaired. Our approach is general, and can be applied to any protein of unknown function. It might also be useful to guide experimental determination of function of uncharacterized proteins.

  20. 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.

  1. Five of Five VHHs Neutralizing Poliovirus Bind the Receptor-Binding Site

    Science.gov (United States)

    Strauss, Mike; Schotte, Lise; Thys, Bert; Filman, David J.

    2016-01-01

    ABSTRACT Nanobodies, or VHHs, that recognize poliovirus type 1 have previously been selected and characterized as candidates for antiviral agents or reagents for standardization of vaccine quality control. In this study, we present high-resolution cryo-electron microscopy reconstructions of poliovirus with five neutralizing VHHs. All VHHs bind the capsid in the canyon at sites that extensively overlap the poliovirus receptor-binding site. In contrast, the interaction involves a unique (and surprisingly extensive) surface for each of the five VHHs. Five regions of the capsid were found to participate in binding with all five VHHs. Four of these five regions are known to alter during the expansion of the capsid associated with viral entry. Interestingly, binding of one of the VHHs, PVSS21E, resulted in significant changes of the capsid structure and thus seems to trap the virus in an early stage of expansion. IMPORTANCE We describe the cryo-electron microscopy structures of complexes of five neutralizing VHHs with the Mahoney strain of type 1 poliovirus at resolutions ranging from 3.8 to 6.3Å. All five VHHs bind deep in the virus canyon at similar sites that overlap extensively with the binding site for the receptor (CD155). The binding surfaces on the VHHs are surprisingly extensive, but despite the use of similar binding surfaces on the virus, the binding surface on the VHHs is unique for each VHH. In four of the five complexes, the virus remains essentially unchanged, but for the fifth there are significant changes reminiscent of but smaller in magnitude than the changes associated with cell entry, suggesting that this VHH traps the virus in a previously undescribed early intermediate state. The neutralizing mechanisms of the VHHs and their potential use as quality control agents for the end game of poliovirus eradication are discussed. PMID:26764003

  2. Specific binding of neoglycoproteins to Toxoplasma gondii tachyzoites.

    Science.gov (United States)

    Robert, R; de la Jarrige, P L; Mahaza, C; Cottin, J; Marot-Leblond, A; Senet, J M

    1991-01-01

    Several studies have shown that protozoa bind to glycoproteins or neoglycoproteins. Here we report that Toxoplasma gondii binds strongly to bovine serum albumin-glucosamide. The binding was rapid, time dependent, partially reversible, saturable, and specific. Scatchard analysis showed about 40,000 molecules of bovine serum albumin-glucosamide per toxoplasma cell. The apparent dissociation constant was found to be 4.46 x 10(-8) M. Images PMID:1937826

  3. RNA-binding specificity of Y-box protein 1.

    Science.gov (United States)

    Dong, Jinjiang; Akcakanat, Argun; Stivers, David N; Zhang, Jiexin; Kim, Doyil; Meric-Bernstam, Funda

    2009-01-01

    Y-box protein 1 (YB-1) is a multifunctional DNA/RNA-binding protein that regulates transcription and translation. The specificity of YB-1's RNA binding and its consequences are unknown. Because expression and subcellular localization of YB-1 have been reported to be important in breast cancer, we determined the specificity and functional impact of YB-1 mRNA-binding in MCF7 breast cancer cells. We used YB-1 antibodies to immunoprecipitate YB-1 and microarray profiling to compare YB-1-bound and total poly(A) RNA. We demonstrated that YB-1 mRNA-binding was preferential. Transcript sequences significantly associated with this binding had high GC content. Selected YB-1 mRNA-binding targets were confirmed by QRT-PCR. However, downregulation of YB-1 levels by siRNA did not affect their RNA or protein expression. Thus, YB-1 has RNA-binding specificity; however, YB-1 binding does not necessarily regulate the stability or translation of its mRNA targets. Further study is needed to determine the functional consequences of selective YB-1 mRNA binding.

  4. Caffeine inhibits glucose transport by binding at the GLUT1 nucleotide-binding site

    Science.gov (United States)

    Sage, Jay M.; Cura, Anthony J.; Lloyd, Kenneth P.

    2015-01-01

    Glucose transporter 1 (GLUT1) is the primary glucose transport protein of the cardiovascular system and astroglia. A recent study proposes that caffeine uncompetitive inhibition of GLUT1 results from interactions at an exofacial GLUT1 site. Intracellular ATP is also an uncompetitive GLUT1 inhibitor and shares structural similarities with caffeine, suggesting that caffeine acts at the previously characterized endofacial GLUT1 nucleotide-binding site. We tested this by confirming that caffeine uncompetitively inhibits GLUT1-mediated 3-O-methylglucose uptake in human erythrocytes [Vmax and Km for transport are reduced fourfold; Ki(app) = 3.5 mM caffeine]. ATP and AMP antagonize caffeine inhibition of 3-O-methylglucose uptake in erythrocyte ghosts by increasing Ki(app) for caffeine inhibition of transport from 0.9 ± 0.3 mM in the absence of intracellular nucleotides to 2.6 ± 0.6 and 2.4 ± 0.5 mM in the presence of 5 mM intracellular ATP or AMP, respectively. Extracellular ATP has no effect on sugar uptake or its inhibition by caffeine. Caffeine and ATP displace the fluorescent ATP derivative, trinitrophenyl-ATP, from the GLUT1 nucleotide-binding site, but d-glucose and the transport inhibitor cytochalasin B do not. Caffeine, but not ATP, inhibits cytochalasin B binding to GLUT1. Like ATP, caffeine renders the GLUT1 carboxy-terminus less accessible to peptide-directed antibodies, but cytochalasin B and d-glucose do not. These results suggest that the caffeine-binding site bridges two nonoverlapping GLUT1 endofacial sites—the regulatory, nucleotide-binding site and the cytochalasin B-binding site. Caffeine binding to GLUT1 mimics the action of ATP but not cytochalasin B on sugar transport. Molecular docking studies support this hypothesis. PMID:25715702

  5. Sequence-Specific DNA Binding by a Short Peptide Dimer

    Science.gov (United States)

    Talanian, Robert V.; McKnight, C. James; Kim, Peter S.

    1990-08-01

    A recently described class of DNA binding proteins is characterized by the "bZIP" motif, which consists of a basic region that contacts DNA and an adjacent "leucine zipper" that mediates protein dimerization. A peptide model for the basic region of the yeast transcriptional activator GCN4 has been developed in which the leucine zipper has been replaced by a disulfide bond. The 34-residue peptide dimer, but not the reduced monomer, binds DNA with nanomolar affinity at 4^circC. DNA binding is sequence-specific as judged by deoxyribonuclease I footprinting. Circular dichroism spectroscopy suggests that the peptide adopts a helical structure when bound to DNA. These results demonstrate directly that the GCN4 basic region is sufficient for sequence-specific DNA binding and suggest that a major function of the GCN4 leucine zipper is simply to mediate protein dimerization. Our approach provides a strategy for the design of short sequence-specific DNA binding peptides.

  6. New insights into a classic aptamer: binding sites, cooperativity and more sensitive adenosine detection.

    Science.gov (United States)

    Zhang, Zijie; Oni, Olatunji; Liu, Juewen

    2017-07-27

    The DNA aptamer for adenosine (also for AMP and ATP) is a highly conserved sequence that has recurred in a few selections. It it a widely used model aptamer for biosensor development, and its nuclear magnetic resonance structure shows that each aptamer binds two AMP molecules. In this work, each binding site was individually removed by rational sequence design, while the remaining site still retained a similar binding affinity and specificity as confirmed by isothermal titration calorimetry. The thermodynamic parameters of binding are presented, and its biochemical implications are discussed. The number of binding sites can also be increased, and up to four sites are introduced in a single DNA sequence. Finally, the different sequences are made into fluorescent biosensors based on the structure-switching signaling aptamer design. The one-site aptamer has 3.8-fold higher sensitivity at lower adenosine concentration with a limit of detection of 9.1 μM adenosine, but weaker fluorescence signal at higher adenosine concentrations, consistent with a moderate cooperativity in the original aptamer. This work has offered insights into a classic aptamer for the relationship between the number of binding sites and sensitivity, and a shorter aptamer for improved biosensor design. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Substance P Receptor Binding Sites are Expressed by Glia in vivo after Neuronal Injury

    Science.gov (United States)

    Mantyh, Patrick W.; Johnson, Donald J.; Boehmer, Christian G.; Catton, Mark D.; Vinters, Harry V.; Maggio, John E.; Too, Heng-Phon; Vigna, Steven R.

    1989-07-01

    In vitro studies have demonstrated that glia can express functional receptors for a variety of neurotransmitters. To determine whether similar neurotransmitter receptors are also expressed by glia in vivo, we examined the glial scar in the transected optic nerve of the albino rabbit by quantitative receptor autoradiography. Receptor binding sites for radiolabeled calcitonin gene-related peptide, cholecystokinin, galanin, glutamate, somatostatin, substance P, and vasoactive intestinal peptide were examined. Specific receptor binding sites for each of these neurotransmitters were identified in the rabbit forebrain but were not detected in the normal optic nerve or tract. In the transected optic nerve and tract, only receptor binding sites for substance P were expressed at detectable levels. The density of substance P receptor binding sites observed in this glial scar is among the highest observed in the rabbit forebrain. Ligand displacement and saturation experiments indicate that the substance P receptor binding site expressed by the glial scar has pharmacological characteristics similar to those of substance P receptors in the rabbit striatum, rat brain, and rat and canine gut. The present study demonstrates that glial cells in vivo express high concentrations of substance P receptor binding sites after transection of retinal ganglion cell axons. Because substance P has been shown to regulate inflammatory and immune responses in peripheral tissues, substance P may also, by analogy, be involved in regulating the glial response to injury in the central nervous system.

  8. Binding site of ribosomal proteins on prokaryotic 5S ribonucleic acids: a study with ribonucleases

    DEFF Research Database (Denmark)

    Douthwaite, S; Christensen, A; Garrett, R A

    1982-01-01

    The binding sites of ribosomal proteins L18 and L25 on 5S RNA from Escherichia coli were probed with ribonucleases A, T1, and T2 and a double helix specific cobra venom endonuclease. The results for the protein-RNA complexes, which were compared with those for the free RNA [Douthwaite, S......., & Garrett, R. A. (1981) Biochemistry 20, 7301--7307], reveal an extensive interaction site for protein L18 and a more localized one for L25. Generally comparable results, with a few important differences, were obtained in a study of the binding sites of the two E. coli proteins on Bacillus...

  9. Processing Binding Relations in Specific Language Impairment

    Science.gov (United States)

    Schwartz, Richard G.; Hestvik, Arild; Seiger-Gardner, Liat; Almodovar, Diana

    2016-01-01

    Purpose: This sentence processing experiment examined the abilities of children with specific language impairment (SLI) and children with typical language development (TD) to establish relations between pronouns or reflexives and their antecedents in real time. Method: Twenty-two children with SLI and 24 age-matched children with TD (7;3-10;11…

  10. Found: Na(+) and K(+) binding sites of the sodium pump.

    Science.gov (United States)

    Rakowski, R F; Sagar, S

    2003-08-01

    Homology modeling and valence mapping have been used to predict the location and structure of Na(+) and K(+) binding sites in the Na(+)-K(+)-ATPase on the basis of the known atomic resolution structure of SERCA. Additional sites are predicted that may be associated with intracellular access and extracellular egress pathways for Na(+). The model predictions are in excellent agreement with previous structure-function and electrical studies.

  11. Defining the plasticity of transcription factor binding sites by Deconstructing DNA consensus sequences: the PhoP-binding sites among gamma/enterobacteria.

    Directory of Open Access Journals (Sweden)

    Oscar Harari

    2010-07-01

    Full Text Available Transcriptional regulators recognize specific DNA sequences. Because these sequences are embedded in the background of genomic DNA, it is hard to identify the key cis-regulatory elements that determine disparate patterns of gene expression. The detection of the intra- and inter-species differences among these sequences is crucial for understanding the molecular basis of both differential gene expression and evolution. Here, we address this problem by investigating the target promoters controlled by the DNA-binding PhoP protein, which governs virulence and Mg(2+ homeostasis in several bacterial species. PhoP is particularly interesting; it is highly conserved in different gamma/enterobacteria, regulating not only ancestral genes but also governing the expression of dozens of horizontally acquired genes that differ from species to species. Our approach consists of decomposing the DNA binding site sequences for a given regulator into families of motifs (i.e., termed submotifs using a machine learning method inspired by the "Divide & Conquer" strategy. By partitioning a motif into sub-patterns, computational advantages for classification were produced, resulting in the discovery of new members of a regulon, and alleviating the problem of distinguishing functional sites in chromatin immunoprecipitation and DNA microarray genome-wide analysis. Moreover, we found that certain partitions were useful in revealing biological properties of binding site sequences, including modular gains and losses of PhoP binding sites through evolutionary turnover events, as well as conservation in distant species. The high conservation of PhoP submotifs within gamma/enterobacteria, as well as the regulatory protein that recognizes them, suggests that the major cause of divergence between related species is not due to the binding sites, as was previously suggested for other regulators. Instead, the divergence may be attributed to the fast evolution of orthologous target

  12. Properties of achetakinin binding sites on malpighian tubule membranes from the house cricket, Acheta domesticus.

    Science.gov (United States)

    Chung, J S; Wheeler, C H; Goldsworthy, G J; Coast, G M

    1995-01-01

    A biologically active 125I-labeled analogue of AK-II (3'-hydroxyphenyl propionic-Gly-Gly-Gly-Phe-Ser-Pro-Trp-Gly-NH2) was used to investigate the properties of achetakinin binding sites on plasma membranes from Malpighian tubules of Acheta domesticus. With optimized conditions, binding was rapid, reversible, and specific, and saturation studies revealed a single class of binding sites with Kd 0.55 nM and Bmax 39.9 fmol/mg membrane protein. The affinities of achetakinins for binding sites on tubule membranes ranked AK-V > AK III > AK-II > AK-I > or = AK-IV, in general agreement with their potencies in functional assays. However, IC50 values were several orders of magnitude higher than corresponding values for EC50, which suggests a considerable receptor reserve.

  13. Identification and polymorphism of Plasmodium vivax RBP-1 peptides which bind specifically to reticulocytes.

    Science.gov (United States)

    Urquiza, Mauricio; Patarroyo, Manuel A; Marí, Viviana; Ocampo, Marisol; Suarez, Jorge; Lopez, Ramses; Puentes, Alvaro; Curtidor, Hernando; García, Javier; Rodríuez, Luis E; Vera, Ricardo; Torres, Angela; Laverde, Marilu; Robles, Ana P; Patarroyo, Manuel E

    2002-12-01

    Plasmodium vivax merozoite preferentially invades reticulocytes probably using PvRBP-1 as ligand. One hundred and ninety-five, 15-mer peptides has been synthesised from PvRBP-1 sequence; tested in reticulocyte- or erythrocyte-binding assays. Twenty-five peptides (K(d)=76-380 nM) specifically defined four reticulocyte-binding regions. It has been reported that a highly conserved Region-I recombinant fragment binds specifically to reticulocytes. HABP-critical residues for reticulocyte-binding were highly conserved in 20 Colombian P. vivax clinical isolates, suggesting an important biological function. There were six overlapping reticulocyte-binding sites for these peptides according to enzyme sensitivity and mutual competition-binding assays; located on 26- and 41-kDa reticulocyte membrane surface proteins.

  14. Protein-binding RNA aptamers affect molecular interactions distantly from their binding sites

    DEFF Research Database (Denmark)

    Dupont, Daniel M; Thuesen, Cathrine K; Bøtkjær, Kenneth A

    2015-01-01

    Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless......, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126) with therapeutic...... potential, both binding to the serine protease urokinase-type plasminogen activator (uPA). We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A) controlling uPA activities. One of the aptamers (upanap-126) binds to the area...

  15. Binding site of ribosomal proteins on prokaryotic 5S ribonucleic acids: a study with ribonucleases

    DEFF Research Database (Denmark)

    Douthwaite, S; Christensen, A; Garrett, R A

    1982-01-01

    The binding sites of ribosomal proteins L18 and L25 on 5S RNA from Escherichia coli were probed with ribonucleases A, T1, and T2 and a double helix specific cobra venom endonuclease. The results for the protein-RNA complexes, which were compared with those for the free RNA [Douthwaite, S., & Garr......The binding sites of ribosomal proteins L18 and L25 on 5S RNA from Escherichia coli were probed with ribonucleases A, T1, and T2 and a double helix specific cobra venom endonuclease. The results for the protein-RNA complexes, which were compared with those for the free RNA [Douthwaite, S...

  16. DNA deformability changes of single base pair mutants within CDE binding sites in S. Cerevisiae centromere DNA correlate with measured chromosomal loss rates and CDE binding site symmetries

    Directory of Open Access Journals (Sweden)

    Marx Kenneth A

    2006-03-01

    Full Text Available Abstract Background The centromeres in yeast (S. cerevisiae are organized by short DNA sequences (125 bp on each chromosome consisting of 2 conserved elements: CDEI and CDEIII spaced by a CDEII region. CDEI and CDEIII are critical sequence specific protein binding sites necessary for correct centromere formation and following assembly with proteins, are positioned near each other on a specialized nucleosome. Hegemann et al. BioEssays 1993, 15: 451–460 reported single base DNA mutants within the critical CDEI and CDEIII binding sites on the centromere of chromosome 6 and quantitated centromere loss of function, which they measured as loss rates for the different chromosome 6 mutants during cell division. Olson et al. Proc Natl Acad Sci USA 1998, 95: 11163–11168 reported the use of protein-DNA crystallography data to produce a DNA dinucleotide protein deformability energetic scale (PD-scale that describes local DNA deformability by sequence specific binding proteins. We have used the PD-scale to investigate the DNA sequence dependence of the yeast chromosome 6 mutants' loss rate data. Each single base mutant changes 2 PD-scale values at that changed base position relative to the wild type. In this study, we have utilized these mutants to demonstrate a correlation between the change in DNA deformability of the CDEI and CDEIII core sites and the overall experimentally measured chromosome loss rates of the chromosome 6 mutants. Results In the CDE I and CDEIII core binding regions an increase in the magnitude of change in deformability of chromosome 6 single base mutants with respect to the wild type correlates to an increase in the measured chromosome loss rate. These correlations were found to be significant relative to 105 Monte Carlo randomizations of the dinucleotide PD-scale applied to the same calculation. A net loss of deformability also tends to increase the loss rate. Binding site position specific, 4 data-point correlations were also

  17. Dangerous connections: on binding site models of infectious disease dynamics.

    Science.gov (United States)

    Leung, Ka Yin; Diekmann, Odo

    2017-02-01

    We formulate models for the spread of infection on networks that are amenable to analysis in the large population limit. We distinguish three different levels: (1) binding sites, (2) individuals, and (3) the population. In the tradition of physiologically structured population models, the formulation starts on the individual level. Influences from the 'outside world' on an individual are captured by environmental variables. These environmental variables are population level quantities. A key characteristic of the network models is that individuals can be decomposed into a number of conditionally independent components: each individual has a fixed number of 'binding sites' for partners. The Markov chain dynamics of binding sites are described by only a few equations. In particular, individual-level probabilities are obtained from binding-site-level probabilities by combinatorics while population-level quantities are obtained by averaging over individuals in the population. Thus we are able to characterize population-level epidemiological quantities, such as [Formula: see text], r, the final size, and the endemic equilibrium, in terms of the corresponding variables.

  18. A Sequence-Based Dynamic Ensemble Learning System for Protein Ligand-Binding Site Prediction.

    Science.gov (United States)

    Chen, Peng; Hu, ShanShan; Zhang, Jun; Gao, Xin; Li, Jinyan; Xia, Junfeng; Wang, Bing

    2016-01-01

    Proteins have the fundamental ability to selectively bind to other molecules and perform specific functions through such interactions, such as protein-ligand binding. Accurate prediction of protein residues that physically bind to ligands is important for drug design and protein docking studies. Most of the successful protein-ligand binding predictions were based on known structures. However, structural information is not largely available in practice due to the huge gap between the number of known protein sequences and that of experimentally solved structures. This paper proposes a dynamic ensemble approach to identify protein-ligand binding residues by using sequence information only. To avoid problems resulting from highly imbalanced samples between the ligand-binding sites and non ligand-binding sites, we constructed several balanced data sets and we trained a random forest classifier for each of them. We dynamically selected a subset of classifiers according to the similarity between the target protein and the proteins in the training data set. The combination of the predictions of the classifier subset to each query protein target yielded the final predictions. The ensemble of these classifiers formed a sequence-based predictor to identify protein-ligand binding sites. Experimental results on two Critical Assessment of protein Structure Prediction datasets and the ccPDB dataset demonstrated that of our proposed method compared favorably with the state-of-the-art. http://www2.ahu.edu.cn/pchen/web/LigandDSES.htm.

  19. Site-specific codon bias in bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Smith, J.M.; Smith, N.H. [Univ. of Sussex, Brighton (United Kingdom)

    1996-03-01

    Sequences of the gapA and ompA genes from 10 genera of enterobacteria have been analyzed. There is strong bias in codon usage, but different synonymous codons are preferred at different sites in the same gene. Site-specific preference for unfavored codons is not confined to the first 100 codons and is usually manifest between two codons utilizing the same tRNA. Statistical analyses, based on conclusions reached in an accompanying paper, show that the use of an unfavored codon at a given site in different genera is not due to common descent and must therefore be caused either by sequence-specific mutation or sequence-specific selection. Reasons are given for thinking that sequence-specific mutation cannot be responsible. We are unable to explain the preference between synonymous codons ending in C or T, but synonymous choice between A and G at third sites is largely explained by avoidance of AG-G (where the hyphen indicates the boundary between codons). We also observed that the preferred codon for proline in Enterobacter cloacea has changed from CCG to CCA. 27 refs., 7 tabs.

  20. Dancing in Place: Site-Specific Work

    Science.gov (United States)

    Metal-Corbin, Josie

    2012-01-01

    In her lecture the 2012 NDA Scholar/Artist, Josie Metal-Corbin, chronicles four decades of working with artists, educators, librarians, and scientists. The kinetic language of dance and the visual impact of specific environments provide provocative opportunities for collaboration, wherein the site becomes the framework or map for the dance design.…

  1. Prospects for site specific weed management

    DEFF Research Database (Denmark)

    Christensen, Svend; Rasmussen, Jesper; Pedersen, Søren Marcus

    2014-01-01

    Research on Site Specific Weed Management (SSWM) started in the late 80's. Since that moment, considerable research has been conducted on different aspects of SSWM, from fundamental studies on the spatial ecology of weeds to the applied development and testing of new technologies for weed detection...

  2. Differential Modulation of Annexin I Binding Sites on Monocytes and Neutrophils

    Directory of Open Access Journals (Sweden)

    H. S. Euzger

    1999-01-01

    Full Text Available Specific binding sites for the anti-inflammatory protein annexin I have been detected on the surface of human monocytes and polymorphonuclear leukocytes (PMN. These binding sites are proteinaceous in nature and are sensitive to cleavage by the proteolytic enzymes trypsin, collagenase, elastase and cathepsin G. When monocytes and PMN were isolated independently from peripheral blood, only the monocytes exhibited constitutive annexin I binding. However PMN acquired the capacity to bind annexin I following co-culture with monocytes. PMN incubation with sodium azide, but not protease inhibitors, partially blocked this process. A similar increase in annexin I binding capacity was also detected in PMN following adhesion to endothelial monolayers. We propose that a juxtacrine activation rather than a cleavage-mediated transfer is involved in this process. Removal of annexin I binding sites from monocytes with elastase rendered monocytes functionally insensitive to full length annexin I or to the annexin I-derived pharmacophore, peptide Ac2-26, assessed as suppression of the respiratory burst. These data indicate that the annexin I binding site on phagocytic cells may have an important function in the feedback control of the inflammatory response and their loss through cleavage could potentiate such responses.

  3. 2( sup 125 I)Iodomelatonin binding sites in spleens of guinea pigs

    Energy Technology Data Exchange (ETDEWEB)

    Poon, A.M.S. (Univ. of Hong Kong, (Hong Kong)); Pang, S.F. (Clarke Inst. of Psychiatry, Toronto, Ontario (Canada))

    1992-01-01

    2-({sup 125}I)Iodomelatonin was found to bind specifically to the membrane preparations of the spleens of guinea pigs with high affinity. The binding was rapid, stable, saturable and reversible. Scatchard analysis of the binding assays revealed an equilibrium dissociation constant (Kd) of 49.8{plus minus}4.12 pmol/l and binding site density (Bmax) of 0.69{plus minus}0.082 fmol/mg protein at mid-light. There was no significant change in the Kd or the Bmax at mid-dark. Kinetic analysis showed a Kd of 23.13{plus minus}4.81 pmol/l, in agreement to that derived from the saturation studies. The 2-({sup 125}I)iodomelatonin binding sites have the following order of potency: 2-iodomelatonin > melatonin > 6-chloromelatonin {much gt} N-acetylserotonin, 6-hydroxymelatonin > 5-methoxytryptamine, 5-methoxytryptophol > serotonin, 5-methoxyindole-3-acetic acid > 5-hydroxytryptophol, 3-acetylindole, 1-acetylindole-3-carboxyaldehyde, L-tryptophan > tryptamine, 5-hydroxyindole-3-acetic acid. Differential centrifugation studies showed that the binding sites are localized mainly in the nuclear fraction, the rest are distributed in the microsomal fraction, mitochondrial fraction and cytosolic fraction. The demonstration of 2-({sup 125}I)iodomelatonin binding sites in the spleen suggests the presence of melatonin receptors and a direct mechanism of action of melatonin on the immune system.

  4. Determination of the toluene diisocyanate binding sites on human serum albumin by tandem mass spectrometry.

    Science.gov (United States)

    Hettick, Justin M; Siegel, Paul D

    2011-07-15

    Diisocyanates are highly reactive chemical compounds widely used in the manufacture of polyurethanes. Although diisocyanates have been identified as causative agents of allergic respiratory diseases, the specific mechanism by which these diseases occur is largely unknown. To better understand the chemical species produced when diisocyanates react with protein, tandem mass spectrometry was employed to unambiguously identify the binding sites of the industrially important isomers, 2,4- and 2,6-toluene diisocyanate, on human serum albumin at varying diisocyanate/protein ratios. The 2,4-isomer results in approximately 2-fold higher conjugation product ion abundances than does the 2,6-isomer, suggesting that the 2,4-isomer has a higher reactivity toward albumin. Both isomers preferentially react with the N-terminal amine of the protein and the ε-NH(2) of lysine. At a low (1:2) diisocyanate/protein ratio, five binding sites are identified, whereas at a high (40:1) ratio, near-stoichiometric conjugation is observed with a maximum of 37 binding sites identified. Binding sites observed at the lowest conjugation ratios are conserved at higher binding ratios, suggesting a subset of 5-10 preferential binding sites on albumin. Diisocyanate-protein conjugation results in a variety of reaction products, including intra- and intermolecular crosslinking, diisocyanate self-polymerization, and diisocyanate hydrolysis. Published by Elsevier Inc.

  5. 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.

  6. The quinone-binding and catalytic site of complex II.

    Science.gov (United States)

    Maklashina, Elena; Cecchini, Gary

    2010-12-01

    The complex II family of proteins includes succinate:quinone oxidoreductase (SQR) and quinol:fumarate oxidoreductase (QFR). In the facultative bacterium Escherichia coli both are expressed as part of the aerobic (SQR) and anaerobic (QFR) respiratory chains. SQR from E. coli is homologous to mitochondrial complex II and has proven to be an excellent model system for structure/function studies of the enzyme. Both SQR and QFR from E. coli are tetrameric membrane-bound enzymes that couple succinate/fumarate interconversion with quinone/quinol reduction/oxidation. Both enzymes are capable of binding either ubiquinone or menaquinone, however, they have adopted different quinone binding sites where catalytic reactions with quinones occur. A comparison of the structures of the quinone binding sites in SQR and QFR reveals how the enzymes have adapted in order to accommodate both benzo- and napthoquinones. A combination of structural, computational, and kinetic studies of members of the complex II family of enzymes has revealed that the catalytic quinone adopts different positions in the quinone-binding pocket. These data suggest that movement of the quinone within the quinone-binding pocket is essential for catalysis. Published by Elsevier B.V.

  7. Mutations and binding sites of human transcription factors

    KAUST Repository

    Kamanu, Frederick Kinyua

    2012-06-01

    Mutations in any genome may lead to phenotype characteristics that determine ability of an individual to cope with adaptation to environmental challenges. In studies of human biology, among the most interesting ones are phenotype characteristics that determine responses to drug treatments, response to infections, or predisposition to specific inherited diseases. Most of the research in this field has been focused on the studies of mutation effects on the final gene products, peptides, and their alterations. Considerably less attention was given to the mutations that may affect regulatory mechanism(s) of gene expression, although these may also affect the phenotype characteristics. In this study we make a pilot analysis of mutations observed in the regulatory regions of 24,667 human RefSeq genes. Our study reveals that out of eight studied mutation types, insertions are the only one that in a statistically significant manner alters predicted transcription factor binding sites (TFBSs). We also find that 25 families of TFBSs have been altered by mutations in a statistically significant manner in the promoter regions we considered. Moreover, we find that the related transcription factors are, for example, prominent in processes related to intracellular signaling; cell fate; morphogenesis of organs and epithelium; development of urogenital system, epithelium, and tube; neuron fate commitment. Our study highlights the significance of studying mutations within the genes regulatory regions and opens way for further detailed investigations on this topic, particularly on the downstream affected pathways. 2012 Kamanu, Medvedeva, Schaefer, Jankovic, Archer and Bajic.

  8. Macrocycles with switchable exo/endo metal binding sites.

    Science.gov (United States)

    Tian, Lei-lei; Wang, Chun; Dawn, Sandipan; Smith, Mark D; Krause, Jeanette A; Shimizu, Linda S

    2009-12-09

    We report herein the synthesis and metal complexation properties of two macrocyclic hosts that contain two 2,2'-bipyridines and two urea groups. These hosts take advantage of the conformationally mobile 5,5'-positions of the bipyridines to give metal binding sites that are dynamic. By simple bond rotation, these hosts can exchange an interior (endo) situated metal binding site for an exterior (exo) binding site. We examine the solid-state structures of the two free hosts and two coordination complexes ([Cd(host 1)(H(2)O)(NO(3))(2)] and [Ag(2)(host 2)](SO(3)CF(3))(2)) using X-ray crystallography. Analysis of these crystal structures suggests that the bipyridine groups within the hosts are able to rotate to access multiple conformations including the desired exo and endo conformations. We also investigate the binding affinity of these new ligands in solution by UV-vis titrations with a series of metal nitrate salts (Ag, Cd, Zn, Ni, Mn, Fe, Co, Cr, and Cu) to afford discrete metal complexes. Some complexes showed a slow subsequent assembly to yield coordination polymers. Thus, these systems may afford unique insights into the process of metal organic framework formation.

  9. Site-Specific PEGylation of Therapeutic Proteins

    Directory of Open Access Journals (Sweden)

    Jonathan K. Dozier

    2015-10-01

    Full Text Available The use of proteins as therapeutics has a long history and is becoming ever more common in modern medicine. While the number of protein-based drugs is growing every year, significant problems still remain with their use. Among these problems are rapid degradation and excretion from patients, thus requiring frequent dosing, which in turn increases the chances for an immunological response as well as increasing the cost of therapy. One of the main strategies to alleviate these problems is to link a polyethylene glycol (PEG group to the protein of interest. This process, called PEGylation, has grown dramatically in recent years resulting in several approved drugs. Installing a single PEG chain at a defined site in a protein is challenging. Recently, there is has been considerable research into various methods for the site-specific PEGylation of proteins. This review seeks to summarize that work and provide background and context for how site-specific PEGylation is performed. After introducing the topic of site-specific PEGylation, recent developments using chemical methods are described. That is followed by a more extensive discussion of bioorthogonal reactions and enzymatic labeling.

  10. Discovering structural motifs using a structural alphabet: Application to magnesium-binding sites

    Directory of Open Access Journals (Sweden)

    Lim Carmay

    2007-03-01

    Full Text Available Abstract Background For many metalloproteins, sequence motifs characteristic of metal-binding sites have not been found or are so short that they would not be expected to be metal-specific. Striking examples of such metalloproteins are those containing Mg2+, one of the most versatile metal cofactors in cellular biochemistry. Even when Mg2+-proteins share insufficient sequence homology to identify Mg2+-specific sequence motifs, they may still share similarity in the Mg2+-binding site structure. However, no structural motifs characteristic of Mg2+-binding sites have been reported. Thus, our aims are (i to develop a general method for discovering structural patterns/motifs characteristic of ligand-binding sites, given the 3D protein structures, and (ii to apply it to Mg2+-proteins sharing 2+-structural motifs are identified as recurring structural patterns. Results The structural alphabet-based motif discovery method has revealed the structural preference of Mg2+-binding sites for certain local/secondary structures: compared to all residues in the Mg2+-proteins, both first and second-shell Mg2+-ligands prefer loops to helices. Even when the Mg2+-proteins share no significant sequence homology, some of them share a similar Mg2+-binding site structure: 4 Mg2+-structural motifs, comprising 21% of the binding sites, were found. In particular, one of the Mg2+-structural motifs found maps to a specific functional group, namely, hydrolases. Furthermore, 2 of the motifs were not found in non metalloproteins or in Ca2+-binding proteins. The structural motifs discovered thus capture some essential biochemical and/or evolutionary properties, and hence may be useful for discovering proteins where Mg2+ plays an important biological role. Conclusion The structural motif discovery method presented herein is general and can be applied to any set of proteins with known 3D structures. This new method is timely considering the increasing number of structures for

  11. Identification of a binding site for quaternary amines in factor Xa.

    Science.gov (United States)

    Monnaie, D; Arosio, D; Griffon, N; Rose, T; Rezaie, A R; Di Cera, E

    2000-05-09

    In the process of characterizing the Na(+)-binding properties of factor Xa, a specific inhibition of this enzyme by quaternary amines was identified, consistent with previous observations. The binding occurs with K(i) in the low millimolar range, with trimethylphenylammonium (TMPA) showing the highest specificity. Binding of TMPA inhibits substrate hydrolysis in a competitive manner, does not inhibit the binding of p-aminobenzamidine to the S1 pocket, and is positively linked to Na(+) binding. Inhibition by TMPA is also seen in thrombin and tissue plasminogen activator (tPA), though to a lesser extent compared to factor Xa. Computer modeling using the crystal structure of factor Xa suggests that TMPA binds to the S2/S3 specificity sites, with its hydrophobic moiety making van der Waals interactions with the side chains of Y99, F174, and W215, and the charged amine coupling electrostatically with the carboxylates of E97. Site-directed mutagenesis of factor Xa, thrombin, and tPA confirms the predictions drawn by docking calculations and reveal a dominant role for residue Y99. Binding of TMPA to factor Xa is drastically (25-fold) reduced by the Y99T replacement. Likewise, the Y99L substitution compromises binding of TMPA to tPA. On the other hand, the affinity of TMPA is enhanced 4-fold in thrombin with the substitution L99Y. The identification of a binding site for quaternary amines in factor Xa has a bearing on the rational design of selective inhibitors of this clotting enzyme.

  12. Nucleotide binding to human UMP-CMP kinase using fluorescent derivatives -- a screening based on affinity for the UMP-CMP binding site.

    Science.gov (United States)

    Topalis, Dimitri; Kumamoto, Hiroki; Amaya Velasco, Maria-Fernanda; Dugué, Laurence; Haouz, Ahmed; Alexandre, Julie Anne C; Gallois-Montbrun, Sarah; Alzari, Pedro Maria; Pochet, Sylvie; Agrofoglio, Luigi André; Deville-Bonne, Dominique

    2007-07-01

    Methylanthraniloyl derivatives of ATP and CDP were used in vitro as fluorescent probes for the donor-binding and acceptor-binding sites of human UMP-CMP kinase, a nucleoside salvage pathway kinase. Like all NMP kinases, UMP-CMP kinase binds the phosphodonor, usually ATP, and the NMP at different binding sites. The reaction results from an in-line phosphotransfer from the donor to the acceptor. The probe for the donor site was displaced by the bisubstrate analogs of the Ap5X series (where X = U, dT, A, G), indicating the broad specificity of the acceptor site. Both CMP and dCMP were competitors for the acceptor site probe. To find antimetabolites for antivirus and anticancer therapies, we have developed a method of screening acyclic phosphonate analogs that is based on the affinity of the acceptor-binding site of the human UMP-CMP kinase. Several uracil vinylphosphonate derivatives had affinities for human UMP-CMP kinase similar to those of dUMP and dCMP and better than that of cidofovir, an acyclic nucleoside phosphonate with a broad spectrum of antiviral activities. The uracil derivatives were inhibitors rather than substrates of human UMP-CMP kinase. Also, the 5-halogen-substituted analogs inhibited the human TMP kinase less efficiently. The broad specificity of the enzyme acceptor-binding site is in agreement with a large substrate-binding pocket, as shown by the 2.1 A crystal structure.

  13. Interactome-Wide Prediction of Protein-Protein Binding Sites Reveals Effects of Protein Sequence Variation in Arabidopsis thaliana

    NARCIS (Netherlands)

    Valentim, F.L.; Neven, F.; Boyen, P.; Dijk, van A.D.J.

    2012-01-01

    The specificity of protein-protein interactions is encoded in those parts of the sequence that compose the binding interface. Therefore, understanding how changes in protein sequence influence interaction specificity, and possibly the phenotype, requires knowing the location of binding sites in

  14. Nanoparticles for Site Specific Genome Editing

    Science.gov (United States)

    McNeer, Nicole Ali

    Triplex-forming peptide nucleic acids (PNAs) can be used to coordinate the recombination of short 50-60 by "donor DNA" fragments into genomic DNA, resulting in site-specific correction of genetic mutations or the introduction of advantageous genetic modifications. Site-specific gene editing in hematopoietic stem and progenitor cells (HSPCs) could result in treatment or cure of inherited disorders of the blood such as beta-thalassemia. Gene editing in HSPCs and differentiated T cells could help combat HIV/AIDs by modifying receptors, such as CCR5, necessary for R5-tropic HIV entry. However, translation of genome modification technologies to clinical practice is limited by challenges in intracellular delivery, especially in difficult-to-transfect hematolymphoid cells. In vivo gene editing could also provide novel treatment for systemic monogenic disorders such as cystic fibrosis, an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane receptor. Here, we have engineered biodegradable nanoparticles to deliver oligonucleotides for site-specific genome editing of disease-relevant genes in human cells, with high efficiency, low toxicity, and editing of clinically relevant cell types. We designed nanoparticles to edit the human beta-globin and CCR5 genes in hematopoietic cells. We show that poly(lactic-co-glycolic acid) (PLGA) nanoparticles can delivery PNA and donor DNA for site-specific gene modification in human hematopoietic cells in vitro and in vivo in NOD-scid IL2rgammanull mice. Nanoparticles delivered by tail vein localized to hematopoietic compartments in the spleen and bone marrow of humanized mice, resulting in modification of the beta-globin and CCR5 genes. Modification frequencies ranged from 0.005 to 20% of cells depending on the organ and cell type, without detectable toxicity. This project developed highly versatile methods for delivery of therapeutics to hematolymphoid cells and hematopoietic stem cells, and will help to

  15. Opioid binding site in EL-4 thymoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Fiorica, E.; Spector, S.

    1988-01-01

    Using EL-4 thymoma cell-line we found a binding site similar to the k opioid receptor of the nervous system. The Scatchard analysis of the binding of (/sup 3/H) bremazocine indicated a single site with a K/sub D/ = 60 +/- 17 nM and Bmax = 2.7 +/- 0.8 pmols/10/sup 6/ cells. To characterize this binding site, competition studies were performed using selective compounds for the various opioid receptors. The k agonist U-50,488H was the most potent displacer of (/sup 3/H) bremazocine with an IC/sub 50/ value = 0.57..mu..M. The two steroisomers levorphanol and dextrorphan showed the same affinity for this site. While morphine, (D-Pen/sup 2/, D-Pen/sup 5/) enkephalin and ..beta..-endorphin failed to displace, except at very high concentrations, codeine demonstrated a IC/sub 50/ = 60..mu..M, that was similar to naloxone. 32 references, 3 figures, 2 tables.

  16. Binding of dinitrogen to an iron-sulfur-carbon site

    Science.gov (United States)

    Čorić, Ilija; Mercado, Brandon Q.; Bill, Eckhard; Vinyard, David J.; Holland, Patrick L.

    2015-10-01

    Nitrogenases are the enzymes by which certain microorganisms convert atmospheric dinitrogen (N2) to ammonia, thereby providing essential nitrogen atoms for higher organisms. The most common nitrogenases reduce atmospheric N2 at the FeMo cofactor, a sulfur-rich iron-molybdenum cluster (FeMoco). The central iron sites that are coordinated to sulfur and carbon atoms in FeMoco have been proposed to be the substrate binding sites, on the basis of kinetic and spectroscopic studies. In the resting state, the central iron sites each have bonds to three sulfur atoms and one carbon atom. Addition of electrons to the resting state causes the FeMoco to react with N2, but the geometry and bonding environment of N2-bound species remain unknown. Here we describe a synthetic complex with a sulfur-rich coordination sphere that, upon reduction, breaks an Fe-S bond and binds N2. The product is the first synthetic Fe-N2 complex in which iron has bonds to sulfur and carbon atoms, providing a model for N2 coordination in the FeMoco. Our results demonstrate that breaking an Fe-S bond is a chemically reasonable route to N2 binding in the FeMoco, and show structural and spectroscopic details for weakened N2 on a sulfur-rich iron site.

  17. Plasmodium falciparum normocyte binding protein (PfNBP-1) peptides bind specifically to human erythrocytes.

    Science.gov (United States)

    Valbuena, John Jairo; Vera, Ricardo; García, Javier; Puentes, Alvaro; Curtidor, Hernando; Ocampo, Marisol; Urquiza, Mauricio; Rivera, Zuly; Guzmán, Fanny; Torres, Elizabeth; Patarroyo, Manuel Elkin

    2003-07-01

    Plasmodium falciparum normocyte binding protein-1 (PfNBP-1), a Plasmodium vivax RBP-1 orthologue is expressed in the apical merozoite area. PfNBP-1 binds directly to human erythrocyte membrane in a sialic acid-dependent but trypsin-resistant way. Erythrocyte binding assays were done with synthetic peptides covering the sequence reported as PfNBP-1. Two specific erythrocyte high activity binding peptides were found: 101VFINDLDTYQYEYFYEWNQ(120), peptide 26332, and 181NTKETYLKELNKKKMLQNKK(200), peptide 26336. These two peptides' binding was saturable and presenting nanomolar affinity constants. The critical binding residues (those residues underlined and highlighted in bold) were determined by competition assays with glycine-scan analogue peptides. These peptides were able to block merozoite in vitro invasion of erythrocytes.

  18. [Functional characteristics of the individual genomic condensin binding sites of Saccharomyces cerevisiae using minichromosome mitotic segregation stability model].

    Science.gov (United States)

    Butylin, P A; Strunnikov, A V

    2008-01-01

    Proper chromatin compaction in mitosis (condensation) is required for equal chromosome distribution and precise genetic information inheritance. Protein complex named condensin is responsible for the mitotic condensation, it also individualizes chromosomes, and ensures chromatin separation between sister chromatids in mitosis as well as proper mitotic spindle tension. Mitotic condensin function depends on recognition of the specific binding sites on the chromosome. Mechanism of condensin binding on the individual sites of the mitotic chromosomes, as well as molecular anatomy of these sites remains to be unclear. Even less known is how condensin binding on the individual sites helps separating chromosomes in anaphase. In current paper using minichromosome test, we analyze seven individual condensin binding sites in Saccharomyces cerevisiae found in previous all-genome CHIP on CHIP screening in our lab. This approach allowed us to find out what was the individual contribution of condensin binding sites in securing mitotic stability of the minichromosomes.

  19. Site-specific weed control technologies

    DEFF Research Database (Denmark)

    Christensen, Svend; Søgaard, Henning Tangen; Kudsk, Per

    2009-01-01

    Site-specific weed control technologies are defined as machinery or equipment embedded with technologies that detect weeds growing in a crop and, taking into account predefined factors such as economics, takes action to maximise the chances of successfully controlling them. In the article, we...... describe the basic parts of site specific weed control technologies, comprising of weed sensing systems, weed management models and precision weed control implements. A review of state-of-the-art technologies shows that several weed sensing systems and precision implements have been developed over the last...... two decades, though barriers prevent their breakthrough. Most important among these is the lack of a truly robust weed recognition method, owing to mutual shading among plants and limitations in the capacity of highly accurate spraying and weeding apparatuses.   Another barrier is the lack...

  20. 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...... potency against class 1 HDACs and are active in tissue culture against various human cancer cell lines. Importantly, enzymological analysis of 26 indicates that the cyclic α3β-tetrapeptide is a fast-on/ off competitive inhibitor of HDACs 1−3 with Ki values of 49, 33, and 37 nM, respectively. Our proof...

  1. Analysis and classification of DNA-binding sites in single-stranded and double-stranded DNA-binding proteins using protein information.

    Science.gov (United States)

    Wang, Wei; Liu, Juan; Xiong, Yi; Zhu, Lida; Zhou, Xionghui

    2014-08-01

    Single-stranded DNA-binding proteins (SSBs) and double-stranded DNA-binding proteins (DSBs) play different roles in biological processes when they bind to single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA). However, the underlying binding mechanisms of SSBs and DSBs have not yet been fully understood. Here, the authors firstly constructed two groups of ssDNA and dsDNA specific binding sites from two non-redundant sets of SSBs and DSBs. They further analysed the relationship between the two classes of binding sites and a newly proposed set of features (residue charge distribution, secondary structure and spatial shape). To assess and utilise the predictive power of these features, they trained a classification model using support vector machine to make predictions about the ssDNA and the dsDNA binding sites. The author's analysis and prediction results indicated that the two classes of binding sites can be distinguishable by the three types of features, and the final classifier using all the features achieved satisfactory performance. In conclusion, the proposed features will deepen their understanding of the specificity of proteins which bind to ssDNA or dsDNA.

  2. PET study of carbon-11-PK 11195 binding to peripheral type benzodiazepine sites in glioblastoma: A case report

    Energy Technology Data Exchange (ETDEWEB)

    Pappata, S.; Cornu, P.; Samson, Y.; Prenant, C.; Benavides, J.; Scatton, B.; Crouzel, C.; Hauw, J.J.; Syrota, A. (INSERM U. 334 Service Hospitalier Frederic Joliot, Paris (France))

    1991-08-01

    The utility of the peripheral type benzodiazepine site ligand 11C-PK 11195, for imaging human glioma in conjunction with Positron Emission Tomography, relies on a high specific binding of the tracer to tumoral peripheral type benzodiazepines sites. In a patient with glioblastoma, the authors found that 11C-PK 11195 binding was two-fold higher in the tumor than in normal gray matter and that 30% of tumoral binding could be displaced by a large excess of unlabeled drug. These findings suggest that tumoral retention of the ligand is due, in part, to specific binding.

  3. 3D-QSAR model of flavonoids binding at benzodiazepine site in GABAA receptors.

    Science.gov (United States)

    Huang, X; Liu, T; Gu, J; Luo, X; Ji, R; Cao, Y; Xue, H; Wong, J T; Wong, B L; Pei, G; Jiang, H; Chen, K

    2001-06-07

    With flavone as a structural template, three-dimensional quantitative structure-activity relationship (3D-QSAR) studies and ab initio calculations were performed on a series of flavonoids. A reasonable pharmacophore model was built through CoMFA, CoMSIA, and HQSAR analyses and electrostatic potential calculations. A plausible binding mode for flavonoids with GABA(A) receptors was rationalized. On the basis of the commonly recognized binding site, the specific S1 and S2 subsites relating to substituent positions were proposed. The different binding affinities could be explained according to the frontier orbitals and electrostatic potential (ESP) maps. The ESP could be used as a novel starting point for designing more selective BZ-binding-site ligands.

  4. Autoradiographic demonstration of oxytocin-binding sites in the macula densa

    Energy Technology Data Exchange (ETDEWEB)

    Stoeckel, M.E.; Freund-Mercier, M.J. (Centre National de la Recherche Scientifique, Strasbourg (France))

    1989-08-01

    Specific oxytocin (OT)-binding sites were localized in the rat kidney with use of a selective {sup 125}I-labeled OT antagonist ({sup 125}I-OTA). High concentrations of OT binding sites were detected on the juxtaglomerular apparatus with use of the conventional film autoradiographic technique. No labeling occurred on other renal structures. The cellular localization of the OT binding sites within the juxtaglomerular apparatus was studied in light microscope autoradiography, on semithin sections from paraformaldehyde-fixed kidney slices incubated in the presence of {sup 125}I-OTA. These preparations revealed selective labeling of the macula densa, mainly concentrated at the basal pole of the cells. Control experiments showed first that {sup 125}I-OTA binding characteristics were not noticeably altered by prior paraformaldehyde fixation of the kidneys and second that autoradiographic detection of the binding sites was not impaired by histological treatments following binding procedures. In view of the role of the macula densa in the tubuloglomerular feedback, the putative OT receptors of this structure might mediate the stimulatory effect of OT on glomerular filtration.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  6. Savannah River Site's Site Specific Plan

    Energy Technology Data Exchange (ETDEWEB)

    1991-08-01

    This Site Specific Plan (SSP) has been prepared by the Savannah River Site (SRS) in order to show the Environmental Restoration and Waste Management activities that were identified during the preparation of the Department of Energy-Headquarters (DOE-HQ) Environmental Restoration and Waste Management Five-Year Plan (FYP) for FY 1992--1996. The SSP has been prepared in accordance with guidance received from DOE-HQ. DOE-SR is accountable to DOE-HQ for the implementation of this plan. The purpose of the SSP is to develop a baseline for policy, budget, and schedules for the DOE Environmental Restoration and Waste Management activities. The plan explains accomplishments since the Fiscal Year (FY) 1990 plan, demonstrates how present and future activities are prioritized, identifies currently funded activities and activities that are planned to be funded in the upcoming fiscal year, and describes future activities that SRS is considering.

  7. Bookmarking by specific and nonspecific binding of FoxA1 pioneer factor to mitotic chromosomes.

    Science.gov (United States)

    Caravaca, Juan Manuel; Donahue, Greg; Becker, Justin S; He, Ximiao; Vinson, Charles; Zaret, Kenneth S

    2013-02-01

    While most transcription factors exit the chromatin during mitosis and the genome becomes silent, a subset of factors remains and "bookmarks" genes for rapid reactivation as cells progress through the cell cycle. However, it is unknown whether such bookmarking factors bind to chromatin similarly in mitosis and how different binding capacities among them relate to function. We compared a diverse set of transcription factors involved in liver differentiation and found markedly different extents of mitotic chromosome binding. Among them, the pioneer factor FoxA1 exhibits the greatest extent of mitotic chromosome binding. Genomically, ~15% of the FoxA1 interphase target sites are bound in mitosis, including at genes that are important for liver differentiation. Biophysical, genome mapping, and mutagenesis studies of FoxA1 reveals two different modes of binding to mitotic chromatin. Specific binding in mitosis occurs at sites that continue to be bound from interphase. Nonspecific binding in mitosis occurs across the chromosome due to the intrinsic chromatin affinity of FoxA1. Both specific and nonspecific binding contribute to timely reactivation of target genes post-mitosis. These studies reveal an unexpected diversity in the mechanisms by which transcription factors help retain cell identity during mitosis.

  8. Specific binding and mineralization of calcified surfaces by small peptides.

    Science.gov (United States)

    Yarbrough, Daniel K; Hagerman, Elizabeth; Eckert, Randal; He, Jian; Choi, Hyewon; Cao, Nga; Le, Karen; Hedger, Jennifer; Qi, Fengxia; Anderson, Maxwell; Rutherford, Bruce; Wu, Ben; Tetradis, Sotiris; Shi, Wenyuan

    2010-01-01

    Several small (dentin phosphoprotein, one of the major noncollagenous proteins thought to be involved in the mineralization of the dentin extracellular matrix during tooth development. These peptides, consisting of multiple repeats of the tripeptide aspartate-serine-serine (DSS), bind with high affinity to calcium phosphate compounds and, when immobilized, can recruit calcium phosphate to peptide-derivatized polystyrene beads or to demineralized human dentin surfaces. The affinity of binding to hydroxyapatite surfaces increases with the number of (DSS)(n) repeats, and though similar repeated sequences-(NTT)(n), (DTT)(n), (ETT)(n), (NSS)(n), (ESS)(n), (DAA)(n), (ASS)(n), and (NAA)(n)-also showed HA binding activity, it was generally not at the same level as the natural sequence. Binding of the (DSS)(n) peptides to sectioned human teeth was shown to be tissue-specific, with high levels of binding to the mantle dentin, lower levels of binding to the circumpulpal dentin, and little or no binding to healthy enamel. Phosphorylation of the serines of these peptides was found to affect the avidity, but not the affinity, of binding. The potential utility of these peptides in the detection of carious lesions, the delivery of therapeutic compounds to mineralized tissues, and the modulation of remineralization is discussed.

  9. Assessing the model transferability for prediction of transcription factor binding sites based on chromatin accessibility.

    Science.gov (United States)

    Liu, Sheng; Zibetti, Cristina; Wan, Jun; Wang, Guohua; Blackshaw, Seth; Qian, Jiang

    2017-07-27

    Computational prediction of transcription factor (TF) binding sites in different cell types is challenging. Recent technology development allows us to determine the genome-wide chromatin accessibility in various cellular and developmental contexts. The chromatin accessibility profiles provide useful information in prediction of TF binding events in various physiological conditions. Furthermore, ChIP-Seq analysis was used to determine genome-wide binding sites for a range of different TFs in multiple cell types. Integration of these two types of genomic information can improve the prediction of TF binding events. We assessed to what extent a model built upon on other TFs and/or other cell types could be used to predict the binding sites of TFs of interest. A random forest model was built using a set of cell type-independent features such as specific sequences recognized by the TFs and evolutionary conservation, as well as cell type-specific features derived from chromatin accessibility data. Our analysis suggested that the models learned from other TFs and/or cell lines performed almost as well as the model learned from the target TF in the cell type of interest. Interestingly, models based on multiple TFs performed better than single-TF models. Finally, we proposed a universal model, BPAC, which was generated using ChIP-Seq data from multiple TFs in various cell types. Integrating chromatin accessibility information with sequence information improves prediction of TF binding.The prediction of TF binding is transferable across TFs and/or cell lines suggesting there are a set of universal "rules". A computational tool was developed to predict TF binding sites based on the universal "rules".

  10. Phenothiazine-binding and attachment sites of CAPP1-calmodulin.

    Science.gov (United States)

    Newton, D L; Klee, C B

    1989-05-02

    In the presence of Ca2+ norchlorpromazine isothiocyanate forms a monocovalent complex with calmodulin: CAPP1-calmodulin (Newton et al, 1983). Trypsin digestion of [3H]CAPP1-calmodulin yields as the major radioactive peptide N epsilon-CAPP-Lys-Met-Lys, corresponding to residues 75-77 of calmodulin. Stoichiometric amounts of all other expected tryptic peptides are also found, indicating that norchlorpromazine isothiocyanate selectively acylates Lys 75. A second molecule of CAPP-NCS can react, albeit slowly, with calmodulin to form CAPP2-calmodulin. Fragments 38-74 and 127-148 are completely missing from the trypsin digests of CAPP2-calmodulin without deliberate exposure to UV irradiation. Possibly the lengthy preparation of CAPP2-calmodulin favors photolysis, caused by room lights, of the putative CAPP-binding domains located in these two peptides. Lys 148, the sole lysyl residue in fragment 127-148, is a probable site of attachment of the second molecule of CAPP. UV irradiation of CAPP1-calmodulin, followed by digestion with trypsin, results in the selective loss of 50% each of peptides containing residues 38-74 and 127-148, suggesting that these peptides contain the hydrophobic amino acids that form the phenothiazine-binding sites. The loss of peptides encompassing residues 38-74 and 127-148, located in the amino and carboxyl halves of calmodulin, respectively, suggests that the hydrophobic rings of CAPP can bind at either one of the two phenothiazine sites. Computer modeling of CAPP1-calmodulin with the X-ray coordinates of calmodulin (Babu et al., 1986) indicates that CAPP attached to Lys 75 cannot interact with the carboxyl-terminal phenothiazine-binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Site-Specific Carbon Isotopes in Organics

    Science.gov (United States)

    Piasecki, A.; Eiler, J. M.

    2012-12-01

    Natural organic molecules exhibit a wide range of internal site-specific isotope variation (i.e., molecules with same isotopic substitution type but different site). Such variations are generally unconstrained by bulk isotopic measurements. If known, site-specific variations might constrain temperatures of equilibrium, mechanisms of formation or consumption reactions, and possibly other details. For example, lipids can exhibit carbon isotope differences of up to 30‰ between adjacent carbon sites as a result of fractionations arising during decarboxylation of pyruvate and other steps in lipid biosynthesis(1). We present a method for site-specific carbon isotope analysis of propane, based on high-resolution, multi-collector gas source mass spectrometry, using a novel prototype instrument - the Thermo MAT 253 Ultra. This machine has an inlet system and electron bombardment ion source resembling those in conventional stable isotope gas source mass spectrometers, and the energy filter, magnet, and detector array resembling those in multi-collector ICPMS and TIMS. The detector array has 7 detector positions, 6 of which are movable, and each of which can collect ions with either a faraday cup (read through amplifiers ranging from 107-1012 ohms) or an SEM. High mass resolving power (up to 27,000, MRP = M/dM definition) is achieved through a narrow entrance slit, adjustable from 250 to 5 μm. Such resolution can cleanly separate isobaric interferences between isotopologues of organic molecules having the same cardinal mass (e.g., 13CH3 and 12CH2D). We use this technology to analyze the isotopologues and fragments of propane, and use such data to solve for the site-specific carbon isotope fractionation. By measuring isotopologues of both the one-carbon (13CH3) and the two-carbon (13C12CH4) fragment ion, we can solve for both bulk δ13C and the difference in δ13C between the terminal and central carbon position. We tested this method by analyzing mixtures between natural

  12. A Conserved Steroid Binding Site in Cytochrome c Oxidase

    Energy Technology Data Exchange (ETDEWEB)

    Qin, Ling; Mills, Denise A.; Buhrow, Leann; Hiser, Carrie; Ferguson-Miller, Shelagh (Michigan)

    2010-09-02

    Micromolar concentrations of the bile salt deoxycholate are shown to rescue the activity of an inactive mutant, E101A, in the K proton pathway of Rhodobacter sphaeroides cytochrome c oxidase. A crystal structure of the wild-type enzyme reveals, as predicted, deoxycholate bound with its carboxyl group at the entrance of the K path. Since cholate is a known potent inhibitor of bovine oxidase and is seen in a similar position in the bovine structure, the crystallographically defined, conserved steroid binding site could reveal a regulatory site for steroids or structurally related molecules that act on the essential K proton path.

  13. Cloud computing for protein-ligand binding site comparison.

    Science.gov (United States)

    Hung, Che-Lun; Hua, Guan-Jie

    2013-01-01

    The proteome-wide analysis of protein-ligand binding sites and their interactions with ligands is important in structure-based drug design and in understanding ligand cross reactivity and toxicity. The well-known and commonly used software, SMAP, has been designed for 3D ligand binding site comparison and similarity searching of a structural proteome. SMAP can also predict drug side effects and reassign existing drugs to new indications. However, the computing scale of SMAP is limited. We have developed a high availability, high performance system that expands the comparison scale of SMAP. This cloud computing service, called Cloud-PLBS, combines the SMAP and Hadoop frameworks and is deployed on a virtual cloud computing platform. To handle the vast amount of experimental data on protein-ligand binding site pairs, Cloud-PLBS exploits the MapReduce paradigm as a management and parallelizing tool. Cloud-PLBS provides a web portal and scalability through which biologists can address a wide range of computer-intensive questions in biology and drug discovery.

  14. Cloud Computing for Protein-Ligand Binding Site Comparison

    Directory of Open Access Journals (Sweden)

    Che-Lun Hung

    2013-01-01

    Full Text Available The proteome-wide analysis of protein-ligand binding sites and their interactions with ligands is important in structure-based drug design and in understanding ligand cross reactivity and toxicity. The well-known and commonly used software, SMAP, has been designed for 3D ligand binding site comparison and similarity searching of a structural proteome. SMAP can also predict drug side effects and reassign existing drugs to new indications. However, the computing scale of SMAP is limited. We have developed a high availability, high performance system that expands the comparison scale of SMAP. This cloud computing service, called Cloud-PLBS, combines the SMAP and Hadoop frameworks and is deployed on a virtual cloud computing platform. To handle the vast amount of experimental data on protein-ligand binding site pairs, Cloud-PLBS exploits the MapReduce paradigm as a management and parallelizing tool. Cloud-PLBS provides a web portal and scalability through which biologists can address a wide range of computer-intensive questions in biology and drug discovery.

  15. Cloud Computing for Protein-Ligand Binding Site Comparison

    Science.gov (United States)

    2013-01-01

    The proteome-wide analysis of protein-ligand binding sites and their interactions with ligands is important in structure-based drug design and in understanding ligand cross reactivity and toxicity. The well-known and commonly used software, SMAP, has been designed for 3D ligand binding site comparison and similarity searching of a structural proteome. SMAP can also predict drug side effects and reassign existing drugs to new indications. However, the computing scale of SMAP is limited. We have developed a high availability, high performance system that expands the comparison scale of SMAP. This cloud computing service, called Cloud-PLBS, combines the SMAP and Hadoop frameworks and is deployed on a virtual cloud computing platform. To handle the vast amount of experimental data on protein-ligand binding site pairs, Cloud-PLBS exploits the MapReduce paradigm as a management and parallelizing tool. Cloud-PLBS provides a web portal and scalability through which biologists can address a wide range of computer-intensive questions in biology and drug discovery. PMID:23762824

  16. A molecular dynamics study of human serum albumin binding sites.

    Science.gov (United States)

    Artali, Roberto; Bombieri, Gabriella; Calabi, Luisella; Del Pra, Antonio

    2005-01-01

    A 2.0 ns unrestrained Molecular Dynamics was used to elucidate the geometric and dynamic properties of the HSA binding sites. The structure is not stress affected and the rmsds calculated from the published crystallographic data are almost constant for all the simulation time, with an averaged value of 2.4A. The major variability is in the C-terminus region. The trajectory analysis of the IIA binding site put in evidence fast oscillations for the Cgamma@Leu203...Cgamma@Leu275 and Cgamma@Leu219...Cgamma@Leu260 distances, with fluctuations around 250 ps, 1000 ps and over for the first, while the second is smoothly increasing with the simulation time from 7 to 10A. These variations are consistent with a volume increase up to 20% confirmed by the inter-domain contacts analysis, in particular for the pair O@Pro148...Ogamma@Ser283, representing the change of distance between IB-h9 and IIA-h6, O@Glu149...Ogamma@Ser189 for sub-domains IB-h9/IIA-h1 and N@Val339...Odelta2@Asp447 sub-domains IIB-h9/IIIA-h1. These inter-domain motions confirm the flexibility of the unfatted HSA with possible binding site pre-formation.

  17. 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.

  18. Angiotensin II binding sites in the conduction system of rat hearts

    Energy Technology Data Exchange (ETDEWEB)

    Saito, K.; Gutkind, J.S.; Saavedra, J.M. (National Institute of Mental Health, Bethesda, MD (USA))

    1987-12-01

    Angiotensin II binding sites were localized and quantified in the conduction system of the rat heart by autoradiography in combination with computerized microdensitometry. Tissue sections, containing cardiac vagus ganglia, sinus node, and atrioventricular node, were incubated with {sup 125}I-Sar{sup 1}-angiotensin II to generate autoradiograms that were compared with other autoradiograms from {sup 125}I-labeled standards. Angiotensin II binding sites were highly localized in both the sinoatrial and atrioventricular nodes and in the cardiac vagus ganglia. In contrast, binding to the angiotensin converting enzyme, determined by incubation of adjacent tissue sections with the specific enzyme inhibitor {sup 125}I-351A, was very low in these areas but high in the cardiac endothelium. The results suggest that angiotensin II may have direct chronotropic effects through stimulation of specific receptors in the conduction system of the heart and in intrinsic parasympathetic ganglia.

  19. Site-directed mutagenesis of substrate binding sites of azoreductase from Rhodobacter sphaeroides.

    Science.gov (United States)

    Liu, Guangfei; Zhou, Jiti; Wang, Jing; Yan, Bin; Li, Jingmei; Lu, Hong; Qu, Yuanyuan; Jin, Ruofei

    2008-05-01

    Comparison of three-dimensional structures of flavin-dependent azoreductases revealed two conserved loops around the flavin mononucleotide (FMN) cofactor. Tyr74, His75 and Lys109 in the two loops of azoreductase AZR from Rhodobacter sphaeroides were replaced with Trp, Asn and Ala/His by site-directed mutagenesis, respectively. The optimal pH values of K109H and H75N were pH 6, and those of K109A and Y74W were pH 9. The optimal temperature (30 degrees C) was not affected by mutation. Positively charged residues at position 109 is critical for the binding of methyl red. K109 might only be involved in the binding of the 2'-phosphate group of NADPH and have no effect on the binding of NADH. Y74W and H75N mutations decreased the binding of methyl red/nitrofurazone and had no affect on the binding of NADPH.

  20. Combining specificity determining and conserved residues improves functional site prediction

    Directory of Open Access Journals (Sweden)

    Gelfand Mikhail S

    2009-06-01

    Full Text Available Abstract Background Predicting the location of functionally important sites from protein sequence and/or structure is a long-standing problem in computational biology. Most current approaches make use of sequence conservation, assuming that amino acid residues conserved within a protein family are most likely to be functionally important. Most often these approaches do not consider many residues that act to define specific sub-functions within a family, or they make no distinction between residues important for function and those more relevant for maintaining structure (e.g. in the hydrophobic core. Many protein families bind and/or act on a variety of ligands, meaning that conserved residues often only bind a common ligand sub-structure or perform general catalytic activities. Results Here we present a novel method for functional site prediction based on identification of conserved positions, as well as those responsible for determining ligand specificity. We define Specificity-Determining Positions (SDPs, as those occupied by conserved residues within sub-groups of proteins in a family having a common specificity, but differ between groups, and are thus likely to account for specific recognition events. We benchmark the approach on enzyme families of known 3D structure with bound substrates, and find that in nearly all families residues predicted by SDPsite are in contact with the bound substrate, and that the addition of SDPs significantly improves functional site prediction accuracy. We apply SDPsite to various families of proteins containing known three-dimensional structures, but lacking clear functional annotations, and discusse several illustrative examples. Conclusion The results suggest a better means to predict functional details for the thousands of protein structures determined prior to a clear understanding of molecular function.

  1. Damage-specific DNA-binding proteins from human cells

    Energy Technology Data Exchange (ETDEWEB)

    Kanjilal, S.

    1992-01-01

    The primary objective of the study was to detect and characterize factors from human cells that bind DNA damaged by ultraviolet radiation. An application of the gel-shift assay was devised in which a DNA probe was UV-irradiated and compared with non-irradiated probe DNA for the ability to bind to such factors in cell extracts. UV-dose dependent binding proteins were identified. Formation of the DNA-protein complexes was independent of the specific sequence, form or source of the DNA. There was a marked preference for lesions on double stranded DNA over those on single stranded DNA. DNA irradiated with gamma rays did not compete with UV-irradiated DNA for the binding activities. Cell lines from patients with genetic diseases associated with disorders of the DNA repair system were screened for the presence of damaged-DNA-binding activities. Simultaneous occurrence of the clinical symptoms of some of these diseases had been previously documented and possible links between the syndromes proposed. However, supporting biochemical or molecular evidence for such associations were lacking. The data from the present investigations indicate that some cases of Xeroderma Pigmentosum group A, Cockayne's Syndrome, Bloom's Syndrome and Ataxia Telangiectasia, all of which exhibit sensitivity to UV or gamma radiation, share an aberrant damaged-DNA-binding factor. These findings support the hypothesis that some of the repair disorder diseases are closely related and may have arisen from a common defect. Partial purification of the binding activities from HeLa cells was achieved. Size-exclusion chromatography resolved the activities into various peaks, one of which was less damage-specific than the others as determined by competition studies using native or UV-irradiated DNA. Some of the activities were further separated by ion-exchange chromatography. On using affinity chromatography methods, the major damage-binding factor could be eluted in the presence of 2 M KCl and 1

  2. Lack of Ligand-Selective Binding of the Aryl Hydrocarbon Receptor to Putative DNA Binding Sites Regulating Expression of Bax and Paraoxonase 1 Genes

    OpenAIRE

    DeGroot, Danica E.; Hayashi, Ai; Michael S Denison

    2013-01-01

    The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates the biological and toxicological effects of structurally diverse chemicals through its ability to bind specific DNA recognition sites (dioxin responsive elements (DREs)), and activate transcription of adjacent genes. While the DRE has a highly conserved consensus sequence, it has been suggested that the nucleotide specificity of AhR DNA binding may be ligand-dependent. The upstream regulatory regions ...

  3. Platelet serotonin-2 receptor binding sites in depression and suicide.

    Science.gov (United States)

    Pandey, G N; Pandey, S C; Janicak, P G; Marks, R C; Davis, J M

    1990-08-01

    In order to examine the role of serotonin-2 (5HT2) receptors in depression and suicide, we determined 5HT2 receptors using 125I-lysergic acid diethylamide (LSD) as the binding ligand in platelets obtained from 20 normal control and 23 drug-free depressed patients. Our results indicate significantly increased 125I-LSD binding sites (Bmax) in the platelets of depressed patients compared with normal control subjects. We also observed that a subgroup of depressed patients with a recent history of suicide attempts or suicidal ideation had significantly higher 5HT2 binding sites as compared with nonsuicidal depressed patients and normal controls. There were no significant differences in the apparent dissociation constant (Kd) values in the platelets of depressed patients compared with normal control subjects. To examine if the baseline 5HT2 receptors are related to either the severity of illness or treatment response, we determined the relationships of the baseline Bmax and Kd with baseline Hamilton Depression Rating Scale (HDRS) and Brief Psychiatric Rating Scale (BPRS) scores and change in scores after treatment. We found no significant correlation between baseline Bmax and Kd with the baseline HDRS or BPRS scores or change in these scores after psychoactive drug treatment. These results thus indicate increased platelet 5HT2 receptors in depression, but much more so in depressed patients with suicidal ideation or attempts.

  4. 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.

  5. 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.

  6. Recognition of AT-Rich DNA Binding Sites by the MogR Repressor

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Aimee; Higgins, Darren E.; Panne, Daniel; (Harvard-Med); (EMBL)

    2009-07-22

    The MogR transcriptional repressor of the intracellular pathogen Listeria monocytogenes recognizes AT-rich binding sites in promoters of flagellar genes to downregulate flagellar gene expression during infection. We describe here the 1.8 A resolution crystal structure of MogR bound to the recognition sequence 5' ATTTTTTAAAAAAAT 3' present within the flaA promoter region. Our structure shows that MogR binds as a dimer. Each half-site is recognized in the major groove by a helix-turn-helix motif and in the minor groove by a loop from the symmetry-related molecule, resulting in a 'crossover' binding mode. This oversampling through minor groove interactions is important for specificity. The MogR binding site has structural features of A-tract DNA and is bent by approximately 52 degrees away from the dimer. The structure explains how MogR achieves binding specificity in the AT-rich genome of L. monocytogenes and explains the evolutionary conservation of A-tract sequence elements within promoter regions of MogR-regulated flagellar genes.

  7. Gamma-aminobutyric acid-modulated benzodiazepine binding sites in bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Lummis, S.C.R.; Johnston, G.A.R. (Univ. of Sydney, New South Wales (Australia)); Nicoletti, G. (Royal Melbourne Inst. of Tech. (Australia)); Holan, G. (CSIRO, Melbourne (Australia))

    1991-01-01

    Benzodiazepine binding sites, which were once considered to exist only in higher vertebrates, are here demonstrated in the bacteria E. coli. The bacterial ({sup 3}H)diazepam binding sites are modulated by GABA; the modulation is dose dependent and is reduced at high concentrations. The most potent competitors of E.Coli ({sup 3}H)diazepam binding are those that are active in displacing ({sup 3}H)benzodiazepines from vertebrate peripheral benzodiazepine binding sites. These vertebrate sites are not modulated by GABA, in contrast to vertebrate neuronal benzodiazepine binding sites. The E.coli benzodiazepine binding sites therefore differ from both classes of vertebrate benzodiazepine binding sites; however the ligand spectrum and GABA-modulatory properties of the E.coli sites are similar to those found in insects. This intermediate type of receptor in lower species suggests a precursor for at least one class of vertebrate benzodiazepine binding sites may have existed.

  8. The effect of drug binding on specific sites in transmembrane helices 4 and 6 of the ABC exporter MsbA studied by DNP-enhanced solid-state NMR.

    Science.gov (United States)

    Spadaccini, Roberta; Kaur, Hundeep; Becker-Baldus, Johanna; Glaubitz, Clemens

    2018-04-01

    MsbA, a homodimeric ABC exporter, translocates its native substrate lipid A as well as a range of smaller, amphiphilic substrates across the membrane. Magic angle sample spinning (MAS) NMR, in combination with dynamic nuclear polarization (DNP) for signal enhancement, has been used to probe two specific sites in transmembrane helices 4 and 6 of full length MsbA embedded in lipid bilayers. Significant chemical shift changes in both sites were observed in the vanadate-trapped state compared to apo state MsbA. The reduced spectral line width indicates a more confined conformational space upon trapping. In the presence of substrates Hoechst 33342 and daunorubicin, further chemical shift changes and line shape alterations mainly in TM6 in the vanadate trapped state were detected. These data illustrate the conformational response of MsbA towards the presence of drugs during the catalytic cycle. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Taxon- and Site-Specific Melatonin Catabolism

    Directory of Open Access Journals (Sweden)

    Rüdiger Hardeland

    2017-11-01

    Full Text Available Melatonin is catabolized both enzymatically and nonenzymatically. Nonenzymatic processes mediated by free radicals, singlet oxygen, other reactive intermediates such as HOCl and peroxynitrite, or pseudoenzymatic mechanisms are not species- or tissue-specific, but vary considerably in their extent. Higher rates of nonenzymatic melatonin metabolism can be expected upon UV exposure, e.g., in plants and in the human skin. Additionally, melatonin is more strongly nonenzymatically degraded at sites of inflammation. Typical products are several hydroxylated derivatives of melatonin and N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK. Most of these products are also formed by enzymatic catalysis. Considerable taxon- and site-specific differences are observed in the main enzymatic routes of catabolism. Formation of 6-hydroxymelatonin by cytochrome P450 subforms are prevailing in vertebrates, predominantly in the liver, but also in the brain. In pineal gland and non-mammalian retina, deacetylation to 5-methoxytryptamine (5-MT plays a certain role. This pathway is quantitatively prevalent in dinoflagellates, in which 5-MT induces cyst formation and is further converted to 5-methoxyindole-3-acetic acid, an end product released to the water. In plants, the major route is catalyzed by melatonin 2-hydroxylase, whose product is tautomerized to 3-acetamidoethyl-3-hydroxy-5-methoxyindolin-2-one (AMIO, which exceeds the levels of melatonin. Formation and properties of various secondary products are discussed.

  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...

  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...

  12. Incorporating evolution of transcription factor binding sites into ...

    Indian Academy of Sciences (India)

    PRAKASH KUMAR

    under the profile Pi, the model respects the initial base composition through position-specific substitution rates. The position-specific transition probabilities at a position i are then given by: where µ is the rate of mutations per site and δ is the Kronecker delta function with δu,v=1 if (u = v) and 0 otherwise. Inserting. PSA u v.

  13. Predicting sequence and structural specificities of RNA binding regions recognized by splicing factor SRSF1

    Directory of Open Access Journals (Sweden)

    Wang Xin

    2011-12-01

    Full Text Available Abstract Background RNA-binding proteins (RBPs play diverse roles in eukaryotic RNA processing. Despite their pervasive functions in coding and noncoding RNA biogenesis and regulation, elucidating the sequence specificities that define protein-RNA interactions remains a major challenge. Recently, CLIP-seq (Cross-linking immunoprecipitation followed by high-throughput sequencing has been successfully implemented to study the transcriptome-wide binding patterns of SRSF1, PTBP1, NOVA and fox2 proteins. These studies either adopted traditional methods like Multiple EM for Motif Elicitation (MEME to discover the sequence consensus of RBP's binding sites or used Z-score statistics to search for the overrepresented nucleotides of a certain size. We argue that most of these methods are not well-suited for RNA motif identification, as they are unable to incorporate the RNA structural context of protein-RNA interactions, which may affect to binding specificity. Here, we describe a novel model-based approach--RNAMotifModeler to identify the consensus of protein-RNA binding regions by integrating sequence features and RNA secondary structures. Results As an example, we implemented RNAMotifModeler on SRSF1 (SF2/ASF CLIP-seq data. The sequence-structural consensus we identified is a purine-rich octamer 'AGAAGAAG' in a highly single-stranded RNA context. The unpaired probabilities, the probabilities of not forming pairs, are significantly higher than negative controls and the flanking sequence surrounding the binding site, indicating that SRSF1 proteins tend to bind on single-stranded RNA. Further statistical evaluations revealed that the second and fifth bases of SRSF1octamer motif have much stronger sequence specificities, but weaker single-strandedness, while the third, fourth, sixth and seventh bases are far more likely to be single-stranded, but have more degenerate sequence specificities. Therefore, we hypothesize that nucleotide specificity and

  14. Acetolactate synthase inhibiting herbicides bind to the regulatory site.

    Science.gov (United States)

    Subramanian, M V; Loney-Gallant, V; Dias, J M; Mireles, L C

    1991-05-01

    Acetolactate synthase from spontaneous mutants of tobacco (Nicotiana tabacum; KS-43 and SK-53) and cotton (Gossypium hirsutum; PS-3, PSH-91, and DO-2) selected in tissue culture for resistance to a triazolopyrimidine sulfonanilide showed varying degrees of insensitivity to feedback inhibitor(s) valine and/or leucine. A similar feature was evident in the enzyme isolated from chlorsulfuron-resistant weed biotypes, Kochia scoparia and Stellaria media. Dual inhibition analyses of triazolopyrimidine sulfonanilide, thifensulfuron, and imazethapyr versus feedback inhibitor leucine revealed that the three herbicides were competitive with the amino acid for binding to acetolactate synthase from wild-type cotton cultures. Acetolactate synthase inhibiting herbicides may bind to the regulatory site on the enzyme.

  15. Isothermal titration calorimetry and surface plasmon resonance allow quantifying substrate binding to different binding sites of Bacillus subtilis xylanase

    DEFF Research Database (Denmark)

    Cuyvers, Sven; Dornez, Emmie; Abou Hachem, Maher

    2012-01-01

    Isothermal titration calorimetry and surface plasmon resonance were tested for their ability to study substrate binding to the active site (AS) and to the secondary binding site (SBS) of Bacillus subtilis xylanase A separately. To this end, three enzyme variants were compared. The first was a cat......Isothermal titration calorimetry and surface plasmon resonance were tested for their ability to study substrate binding to the active site (AS) and to the secondary binding site (SBS) of Bacillus subtilis xylanase A separately. To this end, three enzyme variants were compared. The first...

  16. Pharmacological properties of cardiovascular histamine H1 receptor binding sites: characterisation with 2-phenylhistamines.

    Science.gov (United States)

    Carman-Krzan, M; Krzan, M; Schunack, W

    1997-04-01

    We determined and compared the molecular properties of histamine H1 receptor binding sites in bovine thoracic aorta smooth muscle and guinea pig myocardial membranes from ventricles with saturation and inhibition binding assay, using 3H-mepyramine to label the receptor and specific and selective H1 receptor agonists of the 2-phenylhistamine group as displacers of specific 3H-mepyramine binding. 3H-mepyramine binds in a saturable manner to a homogenous population of binding sites with a K(D) of 5.6 nM and a Bmax of 57 fmol/mg of protein in bovine aorta vascular smooth muscle membranes, whereas in the guinea pig myocardium high and low affinity 3H-mepyramine binding sites exist having the following molecular characteristics: a K(D) of 1.6 nM and a Bmax of 99 fmol/mg of protein (high affinity site) and a K(D) 15.0 nM and a Bmax of 466 fmol/mg of protein (low affinity site). Halogenated 2-phenylhistamines: 2-(3-fluoro-) (2-FPH), 2-(3-trifluoromethyl-) (2-triFMPH), 2-(3-chloro-) (2-CPH), 2-(3-bromo-) (2-BPH) and 2-(3-iodophenyl)histamine (2-IPH), which showed high selectivity and potency for H1 receptors in the functional pharmacological studies, were potent inhibitors of specific radioligand binding in comparison with histamine and parent nonhalogenated 2-phenylhistamine (2-PH). Their rank order of potencies and affinities differ significantly for the vascular and cardiac H1 receptor binding sites: Specific 3H-mepyramine binding to H1 receptors in bovine vascular smooth muscle membranes was displaced in a biphasic manner by 2-(3-fluoro-), 2-(3-trifluoromethyl-), 2-(3-chloro-), 2-(3-bromo-), 2-(3-iodophenyl)histamine and histamine. In guinea pig ventricular myocardium the rank order was 2-(3-iodo-), 2-(3-bromo-), histamine, 2-(3-chloro-), and 2-(3-fluorophenyl)histamine showing better correlation with the lipophilicity of the derivatives than in vascular tissue (order of lipophilicity: 2-triFMPH >2-IPH >2-BPH >2-CPH >2-FPH >2-PH). Displacement of the radioligand binding

  17. Measurement of specific [3H]-ouabain binding to different types of human leucocytes

    DEFF Research Database (Denmark)

    Boon, Arnold; Oh, V M; Taylor, John E.

    1984-01-01

    We have studied the specific binding of [3H]-ouabain to intact mononuclear leucocytes (82% lymphocytes) and polymorphonuclear leucocytes. In both types of cells [3H]-ouabain binding was saturable, confined to a single site of high affinity, slow to reach equilibrium, slow to reverse, temperature...... were expressed per square micron of cell surface area the difference between the two cell types was proportionately greater (83 and 186 sites per micron 2 respectively). We conclude that the [3H]-ouabain binding sites on mononuclear and polymorphonuclear leucocytes are similar in nature, but different...... in both number and density on the cell surface. Measurements of Bmax in mixed cell populations should therefore take account of cell type as well as cell size and number....

  18. Rodent Models of Non-classical Progesterone Action Regulating Ovulation

    Directory of Open Access Journals (Sweden)

    Melinda A. Mittelman-Smith

    2017-07-01

    Full Text Available It is becoming clear that steroid hormones act not only by binding to nuclear receptors that associate with specific response elements in the nucleus but also by binding to receptors on the cell membrane. In this newly discovered manner, steroid hormones can initiate intracellular signaling cascades which elicit rapid effects such as release of internal calcium stores and activation of kinases. We have learned much about the translocation and signaling of steroid hormone receptors from investigations into estrogen receptor α, which can be trafficked to, and signal from, the cell membrane. It is now clear that progesterone (P4 can also elicit effects that cannot be exclusively explained by transcriptional changes. Similar to E2 and its receptors, P4 can initiate signaling at the cell membrane, both through progesterone receptor and via a host of newly discovered membrane receptors (e.g., membrane progesterone receptors, progesterone receptor membrane components. This review discusses the parallels between neurotransmitter-like E2 action and the more recently investigated non-classical P4 signaling, in the context of reproductive behaviors in the rodent.

  19. Computational prediction of cAMP receptor protein (CRP binding sites in cyanobacterial genomes

    Directory of Open Access Journals (Sweden)

    Su Zhengchang

    2009-01-01

    Full Text Available Abstract Background Cyclic AMP receptor protein (CRP, also known as catabolite gene activator protein (CAP, is an important transcriptional regulator widely distributed in many bacteria. The biological processes under the regulation of CRP are highly diverse among different groups of bacterial species. Elucidation of CRP regulons in cyanobacteria will further our understanding of the physiology and ecology of this important group of microorganisms. Previously, CRP has been experimentally studied in only two cyanobacterial strains: Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120; therefore, a systematic genome-scale study of the potential CRP target genes and binding sites in cyanobacterial genomes is urgently needed. Results We have predicted and analyzed the CRP binding sites and regulons in 12 sequenced cyanobacterial genomes using a highly effective cis-regulatory binding site scanning algorithm. Our results show that cyanobacterial CRP binding sites are very similar to those in E. coli; however, the regulons are very different from that of E. coli. Furthermore, CRP regulons in different cyanobacterial species/ecotypes are also highly diversified, ranging from photosynthesis, carbon fixation and nitrogen assimilation, to chemotaxis and signal transduction. In addition, our prediction indicates that crp genes in modern cyanobacteria are likely inherited from a common ancestral gene in their last common ancestor, and have adapted various cellular functions in different environments, while some cyanobacteria lost their crp genes as well as CRP binding sites during the course of evolution. Conclusion The CRP regulons in cyanobacteria are highly diversified, probably as a result of divergent evolution to adapt to various ecological niches. Cyanobacterial CRPs may function as lineage-specific regulators participating in various cellular processes, and are important in some lineages. However, they are dispensable in some other lineages. The

  20. Describing the Peptide Binding Specificity of HLA-C

    DEFF Research Database (Denmark)

    Rasmussen, Michael; Harndahl, Mikkel Nors; Nielsen, Morten

    . We find preference for hydrophobic residues at the peptide C-terminus for all HLA-C molecules. Most molecules were found to have an additional strong anchor at P2 or P3, with auxiliary anchor observed at P1, P2, P3, and P7. The binding affinity is measured for peptides fitting the specificity matrix...

  1. Prediction of DNA-binding specificity in zinc finger proteins

    Indian Academy of Sciences (India)

    2012-06-25

    Jun 25, 2012 ... Zinc finger proteins interact via their individual fingers to three base pair subsites on the target DNA. The four key ... [Roy S, Dutta S, Khanna K, Singla S and Sundar D 2012 Prediction of DNA-binding specificity in zinc finger proteins. J. Biosci. .... well as protection from HIV infection (Reynolds, et al. 2003).

  2. Solubilization of phencyclidine receptors from rat cerebral cortex in an active ligand binding site

    Energy Technology Data Exchange (ETDEWEB)

    McVittie, L.D.; Sibley, D.R.

    1989-01-01

    A phencyclidine (PCP) receptor binding site has been solubilized in an active ligand-binding state from rat cerebral cortical membranes with sodium deoxycholate. Optimal receptor solubilization occurs at a detergent/protein ratio of 0.5 (w/w); for 5 mg protein/ml solubilized with 0.25% sodium deoxycholate, about 60% of the protein and 25% of the receptor is solubilized. Specific binding of either (/sup 3/H)-N-(1-(2-thienyl)cyclohexyl)piperidine ((/sup 3/H)TCP) or (/sup 3/H)MK-801 is measurable by filtration through Sephadex G-50 columns or glass fiber filters; more than 60% of the binding activity is stable after 48 h at 4/degrees/C. In the presence of detergent, (/sup 3/H)TCP binding exhibits a K/sub d/ of 250 nM, a B/sub max/ of 0.56 pmol/mg protein, and a pharmacological profile consistent with that of the membrane-bound PCP receptor, although most drugs bind with affinities 2 to 8 fold lower than in membranes. Upon reduction of detergent concentration, binding parameters approximate those for the membrane-bound receptor (/sup 3/H)TCP binding: K/sub d/ = 48 nM, M/sub max/ = 1.13 pmol/mg protein.

  3. Effect of mifepristone on steroid receptor expression and biotransformation of oestrogen and progesterone in rat uterus and deciduoma.

    Science.gov (United States)

    Vij, Urmila; Kumar, Anand; Sharma, Kanhaiya; Kaushal, Mishi; Mehra, Raj

    2006-01-01

    [corrected] Mifepristone is a synthetic antiprogestin which terminates early pregnancy. Since it interferes with the progesterone maintained decidua, we compared the effect of mifepristone on oestrogen and progesterone receptors, and on the biotransformation of these hormones in normal and deciduous uterus. Ovariectomized rats were treated with an oestrogen-progesterone hormone regimen and deciduoma was induced by trauma in one horn of the rat uterus while the other served as a control under an identical hormonal milieu. Hormone receptor and biotransformation studies were done using radiolabelled oestradiol and progesterone with high specific activity. The artificially formed decidual tissue was comparable with that of early pregnancy. Mifepristone replenished oestrogen and progesterone receptors which were suppressed by progesterone in both the normal and decidualized uterine horns. Inhibition of oestrogen receptors by progesterone correlated with decreased oestradiol levels at the site of action. Metabolism of progesterone to less potent compounds was promoted by mifepristone. The enzymatic activities of 17beta-hydroxysteroid dehydrogenase (which metabolizes oestradiol), and 20alpha-hydroxysteroid dehydrogenase and 5alpha-reductase (which metabolize progesterone) were altered by mifepristone. The effect of mifepristone in varying the hormone receptor population and the availability of different levels of active metabolites of ovarian hormones have an Important role in the antiprogestin action of mifepristone.

  4. Comparison of in silico tools for binding site prediction applied for structure-based design of autolysin inhibitors.

    Science.gov (United States)

    Tibaut, T; Borišek, J; Novič, M; Turk, D

    2016-07-01

    Autolysin E (AtlE) is a bacteriolytic enzyme which plays an important role in division and growth of bacterial cells and therefore represents a promising potential drug target. Its 3D structure has been recently elucidated. We used in silico prediction tools to study substrate or ligand (inhibitor) binding regions of AtlE. We applied several freely available tools and a commercial tool for binding site identification and compared results of the prediction. Calculation time, number of predictions and output data provided by specific software vary according to the different approaches utilized by specific method categories. Despite different approaches, binding sites in similar locations on the protein were predicted. Specific amino acid residues that form these binding sites were predicted as binding residues. The predicted residues, especially those with predicted highest conservation score, could theoretically have catalytic and binding properties. According to our results, we assume that E138, which has the highest conservation score, is the catalytic residue and F161, G162 and Y224, which are also highly conserved, are responsible for substrate binding. Ligands developed with binding specificity towards these residues could inhibit the catalysis and binding of the substrate of AtlE. The molecules with inhibitory potency could therefore represent potential new antibacterial agents.

  5. Protein-Protein Interactions: Structurally Conserved Residues Distinguish between Binding Sites and Exposed Protein Surfaces

    National Research Council Canada - National Science Library

    Buyong Ma; Tal Elkayam; Haim Wolfson; Ruth Nussinov

    2003-01-01

    Polar residue hot spots have been observed at protein-protein binding sites. Here we show that hot spots occur predominantly at the interfaces of macromolecular complexes, distinguishing binding sites from the remainder of the surface...

  6. Leveraging cross-species transcription factor binding site patterns

    DEFF Research Database (Denmark)

    Claussnitzer, Melina; Dankel, Simon N; Klocke, Bernward

    2014-01-01

    to disease susceptibility. We show that integrative computational analysis of phylogenetic conservation with a complexity assessment of co-occurring transcription factor binding sites (TFBS) can identify cis-regulatory variants and elucidate their mechanistic role in disease. Analysis of established type 2...... diabetes risk loci revealed a striking clustering of distinct homeobox TFBS. We identified the PRRX1 homeobox factor as a repressor of PPARG2 expression in adipose cells and demonstrate its adverse effect on lipid metabolism and systemic insulin sensitivity, dependent on the rs4684847 risk allele...

  7. Resistance to Linezolid Caused by Modifications at Its Binding Site on the Ribosome

    DEFF Research Database (Denmark)

    Long, Katherine S.; Vester, Birte

    2012-01-01

    Linezolid is an oxazolidinone antibiotic in clinical use for the treatment of serious infections of resistant Gram-positive bacteria. It inhibits protein synthesis by binding to the peptidyl transferase center on the ribosome. Almost all known resistance mechanisms involve small alterations...... to the linezolid binding site, so this review will therefore focus on the various changes that can adversely affect drug binding and confer resistance. High-resolution structures of linezolid bound to the 50S ribosomal subunit show that it binds in a deep cleft that is surrounded by 23S rRNA nucleotides. Mutation...... of 23S rRNA has for some time been established as a linezolid resistance mechanism. Although ribosomal proteins L3 and L4 are located further away from the bound drug, mutations in specific regions of these proteins are increasingly being associated with linezolid resistance. However, very little...

  8. Characterization of a novel /sup 3/H-5-hydroxytryptamine binding site subtype in bovine brain membranes

    Energy Technology Data Exchange (ETDEWEB)

    Heuring, R.E.; Peroutka, S.J.

    1987-03-01

    /sup 3/H-5-Hydroxytryptamine (5-HT) binding sites were analyzed in bovine brain membranes. The addition of either the 5-HT1A-selective drug 8-OH-DPAT (100 nM) or the 5-HT1C-selective drug mesulergine (100 nM) to the assay resulted in a 5-10% decrease in specific /sup 3/H-5-HT binding. Scatchard analysis revealed that the simultaneous addition of both drugs decreased the Bmax of /sup 3/H-5-HT binding by 10-15% without affecting the KD value (1.8 +/- 0.3 nM). Competition studies using a series of pharmacologic agents revealed that the sites labeled by /sup 3/H-5-HT in bovine caudate in the presence of 100 nM 8-OH-DPAT and 100 nM mesulergine appear to be homogeneous. 5-HT1A selective agents such as 8-OH-DPAT, ipsapirone, and buspirone display micromolar affinities for these sites. RU 24969 and (-)pindolol are approximately 2 orders of magnitude less potent at these sites than at 5-HT1B sites which have been identified in rat brain. Agents displaying nanomolar potencies for 5-HT1C sites such as mianserin and mesulergine are 2-3 orders of magnitude less potent at the /sup 3/H-5-HT binding sites in bovine caudate. In addition, both 5-HT2- and 5-HT3-selective agents are essentially inactive at these binding sites. These /sup 3/H-5-HT sites display nanomolar affinity for 5-carboxyamidotryptamine, 5-methoxytryptamine, metergoline, and 5-HT. Apparent Ki values of 10-100 nM are obtained for d-LSD, RU 24969, methiothepin, tryptamine, methysergide, and yohimbine, whereas I-LSD and corynanthine are significantly less potent. In addition, these /sup 3/H-5-HT labeled sites are regulated by guanine nucleotides and calcium. Regional studies indicate that this class of sites is most dense in the basal ganglia but exists in all regions of bovine brain. These data therefore demonstrate the presence of a homogeneous class of 5-HT1 binding sites in bovine caudate that is pharmacologically distinct from previously defined 5-HT1A, 5-HT1B, 5-HT1C, 5-HT2, and 5-HT3 receptor subtypes

  9. The Multiple Carbohydrate Binding Specificities of Helicobacter pylori

    Science.gov (United States)

    Teneberg, Susann

    Persistent colonization of the human stomach by Helicobacter pylori is a risk factor for the development of peptic ulcer disease and gastric cancer. Adhesion of microbes to the target tissue is an important determinant for successful initiation, establishment and maintenance of infection, and a variety of different candidate carbohydrate receptors for H. pylori have been identified. Here the different the binding specifities, and their potential role in adhesion to human gastric epithelium are described. Finally, recent findings on the roles of sialic acid binding SabA adhesin in interactions with human neutrophils and erythrocytes are discussed.

  10. Characterization of DNA binding sites of the ComE response regulator from Streptococcus mutans.

    Science.gov (United States)

    Hung, David C I; Downey, Jennifer S; Ayala, Eduardo A; Kreth, Jens; Mair, Richard; Senadheera, Dilani B; Qi, Fengxia; Cvitkovitch, Dennis G; Shi, Wenyuan; Goodman, Steven D

    2011-07-01

    In Streptococcus mutans, both competence and bacteriocin production are controlled by ComC and the ComED two-component signal transduction system. Recent studies of S. mutans suggested that purified ComE binds to two 11-bp direct repeats in the nlmC-comC promoter region, where ComE activates nlmC and represses comC. In this work, quantitative binding studies and DNase I footprinting analysis were performed to calculate the equilibrium dissociation constant and further characterize the binding site of ComE. We found that ComE protects sequences inclusive of both direct repeats, has an equilibrium dissociation constant in the nanomolar range, and binds to these two direct repeats cooperatively. Furthermore, similar direct repeats were found upstream of cslAB, comED, comX, ftf, vicRKX, gtfD, gtfB, gtfC, and gbpB. Quantitative binding studies were performed on each of these sequences and showed that only cslAB has a similar specificity and high affinity for ComE as that seen with the upstream region of comC. A mutational analysis of the binding sequences showed that ComE does not require both repeats to bind DNA with high affinity, suggesting that single site sequences in the genome may be targets for ComE-mediated regulation. Based on the mutational analysis and DNase I footprinting analysis, we propose a consensus ComE binding site, TCBTAAAYSGT.

  11. De-novo discovery of differentially abundant transcription factor binding sites including their positional preference.

    Science.gov (United States)

    Keilwagen, Jens; Grau, Jan; Paponov, Ivan A; Posch, Stefan; Strickert, Marc; Grosse, Ivo

    2011-02-10

    Transcription factors are a main component of gene regulation as they activate or repress gene expression by binding to specific binding sites in promoters. The de-novo discovery of transcription factor binding sites in target regions obtained by wet-lab experiments is a challenging problem in computational biology, which has not been fully solved yet. Here, we present a de-novo motif discovery tool called Dispom for finding differentially abundant transcription factor binding sites that models existing positional preferences of binding sites and adjusts the length of the motif in the learning process. Evaluating Dispom, we find that its prediction performance is superior to existing tools for de-novo motif discovery for 18 benchmark data sets with planted binding sites, and for a metazoan compendium based on experimental data from micro-array, ChIP-chip, ChIP-DSL, and DamID as well as Gene Ontology data. Finally, we apply Dispom to find binding sites differentially abundant in promoters of auxin-responsive genes extracted from Arabidopsis thaliana microarray data, and we find a motif that can be interpreted as a refined auxin responsive element predominately positioned in the 250-bp region upstream of the transcription start site. Using an independent data set of auxin-responsive genes, we find in genome-wide predictions that the refined motif is more specific for auxin-responsive genes than the canonical auxin-responsive element. In general, Dispom can be used to find differentially abundant motifs in sequences of any origin. However, the positional distribution learned by Dispom is especially beneficial if all sequences are aligned to some anchor point like the transcription start site in case of promoter sequences. We demonstrate that the combination of searching for differentially abundant motifs and inferring a position distribution from the data is beneficial for de-novo motif discovery. Hence, we make the tool freely available as a component of the open

  12. Quantification of specific bindings of biomolecules by magnetorelaxometry

    Directory of Open Access Journals (Sweden)

    Steinhoff Uwe

    2008-03-01

    Full Text Available Abstract The binding reaction of the biomolecules streptavidin and anti-biotin antibody, both labelled by magnetic nanoparticles (MNP, to biotin coated on agarose beads, was quantified by magnetorelaxometry (MRX. Highly sensitive SQUID-based MRX revealed the immobilization of the MNP caused by the biotin-streptavidin coupling. We found that about 85% of streptavidin-functionalised MNP bound specifically to biotin-agarose beads. On the other hand only 20% of antibiotin-antibody functionalised MNP were specifically bound. Variation of the suspension medium revealed in comparison to phosphate buffer with 0.1% bovine serum albumin a slight change of the binding behaviour in human serum, probably due to the presence of functioning (non heated serum proteins. Furthermore, in human serum an additional non-specific binding occurs, being independent from the serum protein functionality. The presented homogeneous bead based assay is applicable in simple, uncoated vials and it enables the assessment of the binding kinetics in a volume without liquid flow. The estimated association rate constant for the MNP-labelled streptavidin is by about two orders of magnitude smaller than the value reported for free streptavidin. This is probably due to the relatively large size of the magnetic markers which reduces the diffusion of streptavidin. Furthermore, long time non-exponential kinetics were observed and interpreted as agglutination of the agarose beads.

  13. Discovery and information-theoretic characterization of transcription factor binding sites that act cooperatively

    CERN Document Server

    Clifford, Jacob

    2015-01-01

    Transcription factor binding to the surface of DNA regulatory regions is one of the primary causes of regulating gene expression levels. A probabilistic approach to model protein-DNA interactions at the sequence level is through Position Weight Matrices (PWMs) that estimate the joint probability of a DNA binding site sequence by assuming positional independence within the DNA sequence. Here we construct conditional PWMs that depend on the motif signatures in the flanking DNA sequence, by conditioning known binding site loci on the presence or absence of additional binding sites in the flanking sequence of each site's locus. Pooling known sites with similar flanking sequence patterns allows for the estimation of the conditional distribution function over the binding site sequences. We apply our model to the Dorsal transcription factor binding sites active in patterning the Dorsal-Ventral axis of Drosophila development. We find that those binding sites that cooperate with nearby Twist sites on average contain a...

  14. 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.

  15. A Large-Scale Assessment of Nucleic Acids Binding Site Prediction Programs.

    Directory of Open Access Journals (Sweden)

    Zhichao Miao

    2015-12-01

    Full Text Available Computational prediction of nucleic acid binding sites in proteins are necessary to disentangle functional mechanisms in most biological processes and to explore the binding mechanisms. Several strategies have been proposed, but the state-of-the-art approaches display a great diversity in i the definition of nucleic acid binding sites; ii the training and test datasets; iii the algorithmic methods for the prediction strategies; iv the performance measures and v the distribution and availability of the prediction programs. Here we report a large-scale assessment of 19 web servers and 3 stand-alone programs on 41 datasets including more than 5000 proteins derived from 3D structures of protein-nucleic acid complexes. Well-defined binary assessment criteria (specificity, sensitivity, precision, accuracy… are applied. We found that i the tools have been greatly improved over the years; ii some of the approaches suffer from theoretical defects and there is still room for sorting out the essential mechanisms of binding; iii RNA binding and DNA binding appear to follow similar driving forces and iv dataset bias may exist in some methods.

  16. How to use not-always-reliable binding site information in protein-protein docking prediction.

    Science.gov (United States)

    Li, Lin; Huang, Yanzhao; Xiao, Yi

    2013-01-01

    In many protein-protein docking algorithms, binding site information is used to help predicting the protein complex structures. Using correct and accurate binding site information can increase protein-protein docking success rate significantly. On the other hand, using wrong binding sites information should lead to a failed prediction, or, at least decrease the success rate. Recently, various successful theoretical methods have been proposed to predict the binding sites of proteins. However, the predicted binding site information is not always reliable, sometimes wrong binding site information could be given. Hence there is a high risk to use the predicted binding site information in current docking algorithms. In this paper, a softly restricting method (SRM) is developed to solve this problem. By utilizing predicted binding site information in a proper way, the SRM algorithm is sensitive to the correct binding site information but insensitive to wrong information, which decreases the risk of using predicted binding site information. This SRM is tested on benchmark 3.0 using purely predicted binding site information. The result shows that when the predicted information is correct, SRM increases the success rate significantly; however, even if the predicted information is completely wrong, SRM only decreases success rate slightly, which indicates that the SRM is suitable for utilizing predicted binding site information.

  17. 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...

  18. Molecular environment of the phencyclidine binding site in the nicotinic acetylcholine receptor membrane

    Energy Technology Data Exchange (ETDEWEB)

    Palma, A.L.; Wang, H.H. (Department of Biology, University of California, Santa Cruz (United States))

    1991-06-01

    Phencyclidine is a highly specific noncompetitive inhibitor of the nicotinic acetylcholine receptor. In a novel approach to study this site, a spin-labeled analogue of phencyclidine, 4-phenyl-4-(1-piperidinyl)-2,2,6,6-tetramethylpiperidinoxyl (PPT) was synthesized. The binding of PPT inhibits 86Rb flux (IC50 = 6.6 microM), and (3H)phencyclidine binding to both resting and desensitized acetylcholine receptor (IC50 = 17 microM and 0.22 microM, respectively). From an indirect Hill plot of the inhibition of (3H)phencyclidine binding by PPT, a Hill coefficient of approximately one was obtained in the presence of carbamylcholine and 0.8 in alpha-bungarotoxin-treated preparations. Taken together, these results indicate that PPT mimics phencyclidine in its ability to bind to the noncompetitive inhibitor site and is functionally active in blocking ion flux across the acetylcholine receptor channel. Analysis of the electron spin resonance signal of the bound PPT suggests that the environment surrounding the probe within the ion channel is hydrophobic, with a hydrophobicity parameter of 1.09. A dielectric constant for the binding site was estimated to be in the range of 2-3 units.

  19. New fluorescent reagents specific for Ca{sup 2+}-binding proteins

    Energy Technology Data Exchange (ETDEWEB)

    Ben-Hail, Danya; Lemelson, Daniela [Department of Life Sciences and the NIBN, Ben-Gurion University, Beer-Sheva 84105 (Israel); Israelson, Adrian [Department of Physiology, Ben-Gurion University, Beer-Sheva 84105 (Israel); Shoshan-Barmatz, Varda, E-mail: vardasb@bgu.ac.il [Department of Life Sciences and the NIBN, Ben-Gurion University, Beer-Sheva 84105 (Israel)

    2012-09-14

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

  20. Membrane-bound progesterone receptors coupled to G proteins in the fungus Rhizopus nigricans.

    Science.gov (United States)

    Lenasi, Helena; Bavec, Aljosa; Zorko, Matjaz

    2002-07-16

    Steroid binding sites with high affinity for progesterone (Kd=40+/-14 nM determined by binding, and Kd=71+/-22 nM determined by displacement studies) and lower affinity for 21-hydroxyprogesterone and for testosterone, but no affinity for estradiol-17beta, onapristone and alpha-naphthoflavone were detected in the enriched plasma membrane fraction of the fungus Rhizopus nigricans. The amount of steroid binding sites is in accordance with the value of B(max)=744+/-151 fmol (mg protein)(-1). In the membrane fraction, progesterone induced about 30% activation of G proteins over basal level, as determined by GTPase activity (EC50=32+/-8 nM) and by the guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding rate (EC50=61+/-21 nM). The affinity of receptors for progesterone was substantially decreased in the presence of GTPgammaS and of cholera toxin. Our results suggest the existence of progesterone receptors in the membrane of Rhizopus nigricans and their coupling to G proteins.

  1. 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.

  2. Imaging of jasmonic acid binding sites in tissue.

    Science.gov (United States)

    Gao, Yan hong; Yu, Ying; Hu, Xiao gang; Cao, Yu juan; Wu, Jian zhong

    2013-09-15

    Hormones regulate the mechanism of plant growth and development, senescence, and plants' adaptation to the environment; studies of the molecular mechanisms of plant hormone action are necessary for the understanding of these complex phenomena. However, there is no measurable signal for the hormone signal transduction process. We synthesized and applied a quantum dot-based fluorescent probe for the labeling of jasmonic acid (JA) binding sites in plants. This labeling probe was obtained by coupling mercaptoethylamine-modified CdTe quantum dots with JA using N-hydroxysuccinimide (NHS) as a coupling agent. The probe, CdTe-JA, was characterized by transmission electron microscopy, dynamic light scattering, and fluorescent spectrum and applied in labeling JA binding sites in tissue sections of mung bean seedlings and Arabidopsis thaliana root tips. Laser scanning confocal microscopy (LSCM) revealed that the probe selectively labeled JA receptor. The competition assays demonstrated that the CdTe-JA probe retained the original bioactivity of JA. An LSCM three-dimensional reconstruction experiment demonstrated excellent photostability of the probe. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. NAD+ Modulates p53 DNA Binding Specificity and Function

    Science.gov (United States)

    McLure, Kevin G.; Takagi, Masatoshi; Kastan, Michael B.

    2004-01-01

    DNA damage induces p53 DNA binding activity, which affects tumorigenesis, tumor responses to therapies, and the toxicities of cancer therapies (B. Vogelstein, D. Lane, and A. J. Levine, Nature 408:307-310, 2000; K. H. Vousden and X. Lu, Nat. Rev. Cancer 2:594-604, 2002). Both transcriptional and transcription-independent activities of p53 contribute to DNA damage-induced cell cycle arrest, apoptosis, and aneuploidy prevention (M. B. Kastan et al., Cell 71:587-597, 1992; K. H. Vousden and X. Lu, Nat. Rev. Cancer 2:594-604, 2002). Small-molecule manipulation of p53 DNA binding activity has been an elusive goal, but here we show that NAD+ binds to p53 tetramers, induces a conformational change, and modulates p53 DNA binding specificity in vitro. Niacinamide (vitamin B3) increases the rate of intracellular NAD+ synthesis, alters radiation-induced p53 DNA binding specificity, and modulates activation of a subset of p53 transcriptional targets. These effects are likely due to a direct effect of NAD+ on p53, as a molecule structurally related to part of NAD+, TDP, also inhibits p53 DNA binding, and the TDP precursor, thiamine (vitamin B1), inhibits intracellular p53 activity. Niacinamide and thiamine affect two p53-regulated cellular responses to ionizing radiation: rereplication and apoptosis. Thus, niacinamide and thiamine form a novel basis for the development of small molecules that affect p53 function in vivo, and these results suggest that changes in cellular energy metabolism may regulate p53. PMID:15509798

  4. Binding of folates to Dictyostelium discoideum cells. Demonstration of five classes of binding sites and their interconversion

    NARCIS (Netherlands)

    Wit, René J.W. de; Haastert, Peter J.M. van

    1985-01-01

    Studies of the binding of four folate derivatives to the cell surface of Dictyostelium discoideum indicate the existence of five types of sites. About 99% of the total number of binding sites (160000 per cell) belongs to the 'non-selective' type, which recognizes folate, 2-deaminofolate and

  5. RNA Bind-n-Seq: quantitative assessment of the sequence and structural binding specificity of RNA binding proteins

    Science.gov (United States)

    Lambert, Nicole; Robertson, Alex; Jangi, Mohini; McGeary, Sean; Sharp, Phillip A.; Burge, Christopher B.

    2014-01-01

    Summary Specific protein-RNA interactions guide post-transcriptional gene regulation. Here we describe RNA Bind-n-Seq (RBNS), a method that comprehensively characterizes sequence and structural specificity of RNA binding proteins (RBPs), and its application to the developmental alternative splicing factors RBFOX2, CELF1/CUGBP1 and MBNL1. For each factor, we recovered both canonical motifs and additional near-optimal binding motifs. RNA secondary structure inhibits binding of RBFOX2 and CELF1, while MBNL1 favors unpaired Us but tolerates C/G pairing in motifs containing UGC and/or GCU. Dissociation constants calculated from RBNS data using a novel algorithm correlated highly with values measured by surface plasmon resonance. Motifs identified by RBNS were conserved, were bound and active in vivo, and distinguished the subset of motifs enriched by CLIP-Seq that had regulatory activity. Together, our data demonstrate that RBNS complements crosslinking-based methods and show that in vivo binding and activity of these splicing factors is driven largely by intrinsic RNA affinity. PMID:24837674

  6. Alcohol-Binding Sites in Distinct Brain Proteins: The Quest for Atomic Level Resolution

    Science.gov (United States)

    Howard, Rebecca J.; Slesinger, Paul A.; Davies, Daryl L.; Das, Joydip; Trudell, James R.; Harris, R. Adron

    2011-01-01

    Defining the sites of action of ethanol on brain proteins is a major prerequisite to understanding the molecular pharmacology of this drug. The main barrier to reaching an atomic-level understanding of alcohol action is the low potency of alcohols, ethanol in particular, which is a reflection of transient, low-affinity interactions with their targets. These mechanisms are difficult or impossible to study with traditional techniques such as radioligand binding or spectroscopy. However, there has been considerable recent progress in combining X-ray crystallography, structural modeling, and site-directed mutagenesis to define the sites and mechanisms of action of ethanol and related alcohols on key brain proteins. We review such insights for several diverse classes of proteins including inwardly rectifying potassium, transient receptor potential, and neurotransmit-ter-gated ion channels, as well as protein kinase C epsilon. Some common themes are beginning to emerge from these proteins, including hydrogen bonding of the hydroxyl group and van der Waals interactions of the methylene groups of ethanol with specific amino acid residues. The resulting binding energy is proposed to facilitate or stabilize low-energy state transitions in the bound proteins, allowing ethanol to act as a “molecular lubricant” for protein function. We discuss evidence for characteristic, discrete alcohol-binding sites on protein targets, as well as evidence that binding to some proteins is better characterized by an interaction region that can accommodate multiple molecules of ethanol. PMID:21676006

  7. Immobilized sialyloligo-macroligand and its protein binding specificity.

    Science.gov (United States)

    Narla, Satya Nandana; Sun, Xue-Long

    2012-05-14

    We report a chemoenzymatic synthesis of chain-end functionalized sialyllactose-containing glycopolymers with different linkages and their oriented immobilization for glycoarray and SPR-based glyco-biosensor applications. Specifically, O-cyanate chain-end functionalized sialyllactose-containing glycopolymers were synthesized by enzymatic α2,3- and α2,6-sialylation of a lactose-containing glycopolymer that was synthesized by cyanoxyl-mediated free radical polymerization. (1)H NMR showed almost quantitative α2,3- and α2,6-sialylation. The O-cyanate chain-end functionalized sialyllactose-containing glycopolymers were printed onto amine-functionalized glass slides via isourea bond formation for glycoarray formation. Specific protein binding activity of the arrays was confirmed with α2,3- and α2,6-sialyl specific binding lectins together with inhibition assays. Further, immobilizing O-cyanate chain-end functionalized sialyllactose-containing glycopolymers onto amine-modified SPR chip via isourea bond formation afforded SPR-based glyco-biosensor, which showed specific binding activity for lectins and influenza viral hemagglutinins (HA). These sialyloligo-macroligand derived glycoarray and SPR-based glyco-biosensor are closely to mimic 3D nature presentation of sialyloligosaccharides and will provide important high-throughput tools for virus diagnosis and potential antiviral drug candidates screening applications.

  8. Comprehensive human transcription factor binding site map for combinatory binding motifs discovery.

    Directory of Open Access Journals (Sweden)

    Arnoldo J Müller-Molina

    Full Text Available To know the map between transcription factors (TFs and their binding sites is essential to reverse engineer the regulation process. Only about 10%-20% of the transcription factor binding motifs (TFBMs have been reported. This lack of data hinders understanding gene regulation. To address this drawback, we propose a computational method that exploits never used TF properties to discover the missing TFBMs and their sites in all human gene promoters. The method starts by predicting a dictionary of regulatory "DNA words." From this dictionary, it distills 4098 novel predictions. To disclose the crosstalk between motifs, an additional algorithm extracts TF combinatorial binding patterns creating a collection of TF regulatory syntactic rules. Using these rules, we narrowed down a list of 504 novel motifs that appear frequently in syntax patterns. We tested the predictions against 509 known motifs confirming that our system can reliably predict ab initio motifs with an accuracy of 81%-far higher than previous approaches. We found that on average, 90% of the discovered combinatorial binding patterns target at least 10 genes, suggesting that to control in an independent manner smaller gene sets, supplementary regulatory mechanisms are required. Additionally, we discovered that the new TFBMs and their combinatorial patterns convey biological meaning, targeting TFs and genes related to developmental functions. Thus, among all the possible available targets in the genome, the TFs tend to regulate other TFs and genes involved in developmental functions. We provide a comprehensive resource for regulation analysis that includes a dictionary of "DNA words," newly predicted motifs and their corresponding combinatorial patterns. Combinatorial patterns are a useful filter to discover TFBMs that play a major role in orchestrating other factors and thus, are likely to lock/unlock cellular functional clusters.

  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. Inositol 1,4,5-trisphosphate binds to a specific receptor and releases microsomal calcium in the arterior pituitary gland

    Energy Technology Data Exchange (ETDEWEB)

    Guillemette, G.; Balla, T.; Baukal, A.J.; Catt, K.J.

    1987-12-01

    The properties of inositol 1,4,5-trisphosphate (InsP/sub 3/) receptor sites in the anterior pituitary were evaluated by binding studies with InsP/sub 3/ labeled with /sup 32/P to high specific radioactivity. Specific binding of Ins(/sup 32/P)P/sub 3/ was demonstrable in pituitary membrane preparations and was linearly proportional to the amount of membrane added over the range 0.5-2 mg of protein. Kinetic studies showed that specific InsP/sub 3/ binding was half-maximal in about 40 sec and reached a plateau after 15 min at 0/sup 0/C. Scatchard analysis of the binding data was consistent with a single set of high affinity sites. The specificity of Ins(/sup 32/P)P/sub 3/ binding to these sites was illustrated by the much weaker affinity for structural analogs such as inositol 1-phosphate, phytic acid, 2,3-bisphosphoglycerate, and fructose 1,6-bisphosphate. To assess the functional relevance of the InsP/sub 3/ binding sites, the Ca/sup 2 +/-releasing activity of InsP/sub 3/ was measured in pituitary membrane preparations. Under physiological conditions within the cytosol, the high-affinity InsP/sub 3/ binding sites characterized in pituitary membranes could serve as the putative receptors through which InsP/sub 3/ triggers Ca/sup 2 +/ mobilization in the anterior pituitary gland.

  11. Identification of novel allosteric modulator binding sites in NMDA receptors: A molecular modeling study.

    Science.gov (United States)

    Kane, Lucas T; Costa, Blaise M

    2015-09-01

    The dysfunction of N-methyl-d-Aspartate receptors (NMDARs), a subtype of glutamate receptors, is correlated with schizophrenia, stroke, and many other neuropathological disorders. However, not all NMDAR subtypes equally contribute towards these disorders. Since NMDARs composed of different GluN2 subunits (GluN2A-D) confer varied physiological properties and have different distributions in the brain, pharmacological agents that target NMDARs with specific GluN2 subunits have significant potential for therapeutic applications. In our previous research, we have identified a family of novel allosteric modulators that differentially potentiate and/or inhibit NMDARs of differing GluN2 subunit composition. To further elucidate their molecular mechanisms, in the present study, we have identified four potential binding sites for novel allosteric modulators by performing molecular modeling, docking, and in silico mutations. The molecular determinants of the modulator binding sites (MBS), analysis of particular MBS electrostatics, and the specific loss or gain of binding after mutations have revealed modulators that have strong potential affinities for specific MBS on given subunits and the role of key amino acids in either promoting or obstructing modulator binding. These findings will help design higher affinity GluN2 subunit-selective pharmaceuticals, which are currently unavailable to treat psychiatric and neurological disorders. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Wide-scale analysis of human functional transcription factor binding reveals a strong bias towards the transcription start site.

    Directory of Open Access Journals (Sweden)

    Yuval Tabach

    Full Text Available BACKGROUND: Transcription factors (TF regulate expression by binding to specific DNA sequences. A binding event is functional when it affects gene expression. Functionality of a binding site is reflected in conservation of the binding sequence during evolution and in over represented binding in gene groups with coherent biological functions. Functionality is governed by several parameters such as the TF-DNA binding strength, distance of the binding site from the transcription start site (TSS, DNA packing, and more. Understanding how these parameters control functionality of different TFs in different biological contexts is a must for identifying functional TF binding sites and for understanding regulation of transcription. METHODOLOGY/PRINCIPAL FINDINGS: We introduce a novel method to screen the promoters of a set of genes with shared biological function (obtained from the functional Gene Ontology (GO classification against a precompiled library of motifs, and find those motifs which are statistically over-represented in the gene set. More than 8,000 human (and 23,000 mouse genes, were assigned to one of 134 GO sets. Their promoters were searched (from 200 bp downstream to 1,000 bp upstream the TSS for 414 known DNA motifs. We optimized the sequence similarity score threshold, independently for every location window, taking into account nucleotide heterogeneity along the promoters of the target genes. The method, combined with binding sequence and location conservation between human and mouse, identifies with high probability functional binding sites for groups of functionally-related genes. We found many location-sensitive functional binding events and showed that they clustered close to the TSS. Our method and findings were tested experimentally. CONCLUSIONS/SIGNIFICANCE: We identified reliably functional TF binding sites. This is an essential step towards constructing regulatory networks. The promoter region proximal to the TSS is of central

  13. NMR mapping of the IFNAR1-EC binding site on IFNalpha2 reveals allosteric changes in the IFNAR2-EC binding site.

    Science.gov (United States)

    Akabayov, Sabine Ruth; Biron, Zohar; Lamken, Peter; Piehler, Jacob; Anglister, Jacob

    2010-02-02

    All type I interferons (IFNs) bind to a common cell-surface receptor consisting of two subunits. IFNs initiate intracellular signal transduction cascades by simultaneous interaction with the extracellular domains of its receptor subunits, IFNAR1 and IFNAR2. In this study, we mapped the surface of IFNalpha2 interacting with the extracellular domain of IFNAR1 (IFNAR1-EC) by following changes in or the disappearance of the (1)H-(15)N TROSY-HSQC cross peaks of IFNalpha2 caused by the binding of the extracellular domain of IFNAR1 (IFNAR1-EC) to the binary complex of IFNalpha2 with IFNAR2-EC. The NMR study of the 89 kDa complex was conducted at pH 8 and 308 K using an 800 MHz spectrometer. IFNAR1 binding affected a total of 47 of 165 IFNalpha2 residues contained in two large patches on the face of the protein opposing the binding site for IFNAR2 and in a third patch located on the face containing the IFNAR2 binding site. The first two patches form the IFNAR1 binding site, and one of these matches the IFNAR1 binding site previously identified by site-directed mutagenesis. The third patch partially matches the IFNalpha2 binding site for IFNAR2-EC, indicating allosteric communication between the binding sites for the two receptor subunits.

  14. Plasmodium vivax MSP-1 peptides have high specific binding activity to human reticulocytes.

    Science.gov (United States)

    Rodríguez, Luis Eduardo; Urquiza, Mauricio; Ocampo, Marisol; Curtidor, Hernando; Suárez, Jorge; García, Javier; Vera, Ricardo; Puentes, Alvaro; López, Ramses; Pinto, Martha; Rivera, Zuly; Patarroyo, Manuel Elkin

    2002-01-31

    Plasmodium vivax merozoites have high preferential ability to interact with and invade reticulocytes, although these cells correspond to only 2% of the red blood cells (RBC) population. P. vivax merozoite surface protein-1 (Pv-MSP-1) is believed to have an important role in attachment and invasion process. Using 88 non-overlapping 20-mer peptides, covering the entire Pv-MSP-1 Belem strain sequence, RBC and reticulocyte binding assays were performed. Fourteen sequences were identified with high specific binding activity to reticulocytes, but only three had high specific binding activity to mature erythrocytes. These peptides showed affinity constant values between 20 and 150nM, indicating a strong interaction between these sequences and reticulocyte receptors. Critical residues in binding to reticulocytes for these peptides were determined by competition binding assays with glycine scanning analogues. All high binding peptides bind to reticulocyte surface proteins having a molecular mass of around 18-20kDa which are not present in mature RBC. Interestingly, some high activity binding peptides (HABPs) are located close to the hypothesised 42 and 19kDa fragment cleavage sites for this protein, suggesting that these sequences have an important role in target cell attachment and invasion process by Pv-MSP-1.HABPs may be clustered in two regions, with region I being located between amino acids 280-719, and region II between amino acids 1060-1599 with higher than 25% identity level. A P. falciparum MSP-1 antigenic domain binds to RBCs and inhibits parasite invasion. Peptides 1721 and 1724 bind with high activity to reticulocytes in homologous Pv-MSP-1, suggesting similar functions for these two sequences.

  15. Europium ion as a probe for binding sites to carrageenans

    Energy Technology Data Exchange (ETDEWEB)

    Ramos, Ana P.; Goncalves, Rogeria R.; Serra, Osvaldo A. [Departamento de Quimica, Faculdade de Filosofia, Ciencias e Letras de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, Sao Paulo 14040-901 (Brazil); Zaniquelli, Maria Elisabete D. [Departamento de Quimica, Faculdade de Filosofia, Ciencias e Letras de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, Sao Paulo 14040-901 (Brazil)], E-mail: medzaniquelli@ffclrp.usp.br; Wong, Kenneth [Laboratorio de Fisico-Quimica, Centro de Pesquisas de Paulinia, Rhodia Brasil, Paulinia, Sao Paulo (Brazil)

    2007-12-15

    Carrageenans, sulfated polysaccharides extracted from red algae, present a coil-helix transition and helix aggregation dependence on the type and concentration of counterions. In this study, we focus attention on a mixed valence counterion system: Eu{sup 3+}/Na{sup +} or K{sup +} with different gel-forming carrageenans: kappa, iota, and kappa-2. Results of stationary and time-dependent luminescence showed to be a suitable tool to probe ion binding to both the negatively charged sulfate group and the hydroxyl groups present in the biopolymer. For lower europium ion concentrations, a single longer decay emission lifetime was detected, which was attributed to the binding of europium ion to the carrageenan sulfate groups. An additional decay ascribed to europium binding to hydroxyl groups was observed above a threshold concentration, and this decay was dependent on the carrageenan charge density. Symmetry of the europium ion microenvironment was estimated by the ratio between the intensities of its emission bands, which has been shown to depend on the concentration of europium ions and on the specificity of the monovalent counterion bound to the carrageenan.

  16. Factors influencing the specificity of inhibitor binding to the human and malaria parasite dihydroorotate dehydrogenases.

    Science.gov (United States)

    Bedingfield, Paul T P; Cowen, Deborah; Acklam, Paul; Cunningham, Fraser; Parsons, Mark R; McConkey, Glenn A; Fishwick, Colin W G; Johnson, A Peter

    2012-06-28

    The de novo pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase is an emerging drug target for the treatment of malaria. In this context a key property of Plasmodium falciparum DHODH (PfDHODH) is that it can be selectively inhibited over its human homologue (HsDHODH). However, HsDHODH is also a validated drug target for autoimmune diseases such as arthritis. Here a series of novel inhibitors is described that includes compounds that switch specificity between the two enzymes as a result of small alterations in chemical structure. Structure-activity relationship (SAR), crystallography, docking, and mutagenesis studies are used to examine the binding modes of the compounds within the two enzymes and to reveal structural changes induced by inhibitor binding. Within this series, compounds with therapeutically relevant HsDHODH activity are described and their binding modes characterized using X-ray crystallography, which reveals a novel conformational shift within the inhibitor binding site.

  17. Dinucleotide weight matrices for predicting transcription factor binding sites: generalizing the position weight matrix.

    Directory of Open Access Journals (Sweden)

    Rahul Siddharthan

    Full Text Available BACKGROUND: Identifying transcription factor binding sites (TFBS in silico is key in understanding gene regulation. TFBS are string patterns that exhibit some variability, commonly modelled as "position weight matrices" (PWMs. Though convenient, the PWM has significant limitations, in particular the assumed independence of positions within the binding motif; and predictions based on PWMs are usually not very specific to known functional sites. Analysis here on binding sites in yeast suggests that correlation of dinucleotides is not limited to near-neighbours, but can extend over considerable gaps. METHODOLOGY/PRINCIPAL FINDINGS: I describe a straightforward generalization of the PWM model, that considers frequencies of dinucleotides instead of individual nucleotides. Unlike previous efforts, this method considers all dinucleotides within an extended binding region, and does not make an attempt to determine a priori the significance of particular dinucleotide correlations. I describe how to use a "dinucleotide weight matrix" (DWM to predict binding sites, dealing in particular with the complication that its entries are not independent probabilities. Benchmarks show, for many factors, a dramatic improvement over PWMs in precision of predicting known targets. In most cases, significant further improvement arises by extending the commonly defined "core motifs" by about 10 bp on either side. Though this flanking sequence shows no strong motif at the nucleotide level, the predictive power of the dinucleotide model suggests that the "signature" in DNA sequence of protein-binding affinity extends beyond the core protein-DNA contact region. CONCLUSION/SIGNIFICANCE: While computationally more demanding and slower than PWM-based approaches, this dinucleotide method is straightforward, both conceptually and in implementation, and can serve as a basis for future improvements.

  18. Antibody-antigen interactions: contact analysis and binding site topography.

    Science.gov (United States)

    MacCallum, R M; Martin, A C; Thornton, J M

    1996-10-11

    We have analysed antigen-contacting residues and combining site shape in the antibody Fv and Fab crystal structures now available from the Protein Data Bank. Antigen-contacting propensities are presented for each antibody residue, allowing a new definition for the complementarity determining regions (CDRs) to be proposed based on observed antigen contacts. Contacts are more common at CDR residues which are located centrally within the combining site; some less central CDR residues are only contacted by large antigens. Non-contacting residues within the CDRs coincide with residues identified by Chothia and co-workers as important in defining "canonical" conformations. An objective means of classifying protein surfaces by gross topography has been developed and applied to the antibody combining site surfaces. The surfaces have been clustered into four topographic classes: concave and moderately concave (mostly hapten binders), ridged (mostly peptide binders) and planar (mostly protein binders). We have determined the topographic classes for ten pairs of complexed and uncomplexed antibody-antigen crystal structures; four change topographic class on complexation. The results will be of use in antibody engineering, antigen docking and in clinical immunology. To demonstrate one application, we show how the data can be used to locate the antigen binding pocket on antibody structures.

  19. RNA-binding specificity landscape of the pentatricopeptide repeat protein PPR10.

    Science.gov (United States)

    Miranda, Rafael G; Rojas, Margarita; Montgomery, Michael P; Gribbin, Kyle P; Barkan, Alice

    2017-04-01

    Pentatricopeptide repeat (PPR) proteins comprise a large family of helical repeat proteins that influence gene expression in mitochondria and chloroplasts. PPR tracts can bind RNA via a modular one repeat-one nucleotide mechanism in which the nucleotide is specified by the identities of several amino acids in each repeat. This mode of recognition, the so-called PPR code, offers opportunities for the prediction of native PPR binding sites and the design of proteins to bind specified RNAs. However, a deep understanding of the parameters that dictate the affinity and specificity of PPR-RNA interactions is necessary to realize these goals. We report a comprehensive analysis of the sequence specificity of PPR10, a protein that binds similar RNA sequences of ∼18 nucleotides (nt) near the chloroplast atpH and psaJ genes in maize. We assessed the contribution of each nucleotide in the atpH binding site to PPR10 affinity in vitro by analyzing the effects of single-nucleotide changes at each position. In a complementary approach, the RNAs bound by PPR10 from partially randomized RNA pools were analyzed by deep sequencing. The results revealed three patches in which nucleotide identity has a major impact on binding affinity. These include 5 nt for which protein contacts were not observed in a PPR10-RNA crystal structure and 4 nt that are not explained by current views of the PPR code. These findings highlight aspects of PPR-RNA interactions that pose challenges for binding site prediction and design. © 2017 Miranda et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  20. Statistical and Economic Techniques for Site-specific Nematode Management.

    Science.gov (United States)

    Liu, Zheng; Griffin, Terry; Kirkpatrick, Terrence L

    2014-03-01

    Recent advances in precision agriculture technologies and spatial statistics allow realistic, site-specific estimation of nematode damage to field crops and provide a platform for the site-specific delivery of nematicides within individual fields. This paper reviews the spatial statistical techniques that model correlations among neighboring observations and develop a spatial economic analysis to determine the potential of site-specific nematicide application. The spatial econometric methodology applied in the context of site-specific crop yield response contributes to closing the gap between data analysis and realistic site-specific nematicide recommendations and helps to provide a practical method of site-specifically controlling nematodes.

  1. Binding of human urokinase-type plasminogen activator to its receptor: residues involved in species specificity and binding.

    Science.gov (United States)

    Quax, P H; Grimbergen, J M; Lansink, M; Bakker, A H; Blatter, M C; Belin, D; van Hinsbergh, V W; Verheijen, J H

    1998-05-01

    Urokinase-type plasminogen activator (UPA), particularly when bound to its receptor (UPAR), is thought to play a major role in local proteolytic processes, thus facilitating cell migration as may occur during angiogenesis, neointima and atherosclerotic plaque formation, and tumor cell invasion. To facilitate understanding of the need and function of the UPA/UPAR interaction in cell migration and vascular remodeling, we changed several amino acid residues in UPA so as to interfere with its interaction with its receptor. The receptor-binding domain of UPA has been localized to a region in the growth factor domain between residues 20 and 32. Since the binding of UPA to UPAR appears to be species specific, we used the differences in amino acid sequences in the growth factor domain of UPA between various species to construct a human UPA variant that does not bind to the human UPAR. We substituted Asn22 for its mouse equivalent Tyr by site-directed mutagenesis. This mutant UPA had similar plasminogen activator characteristics as wild-type UPA, including its specific activity and interaction with plasminogen activator inhibitor-1. However, no UPA/UPAR complexes could be observed in cross-linking experiments using DFP-treated 125I-labeled mutant UPA and lysates of various cells, including U937 histiocytic lymphoma cells, phorbol myristate acetate-treated human ECs, and mouse LB6 cells transfected with human UPAR cDNA. In direct binding experiments, DFP-treated 125I-labeled mutant UPA could not bind to phorbol myristate acetate-treated ECs, whereas wild-type UPA did bind. Furthermore, a 25-fold excess of wild-type UPA completely prevented the binding of DFP-treated 125I-labeled wild-type UPA to the human receptor on transfected LB6 cells, whereas an equal amount of mutant UPA had only a very small effect. In ligand blotting assays, very weak binding of mutant UPA to human UPAR could be observed. Changing Asn22 into the other amino acid residues alanine or glutamine had no

  2. Methods and systems for identifying ligand-protein binding sites

    KAUST Repository

    Gao, Xin

    2016-05-06

    The invention provides a novel integrated structure and system-based approach for drug target prediction that enables the large-scale discovery of new targets for existing drugs Novel computer-readable storage media and computer systems are also provided. Methods and systems of the invention use novel sequence order-independent structure alignment, hierarchical clustering, and probabilistic sequence similarity techniques to construct a probabilistic pocket ensemble (PPE) that captures even promiscuous structural features of different binding sites for a drug on known targets. The drug\\'s PPE is combined with an approximation of the drug delivery profile to facilitate large-scale prediction of novel drug- protein interactions with several applications to biological research and drug development.

  3. Mapping protein binding sites on the biomolecular corona of nanoparticles

    Science.gov (United States)

    Kelly, Philip M.; Åberg, Christoffer; Polo, Ester; O'Connell, Ann; Cookman, Jennifer; Fallon, Jonathan; Krpetić, Željka; Dawson, Kenneth A.

    2015-05-01

    Nanoparticles in a biological milieu are known to form a sufficiently long-lived and well-organized ‘corona’ of biomolecules to confer a biological identity to the particle. Because this nanoparticle-biomolecule complex interacts with cells and biological barriers, potentially engaging with different biological pathways, it is important to clarify the presentation of functional biomolecular motifs at its interface. Here, we demonstrate that by using antibody-labelled gold nanoparticles, differential centrifugal sedimentation and various imaging techniques it is possible to identify the spatial location of proteins, their functional motifs and their binding sites. We show that for transferrin-coated polystyrene nanoparticles only a minority of adsorbed proteins exhibit functional motifs and the spatial organization appears random, which is consistent, overall, with a stochastic and irreversible adsorption process. Our methods are applicable to a wide array of nanoparticles and can offer a microscopic molecular description of the biological identity of nanoparticles.

  4. CSI-Tree: a regression tree approach for modeling binding properties of DNA-binding molecules based on cognate site identification (CSI) data.

    Science.gov (United States)

    Keleş, Sündüz; Warren, Christopher L; Carlson, Clayton D; Ansari, Aseem Z

    2008-06-01

    The identification and characterization of binding sites of DNA-binding molecules, including transcription factors (TFs), is a critical problem at the interface of chemistry, biology and molecular medicine. The Cognate Site Identification (CSI) array is a high-throughput microarray platform for measuring comprehensive recognition profiles of DNA-binding molecules. This technique produces datasets that are useful not only for identifying binding sites of previously uncharacterized TFs but also for elucidating dependencies, both local and nonlocal, between the nucleotides at different positions of the recognition sites. We have developed a regression tree technique, CSI-Tree, for exploring the spectrum of binding sites of DNA-binding molecules. Our approach constructs regression trees utilizing the CSI data of unaligned sequences. The resulting model partitions the binding spectrum into homogeneous regions of position specific nucleotide effects. Each homogeneous partition is then summarized by a position weight matrix (PWM). Hence, the final outcome is a binding intensity rank-ordered collection of PWMs each of which spans a different region in the binding spectrum. Nodes of the regression tree depict the critical position/nucleotide combinations. We analyze the CSI data of the eukaryotic TF Nkx-2.5 and two engineered small molecule DNA ligands and obtain unique insights into their binding properties. The CSI tree for Nkx-2.5 reveals an interaction between two positions of the binding profile and elucidates how different nucleotide combinations at these two positions lead to different binding affinities. The CSI trees for the engineered DNA ligands exhibit a common preference for the dinucleotide AA in the first two positions, which is consistent with preference for a narrow and relatively flat minor groove. We carry out a reanalysis of these data with a mixture of PWMs approach. This approach is an advancement over the simple PWM model and accommodates position

  5. Interaction of D-LSD with binding sites in brain: a study in vivo and in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Ebersole, B.L.J.

    1985-01-01

    The localization of (/sup 3/H)-d-lysergic acid diethylamide ((/sup 3/H)LSD) binding sites in the mouse brain was compared in vivo and in vitro. Radioautography of brain sections incubated with (/sup 3/H)LSD in vitro revealed substantial specific (/sup 3/H)LSD binding in cortical layers III-IV and areas CA1 and dentate gyrus in hippocampus. In contrast, in brain sections from animals that received (/sup 3/H)LSD in vivo, binding in hippocampus was scant and diffuse, although the pattern of labeling in cortex was similar to that seen in vitro. The low specific binding in hippocampus relative to cortex was confirmed by homogenate filtration studies of brain areas from mice that received injections of (/sup 3/H)LSD. Time-course studies established that peak specific binding at ten minutes was the same in cortex and hippocampus. At all times, binding in hippocampus was about one-third of that in cortex; in contrast, the concentration of free (/sup 3/H)LSD did not vary between regions. This finding was unexpected, because binding studies in vitro in membrane preparations indicated that the density and affinity of (/sup 3/H)LSD binding sites were similar in both brain regions. Saturation binding studies in vivo showed that the lower amount of (/sup 3/H)LSD binding in hippocampus was attributable to a lower density of sites labeled by (/sup 3/H)LSD. The pharmacological identify of (/sub 3/H)LSD binding sites in vivo may be relevant to the hallucinogenic properties of LSD and of other related hallucinogens.

  6. Site-Specific Seismic Site Response Model for the Waste Treatment Plant, Hanford, Washington

    Energy Technology Data Exchange (ETDEWEB)

    Rohay, Alan C.; Reidel, Steve P.

    2005-02-24

    This interim report documents the collection of site-specific geologic and geophysical data characterizing the Waste Treatment Plant site and the modeling of the site-specific structure response to earthquake ground motions.

  7. Determining DNA Sequence Specificity of Natural and Artificial Transcription Factors by Cognate Site Identifier Analysis

    Science.gov (United States)

    Ozers, Mary S.; Warren, Christopher L.; Ansari, Aseem Z.

    Artificial transcription factors (ATFs) are designed to mimic natural transcription factors in the control of gene expression and are comprised of domains for DNA binding and gene regulation. ATF domains are modular, interchangeable, and can be composed of protein-based or nonpeptidic moieties, yielding DNA-interacting regulatory molecules that can either activate or inhibit transcription. Sequence-specific targeting is a key determinant in ATF activity, and DNA-binding domains such as natural zinc fingers and synthetic polyamides have emerged as useful DNA targeting molecules. Defining the comprehensive DNA binding specificity of these targeting molecules for accurate manipulations of the genome can be achieved using cognate site identifier DNA microarrays to explore the entire sequence space of binding sites. Design of ATFs that regulate gene expression with temporal control will generate important molecular tools to probe cell- and tissue-specific gene regulation and to function as potential therapeutic agents.

  8. 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

    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......, (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...... 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...

  9. [Progesterone and prevention of preterm birth].

    Science.gov (United States)

    Fuchs, F; Senat, M-V

    2015-10-01

    The literature confirms the interest of progesterone for prevention of preterm delivery in specific indications for patients carrying a singleton pregnancy. In contrast, randomized trials have shown no benefit using progesterone in the prevention of prematurity in twins and even an adverse effect. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  10. PROGESTERONE: BIOSYNTHESIS FROM PREGNENOLONE IN HOLARRHENA FLORIBUNDA.

    Science.gov (United States)

    BENNETT, R D; HEFTMANN, E

    1965-08-06

    After administration of pregnenolone-4-C(14) to Holarrhena floribunda leaves, radioactive progesterone was isolated and purified to constant specific activity by chromatography, conversion to a derivative, and recrystallization. The result suggests that the biogenetic sequence leading to progesterone is the same in plants as in animals.

  11. Propofol Inhibits SIRT2 Deacetylase through a Conformation-specific, Allosteric Site*

    Science.gov (United States)

    Weiser, Brian P.; Eckenhoff, Roderic G.

    2015-01-01

    meta-Azi-propofol (AziPm) is a photoactive analog of the general anesthetic propofol. We photolabeled a myelin-enriched fraction from rat brain with [3H]AziPm and identified the sirtuin deacetylase SIRT2 as a target of the anesthetic. AziPm photolabeled three SIRT2 residues (Tyr139, Phe190, and Met206) that are located in a single allosteric protein site, and propofol inhibited [3H]AziPm photolabeling of this site in myelin SIRT2. Structural modeling and in vitro experiments with recombinant human SIRT2 determined that propofol and [3H]AziPm only bind specifically and competitively to the enzyme when co-equilibrated with other substrates, which suggests that the anesthetic site is either created or stabilized in enzymatic conformations that are induced by substrate binding. In contrast to SIRT2, specific binding of [3H]AziPm or propofol to recombinant human SIRT1 was not observed. Residues that line the propofol binding site on SIRT2 contact the sirtuin co-substrate NAD+ during enzymatic catalysis, and assays that measured SIRT2 deacetylation of acetylated α-tubulin revealed that propofol inhibits enzymatic function. We conclude that propofol inhibits the mammalian deacetylase SIRT2 through a conformation-specific, allosteric protein site that is unique from the previously described binding sites of other inhibitors. This suggests that propofol might influence cellular events that are regulated by protein acetylation state. PMID:25666612

  12. X-ray structures of progesterone receptor ligand binding domain in its agonist state reveal differing mechanisms for mixed profiles of 11beta-substituted steroids.

    NARCIS (Netherlands)

    Lusher, S.J.; Raaijmakers, H.C.A.; Vu-Pham, D.; Kazemier, B.; Bosch, R.; McGuire, R.; Azevedo, R.; Hamersma, H.; Dechering, K.; Oubrie, A.; Duin, M. van; Vlieg, J. de

    2012-01-01

    We present here the x-ray structures of the progesterone receptor (PR) in complex with two mixed profile PR modulators whose functional activity results from two differing molecular mechanisms. The structure of Asoprisnil bound to the agonist state of PR demonstrates the contribution of the ligand

  13. Molecular characterization of the microsomal tamoxifen binding site.

    Science.gov (United States)

    Kedjouar, Blandine; de Médina, Philippe; Oulad-Abdelghani, Mustapha; Payré, Bruno; Silvente-Poirot, Sandrine; Favre, Gilles; Faye, Jean-Charles; Poirot, Marc

    2004-08-06

    Tamoxifen is a selective estrogen receptor modulator widely used for the prophylactic treatment of breast cancer. In addition to the estrogen receptor (ER), tamoxifen binds with high affinity to the microsomal antiestrogen binding site (AEBS), which is involved in ER-independent effects of tamoxifen. In the present study, we investigate the modulation of the biosynthesis of cholesterol in tumor cell lines by AEBS ligands. As a consequence of the treatment with the antitumoral drugs tamoxifen or PBPE, a selective AEBS ligand, we show that tumor cells produced a significant concentration- and time-dependent accumulation of cholesterol precursors. Sterols have been purified by HPLC and gas chromatography, and their chemical structures determined by mass spectrometric analysis. The major metabolites identified were 5alpha-cholest-8-en-3beta-ol for tamoxifen treatment and 5alpha-cholest-8-en-3beta-ol and cholesta-5,7-dien-3beta-ol, for PBPE treatment, suggesting that these AEBS ligands affect at least two enzymatic steps: the 3beta-hydroxysterol-Delta8-Delta7-isomerase and the 3beta-hydroxysterol-Delta7-reductase. Steroidal antiestrogens such as ICI 182,780 and RU 58,668 did not affect these enzymatic steps, because they do not bind to the AEBS. Transient co-expression of human 3beta-hydroxysterol-Delta8-Delta7-isomerase and 3beta-hydroxysterol-Delta7-reductase and immunoprecipitation experiments showed that both enzymes were required to reconstitute the AEBS in mammalian cells. Altogether, these data provide strong evidence that the AEBS is a hetero-oligomeric complex including 3beta-hydroxysterol-Delta8-Delta7-isomerase and the 3beta-hydroxysterol-Delta7-reductase as subunits that are necessary and sufficient for tamoxifen binding in mammary cells. Furthermore, because selective AEBS ligands are antitumoral compounds, these data suggest a link between cholesterol metabolism at a post-lanosterol step and tumor growth control. These data afford both the identification

  14. Demonstration: Fra bodystorm til Site Specific

    DEFF Research Database (Denmark)

    Andersen, Kirsten Vagn

    2016-01-01

    Ved at arbejde ‘site specificc’ åbner vi opmærksomheden over for impulser fra rummet. Artiklen beskriver en workshop og project-eksempler......Ved at arbejde ‘site specificc’ åbner vi opmærksomheden over for impulser fra rummet. Artiklen beskriver en workshop og project-eksempler...

  15. A conserved chloramphenicol binding site at the entrance to the ribosomal peptide exit tunnel

    DEFF Research Database (Denmark)

    Long, Katherine S; Porse, Bo T

    2003-01-01

    , of E.coli 23S rRNA and G2084 (2058 in E.coli numbering) in domain V of H.halobium 23S rRNA. The modification sites overlap with a portion of the macrolide binding site and cluster at the entrance to the peptide exit tunnel. The data correlate with the recently reported chloramphenicol binding site...... on an archaeal ribosome and suggest that a similar binding site is present on the E.coli ribosome....

  16. Developmental changes in the distribution of cecal lectin-binding sites of Balb-c mice.

    Science.gov (United States)

    Doehrn, S; Breipohl, W; Lierse, W; Romaniuk, K; Young, W

    1992-01-01

    The existence of lectin-binding sites was investigated in the cecum of Balb-c mice at seven developmental stages ranging from 18 days post conception (p.c.) to 8 weeks after birth. Nine horseradish-peroxidase-conjugated lectins (concanavalin A, Triticum vulgaris, Dolichus biflorus, Helix pomatia, Arachis hypogaea, Glycine maximus, Lotus tetragonolobus, Ulex europaeus, Limulus polyphemus) were applied to 5- to 7-microns thin paraffin sections of Bouin-fixed tissue. After DAB staining the sections were evaluated by light microscopy. It was shown that each lectin exhibits a unique developmental pattern. The adult binding patterns were established at the age of 3-4 weeks with only minor changes occurring thereafter. Considerable differences in binding patterns occurred not only between lectins of different groups but also between lectins with the same nominal monosaccharide specificity.

  17. 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

  18. The Escherichia coli Tus-Ter replication fork barrier causes site-specific DNA replication perturbation in yeast

    DEFF Research Database (Denmark)

    Larsen, Nicolai B; Sass, Ehud; Suski, Catherine

    2014-01-01

    Replication fork (RF) pausing occurs at both 'programmed' sites and non-physiological barriers (for example, DNA adducts). Programmed RF pausing is required for site-specific DNA replication termination in Escherichia coli, and this process requires the binding of the polar terminator protein, Tus...... as a versatile, site-specific, heterologous DNA replication-perturbing system, with a variety of potential applications....

  19. Discovery and information-theoretic characterization of transcription factor binding sites that act cooperatively

    Science.gov (United States)

    Clifford, Jacob; Adami, Christoph

    2015-10-01

    Transcription factor binding to the surface of DNA regulatory regions is one of the primary causes of regulating gene expression levels. A probabilistic approach to model protein-DNA interactions at the sequence level is through position weight matrices (PWMs) that estimate the joint probability of a DNA binding site sequence by assuming positional independence within the DNA sequence. Here we construct conditional PWMs that depend on the motif signatures in the flanking DNA sequence, by conditioning known binding site loci on the presence or absence of additional binding sites in the flanking sequence of each site's locus. Pooling known sites with similar flanking sequence patterns allows for the estimation of the conditional distribution function over the binding site sequences. We apply our model to the Dorsal transcription factor binding sites active in patterning the Dorsal-Ventral axis of Drosophila development. We find that those binding sites that cooperate with nearby Twist sites on average contain about 0.5 bits of information about the presence of Twist transcription factor binding sites in the flanking sequence. We also find that Dorsal binding site detectors conditioned on flanking sequence information make better predictions about what is a Dorsal site relative to background DNA than detection without information about flanking sequence features.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-07-27

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

  1. Identification of tubulin drug binding sites and prediction of relative differences in binding affinities to tubulin isotypes using digital signal processing.

    Science.gov (United States)

    Chen, Ke; Huzil, J Torin; Freedman, Holly; Ramachandran, Parameswaran; Antoniou, Andreas; Tuszynski, Jack A; Kurgan, Lukasz

    2008-11-01

    Microtubules are involved in numerous cellular processes including chromosome segregation during mitosis and, as a result, their constituent protein, tubulin, has become a successful target of several chemotherapeutic drugs. In general, these drugs bind indiscriminately to tubulin within both cancerous and healthy cells, resulting in unwanted side effects. However, differences between beta-tubulin isotypes expressed in a wide range of cell types may aid in the development of anti-tubulin drugs having increased specificity for only certain types of cells. Here, we describe a digital signal processing (DSP) method that is capable of predicting hot spots for the tubulin family of proteins as well as determining relative differences in binding affinities to these hot spots based only on the primary sequence of 10 human tubulin isotypes. Due to the fact that several drug binding sites have already been characterized within beta-tubulin, we are able to correlate hot spots with the binding sites for known chemotherapy drugs. We have also verified the accuracy of this method using the correlation between the binding affinities of characterized drugs and the tubulin isotypes. Additionally, the DSP method enables the rapid estimation of relative differences in binding affinities within the binding sites of tubulin isotypes that are yet to be experimentally determined.

  2. Deciphering common recognition principles of nucleoside mono/di and tri-phosphates binding in diverse proteins via structural matching of their binding sites.

    Science.gov (United States)

    Bhagavat, Raghu; Srinivasan, Narayanaswamy; Chandra, Nagasuma

    2017-09-01

    Nucleoside triphosphate (NTP) ligands are of high biological importance and are essential for all life forms. A pre-requisite for them to participate in diverse biochemical processes is their recognition by diverse proteins. It is thus of great interest to understand the basis for such recognition in different proteins. Towards this, we have used a structural bioinformatics approach and analyze structures of 4677 NTP complexes available in Protein Data Bank (PDB). Binding sites were extracted and compared exhaustively using PocketMatch, a sensitive in-house site comparison algorithm, which resulted in grouping the entire dataset into 27 site-types. Each of these site-types represent a structural motif comprised of two or more residue conservations, derived using another in-house tool for superposing binding sites, PocketAlign. The 27 site-types could be grouped further into 9 super-types by considering partial similarities in the sites, which indicated that the individual site-types comprise different combinations of one or more site features. A scan across PDB using the 27 structural motifs determined the motifs to be specific to NTP binding sites, and a computational alanine mutagenesis indicated that residues identified to be highly conserved in the motifs are also most contributing to binding. Alternate orientations of the ligand in several site-types were observed and rationalized, indicating the possibility of some residues serving as anchors for NTP recognition. The presence of multiple site-types and the grouping of multiple folds into each site-type is strongly suggestive of convergent evolution. Knowledge of determinants obtained from this study will be useful for detecting function in unknown proteins. Proteins 2017; 85:1699-1712. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  3. Structure-based prediction of RNA-binding domains and RNA-binding sites and application to structural genomics targets.

    Science.gov (United States)

    Zhao, Huiying; Yang, Yuedong; Zhou, Yaoqi

    2011-04-01

    Mechanistic understanding of many key cellular processes often involves identification of RNA binding proteins (RBPs) and RNA binding sites in two separate steps. Here, they are predicted simultaneously by structural alignment to known protein-RNA complex structures followed by binding assessment with a DFIRE-based statistical energy function. This method achieves 98% accuracy and 91% precision for predicting RBPs and 93% accuracy and 78% precision for predicting RNA-binding amino-acid residues for a large benchmark of 212 RNA binding and 6761 non-RNA binding domains (leave-one-out cross-validation). Additional tests revealed that the method makes no false positive prediction from 311 DNA binding domains but correctly detects six domains binding with both DNA and RNA. In addition, it correctly identified 31 of 75 unbound RNA-binding domains with 92% accuracy and 65% precision for predicted binding residues and achieved 86% success rate in its application to SCOP RNA binding domain superfamily (Structural Classification Of Proteins). It further predicts 25 targets as RBPs in 2076 structural genomics targets: 20 of 25 predicted ones (80%) are putatively RNA binding. The superior performance over existing methods indicates the importance of dividing structures into domains, using a Z-score to measure relative structural similarity, and a statistical energy function to measure protein-RNA binding affinity.

  4. Characteristics and retention of luteal structures, extended postinsemination cycle, progesterone, and pregnancy-specific protein B in serum after human chorionic gonadotropin treatment of dairy cows.

    Science.gov (United States)

    Stevenson, J S; Pulley, S L

    2012-08-01

    and 28 after AI were intermediate between nonpregnant cows that returned to estrus by d 25 and all pregnant cows. Concentrations of pregnancy-specific protein B were elevated in some of these nonpregnant, CL-retained cows, indicating early pregnancy loss. Retention of original luteal tissue in nonpregnant cows to d 28 after AI indicated that pregnancy had been initiated but failed, as verified by concentrations of progesterone and pregnancy-specific protein B. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  5. Form, symmetry and packing of biomacromolecules. III. Antigenic, receptor and contact binding sites in picornaviruses.

    Science.gov (United States)

    Janner, A

    2011-03-01

    The relation between serotype differentiation and crystallographic symmetry, revealed by the contact fingerprint diagrams investigated in Part II [Janner (2010). Acta Cryst. A66, 312-326] for the human rhinovirus, is extended to the Picornaviridae family. The approach, outlined in Part I [Janner (2010). Acta Cryst. A66, 301-311] and Part II for biomacromolecules packed in a crystal and based on concepts such as packing lattice, kissing points and crystal-packing parameters, can directly be applied to the picornaviruses. In particular, the contact fingerprint diagrams of 20 different virus strains have been derived. In these cases, as for the rhinovirus, these diagrams are serotype/strain specific, justifying the name fingerprint. The molecular basis for the serotype variability, and the associated conservation requirements, is usually analysed by considering antigenic sites, where capsid residues bind with antibodies, and receptor sites, where other residues bind with molecular receptors of the host cell membrane. Both the antigenic variation and the receptor conservation allow repeated infection of the host cells of the given animals. The graphical description of these sites is usually done by footprints and roadmap diagrams, mapping properties of the capsid surface and using the icosahedral symmetry of the capsid. The alternative fingerprint diagrammatic description, based on the crystal symmetry, adopted in Part II for the contact sites, where a capsid is bound to the next one in the crystal packing, is extended to the antigenic and receptor binding sites. Again, the antigenic/receptor fingerprints are specific, at least for the nine picornaviruses investigated so far, despite the more than a factor of ten larger coarse graining with respect to the corresponding footprint and roadmap diagrams. The latter are based on a grid spacing of about 2 Å, whereas the spacing implied by the packing-lattice approximation adopted in fingerprints varies typically from 20 to

  6. Achieving peptide binding specificity and promiscuity by loops: case of the forkhead-associated domain.

    Science.gov (United States)

    Huang, Yu-Ming M; Chang, Chia-En A

    2014-01-01

    The regulation of a series of cellular events requires specific protein-protein interactions, which are usually mediated by modular domains to precisely select a particular sequence from diverse partners. However, most signaling domains can bind to more than one peptide sequence. How do proteins create promiscuity from precision? Moreover, these complex interactions typically occur at the interface of a well-defined secondary structure, α helix and β sheet. However, the molecular recognition primarily controlled by loop architecture is not fully understood. To gain a deep understanding of binding selectivity and promiscuity by the conformation of loops, we chose the forkhead-associated (FHA) domain as our model system. The domain can bind to diverse peptides via various loops but only interact with sequences containing phosphothreonine (pThr). We applied molecular dynamics (MD) simulations for multiple free and bound FHA domains to study the changes in conformations and dynamics. Generally, FHA domains share a similar folding structure whereby the backbone holds the overall geometry and the variety of sidechain atoms of multiple loops creates a binding surface to target a specific partner. FHA domains determine the specificity of pThr by well-organized binding loops, which are rigid to define a phospho recognition site. The broad range of peptide recognition can be attributed to different arrangements of the loop interaction network. The moderate flexibility of the loop conformation can help access or exclude binding partners. Our work provides insights into molecular recognition in terms of binding specificity and promiscuity and helpful clues for further peptide design.

  7. γ-Secretase Modulator (GSM) Photoaffinity Probes Reveal Distinct Allosteric Binding Sites on Presenilin*

    Science.gov (United States)

    Pozdnyakov, Nikolay; Murrey, Heather E.; Crump, Christina J.; Pettersson, Martin; Ballard, T. Eric; am Ende, Christopher W.; Ahn, Kwangwook; Li, Yue-Ming; Bales, Kelly R.; Johnson, Douglas S.

    2013-01-01

    γ-Secretase is an intramembrane aspartyl protease that cleaves the amyloid precursor protein to produce neurotoxic β-amyloid peptides (i.e. Aβ42) that have been implicated in the pathogenesis of Alzheimer disease. Small molecule γ-secretase modulators (GSMs) have emerged as potential disease-modifying treatments for Alzheimer disease because they reduce the formation of Aβ42 while not blocking the processing of γ-secretase substrates. We developed clickable GSM photoaffinity probes with the goal of identifying the target of various classes of GSMs and to better understand their mechanism of action. Here, we demonstrate that the photoaffinity probe E2012-BPyne specifically labels the N-terminal fragment of presenilin-1 (PS1-NTF) in cell membranes as well as in live cells and primary neuronal cultures. The labeling is competed in the presence of the parent imidazole GSM E2012, but not with acid GSM-1, allosteric GSI BMS-708163, or substrate docking site peptide inhibitor pep11, providing evidence that these compounds have distinct binding sites. Surprisingly, we found that the cross-linking of E2012-BPyne to PS1-NTF is significantly enhanced in the presence of the active site-directed GSI L-685,458 (L458). In contrast, L458 does not affect the labeling of the acid GSM photoprobe GSM-5. We also observed that E2012-BPyne specifically labels PS1-NTF (active γ-secretase) but not full-length PS1 (inactive γ-secretase) in ANP.24 cells. Taken together, our results support the hypothesis that multiple binding sites within the γ-secretase complex exist, each of which may contribute to different modes of modulatory action. Furthermore, the enhancement of PS1-NTF labeling by E2012-BPyne in the presence of L458 suggests a degree of cooperativity between the active site of γ-secretase and the modulatory binding site of certain GSMs. PMID:23396974

  8. γ-Secretase modulator (GSM) photoaffinity probes reveal distinct allosteric binding sites on presenilin.

    Science.gov (United States)

    Pozdnyakov, Nikolay; Murrey, Heather E; Crump, Christina J; Pettersson, Martin; Ballard, T Eric; Am Ende, Christopher W; Ahn, Kwangwook; Li, Yue-Ming; Bales, Kelly R; Johnson, Douglas S

    2013-04-05

    γ-Secretase is an intramembrane aspartyl protease that cleaves the amyloid precursor protein to produce neurotoxic β-amyloid peptides (i.e. Aβ42) that have been implicated in the pathogenesis of Alzheimer disease. Small molecule γ-secretase modulators (GSMs) have emerged as potential disease-modifying treatments for Alzheimer disease because they reduce the formation of Aβ42 while not blocking the processing of γ-secretase substrates. We developed clickable GSM photoaffinity probes with the goal of identifying the target of various classes of GSMs and to better understand their mechanism of action. Here, we demonstrate that the photoaffinity probe E2012-BPyne specifically labels the N-terminal fragment of presenilin-1 (PS1-NTF) in cell membranes as well as in live cells and primary neuronal cultures. The labeling is competed in the presence of the parent imidazole GSM E2012, but not with acid GSM-1, allosteric GSI BMS-708163, or substrate docking site peptide inhibitor pep11, providing evidence that these compounds have distinct binding sites. Surprisingly, we found that the cross-linking of E2012-BPyne to PS1-NTF is significantly enhanced in the presence of the active site-directed GSI L-685,458 (L458). In contrast, L458 does not affect the labeling of the acid GSM photoprobe GSM-5. We also observed that E2012-BPyne specifically labels PS1-NTF (active γ-secretase) but not full-length PS1 (inactive γ-secretase) in ANP.24 cells. Taken together, our results support the hypothesis that multiple binding sites within the γ-secretase complex exist, each of which may contribute to different modes of modulatory action. Furthermore, the enhancement of PS1-NTF labeling by E2012-BPyne in the presence of L458 suggests a degree of cooperativity between the active site of γ-secretase and the modulatory binding site of certain GSMs.

  9. Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7

    KAUST Repository

    Lee, Seung-Joo

    2010-03-28

    DNA primases catalyze the synthesis of the oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Biochemical studies have elucidated the mechanism for the sequence-specific synthesis of primers. However, the physical interactions of the primase with the DNA template to explain the basis of specificity have not been demonstrated. Using a combination of surface plasmon resonance and biochemical assays, we show that T7 DNA primase has only a slightly higher affinity for DNA containing the primase recognition sequence (5\\'-TGGTC-3\\') than for DNA lacking the recognition site. However, this binding is drastically enhanced by the presence of the cognate Nucleoside triphosphates (NTPs), Adenosine triphosphate (ATP) and Cytosine triphosphate (CTP) that are incorporated into the primer, pppACCA. Formation of the dimer, pppAC, the initial step of sequence-specific primer synthesis, is not sufficient for the stable binding. Preformed primers exhibit significantly less selective binding than that observed with ATP and CTP. Alterations in subdomains of the primase result in loss of selective DNA binding. We present a model in which conformational changes induced during primer synthesis facilitate contact between the zinc-binding domain and the polymerase domain. The Author(s) 2010. Published by Oxford University Press.

  10. A Gata2-Dependent Transcription Network Regulates Uterine Progesterone Responsiveness and Endometrial Function

    Directory of Open Access Journals (Sweden)

    Cory A. Rubel

    2016-10-01

    Full Text Available Altered progesterone responsiveness leads to female infertility and cancer, but underlying mechanisms remain unclear. Mice with uterine-specific ablation of GATA binding protein 2 (Gata2 are infertile, showing failures in embryo implantation, endometrial decidualization, and uninhibited estrogen signaling. Gata2 deficiency results in reduced progesterone receptor (PGR expression and attenuated progesterone signaling, as evidenced by genome-wide expression profiling and chromatin immunoprecipitation. GATA2 not only occupies at and promotes expression of the Pgr gene but also regulates downstream progesterone responsive genes in conjunction with the PGR. Additionally, Gata2 knockout uteri exhibit abnormal luminal epithelia with ectopic TRP63 expressing squamous cells and a cancer-related molecular profile in a progesterone-independent manner. Lastly, we found a conserved GATA2-PGR regulatory network in both human and mice based on gene signature and path analyses using gene expression profiles of human endometrial tissues. In conclusion, uterine Gata2 regulates a key regulatory network of gene expression for progesterone signaling at the early pregnancy stage.

  11. ck2-Dependent Phosphorylation of Progesterone Receptors (PR) on Ser81 Regulates PR-B Isoform-Specific Target Gene Expression in Breast Cancer Cells ▿

    Science.gov (United States)

    Hagan, Christy R.; Regan, Tarah M.; Dressing, Gwen E.; Lange, Carol A.

    2011-01-01

    Progesterone receptors (PR) are critical mediators of mammary gland development and contribute to breast cancer progression. Progestin-induced rapid activation of cytoplasmic protein kinases leads to selective regulation of growth-promoting genes by phospho-PR species. Herein, we show that phosphorylation of PR Ser81 is ck2 dependent and progestin regulated in intact cells but also occurs in the absence of PR ligands when cells enter the G1/S phase of the cell cycle. T47D breast cancer cells stably expressing a PR-B mutant receptor that cannot be phosphorylated at Ser79/81 (S79/81A) formed fewer soft agar colonies. Regulation of selected genes by PR-B, but not PR-A, also required Ser79/81 phosphorylation for basal and/or progestin-regulated (BIRC3, HSD11β2, and HbEGF) expression. Additionally, wild-type (wt) PR-B, but not S79/81A mutant PR, was robustly recruited to a progesterone response element (PRE)-containing transcriptional enhancer region of BIRC3; abundant ck2 also associated with this region in cells expressing wt but not S79/81A PR. We conclude that phospho-Ser81 PR provides a platform for ck2 recruitment and regulation of selected PR-B target genes. Understanding how ligand-independent PRs function in the context of high levels of kinase activities characteristic of breast cancer is critical to understanding the basis of tumor-specific changes in gene expression and will speed the development of highly selective treatments. PMID:21518957

  12. The chondroitin sulfate A-binding site of the VAR2CSA protein involves multiple N-terminal domains

    DEFF Research Database (Denmark)

    Dahlbäck, Madeleine; Jørgensen, Lars M; Nielsen, Morten A

    2011-01-01

    shown that full-length recombinant VAR2CSA binds specifically to CSA with high affinity, however to date no sub-fragment of VAR2CSA has been shown to interact with CSA with similar affinity or specificity. In this study, we used a biosensor technology to examine the binding properties of a panel...... of truncated VAR2CSA proteins. The experiments indicate that the core of the CSA-binding site is situated in three domains, DBL2X-CIDR(PAM) and a flanking domain, located in the N-terminal part of VAR2CSA. Furthermore, recombinant VAR2CSA subfragments containing this region elicit antibodies with high parasite...

  13. Mutated primer binding sites interacting with different tRNAs allow efficient murine leukemia virus replication

    DEFF Research Database (Denmark)

    Lund, Anders Henrik; Duch, M; Lovmand, J

    1993-01-01

    Two Akv murine leukemia virus-based retroviral vectors with primer binding sites matching tRNA(Gln-1) and tRNA(Lys-3) were constructed. The transduction efficiency of these mutated vectors was found to be comparable to that of a vector carrying the wild-type primer binding site matching t......RNA(Pro). Polymerase chain reaction amplification and sequence analysis of transduced proviruses confirmed the transfer of vectors with mutated primer binding sites and further showed that tRNA(Gln-2) may act efficiently in conjunction with the tRNA(Gln-1) primer binding site. We conclude that murine leukemia virus...... can replicate by using various tRNA molecules as primers and propose primer binding site-tRNA primer interactions to be of major importance for tRNA primer selection. However, efficient primer selection does not require perfect Watson-Crick base pairing at all 18 positions of the primer binding site....

  14. Eosinophil cationic protein (ECP) can bind heparin and other glycosaminoglycans through its RNase active site.

    Science.gov (United States)

    Torrent, Marc; Nogués, M Victòria; Boix, Ester

    2011-01-01

    The eosinophil cationic protein (ECP) is an eosinophil-secreted RNase involved in the immune host defense, with a cytotoxic activity against a wide range of pathogens. During inflammation and eosinophilia disorders, ECP is secreted to the inflammation area, where it would contribute to the immune response. ECP secretion causes also severe damage to the host own tissues. ECP presents a high affinity for heparin and this property might be crucial for its immunomodulating properties, antipathogen action, and its toxicity against eukaryotic cells. ECP, also known as human RNase 3, belongs to the mammalian RNase A superfamily and its RNase activity is required for some of its biological properties. We have now proven that ECP heparin binding affinity depends on its RNase catalytic site, as the enzymatic activity is blocked by heparin. We have applied molecular modeling to analyze ECP binding to heparin representative probes, and identified protein residues at the catalytic and substrate binding sites that could contribute to the interaction. ECP affinity for heparin and other negatively charged glycosaminoglycans (GAGs) can explain not only its binding to the eukaryote cells glycocalix but also the reported high affinity for the specific carbohydrates at bacteria cell wall, promoting its antimicrobial action. 2010 John Wiley & Sons, Ltd.

  15. Nature and function of insulator protein binding sites in the Drosophila genome.

    Science.gov (United States)

    Schwartz, Yuri B; Linder-Basso, Daniela; Kharchenko, Peter V; Tolstorukov, Michael Y; Kim, Maria; Li, Hua-Bing; Gorchakov, Andrey A; Minoda, Aki; Shanower, Gregory; Alekseyenko, Artyom A; Riddle, Nicole C; Jung, Youngsook L; Gu, Tingting; Plachetka, Annette; Elgin, Sarah C R; Kuroda, Mitzi I; Park, Peter J; Savitsky, Mikhail; Karpen, Gary H; Pirrotta, Vincenzo

    2012-11-01

    Chromatin insulator elements and associated proteins have been proposed to partition eukaryotic genomes into sets of independently regulated domains. Here we test this hypothesis by quantitative genome-wide analysis of insulator protein binding to Drosophila chromatin. We find distinct combinatorial binding of insulator proteins to different classes of sites and uncover a novel type of insulator element that binds CP190 but not any other known insulator proteins. Functional characterization of different classes of binding sites indicates that only a small fraction act as robust insulators in standard enhancer-blocking assays. We show that insulators restrict the spreading of the H3K27me3 mark but only at a small number of Polycomb target regions and only to prevent repressive histone methylation within adjacent genes that are already transcriptionally inactive. RNAi knockdown of insulator proteins in cultured cells does not lead to major alterations in genome expression. Taken together, these observations argue against the concept of a genome partitioned by specialized boundary elements and suggest that insulators are reserved for specific regulation of selected genes.

  16. Nature and function of insulator protein binding sites in the Drosophila genome

    Science.gov (United States)

    Schwartz, Yuri B.; Linder-Basso, Daniela; Kharchenko, Peter V.; Tolstorukov, Michael Y.; Kim, Maria; Li, Hua-Bing; Gorchakov, Andrey A.; Minoda, Aki; Shanower, Gregory; Alekseyenko, Artyom A.; Riddle, Nicole C.; Jung, Youngsook L.; Gu, Tingting; Plachetka, Annette; Elgin, Sarah C.R.; Kuroda, Mitzi I.; Park, Peter J.; Savitsky, Mikhail; Karpen, Gary H.; Pirrotta, Vincenzo

    2012-01-01

    Chromatin insulator elements and associated proteins have been proposed to partition eukaryotic genomes into sets of independently regulated domains. Here we test this hypothesis by quantitative genome-wide analysis of insulator protein binding to Drosophila chromatin. We find distinct combinatorial binding of insulator proteins to different classes of sites and uncover a novel type of insulator element that binds CP190 but not any other known insulator proteins. Functional characterization of different classes of binding sites indicates that only a small fraction act as robust insulators in standard enhancer-blocking assays. We show that insulators restrict the spreading of the H3K27me3 mark but only at a small number of Polycomb target regions and only to prevent repressive histone methylation within adjacent genes that are already transcriptionally inactive. RNAi knockdown of insulator proteins in cultured cells does not lead to major alterations in genome expression. Taken together, these observations argue against the concept of a genome partitioned by specialized boundary elements and suggest that insulators are reserved for specific regulation of selected genes. PMID:22767387

  17. Epidemiology and site specificity of stress fractures.

    Science.gov (United States)

    Bennell, K L; Brukner, P D

    1997-04-01

    Clinically, stress fractures appear to be a common overuse injury among athletes and in military recruits undertaking basic training; however, there is a lack of sound epidemiologic studies describing stress fracture occurrence in athletes. Few have directly compared stress fracture rates between sports to establish which poses the greatest risk for this injury. Furthermore, incidence rates, expressed in terms of exposure, have rarely been reported for stress fractures in athletes. Nevertheless, available data suggest that runners and ballet dancers are at relatively high risk for stress fractures. Although a gender difference in rates is clearly evident in military populations, this is less apparent in athletes. Other participant characteristics, such as age and race, may also influence stress fracture risk. The most common site of stress fracture in athletes is the tibia, although the site reflects the nature of the load applied to the skeleton. Stress fracture morbidity, expressed as the time until return to sport or activity, varies depending on the site. Generally, a period of 6 to 8 weeks is needed for healing; however, stress fractures at certain sites, such as the navicular and anterior tibial cortex, are often associated with protracted recovery and, in some cases, termination of sporting pursuits.

  18. Development of recombinant Aleuria aurantia lectins with altered binding specificities to fucosylated glycans.

    Science.gov (United States)

    Romano, Patrick R; Mackay, Andrew; Vong, Minh; DeSa, Johann; Lamontagne, Anne; Comunale, Mary Ann; Hafner, Julie; Block, Timothy; Lec, Ryszard; Mehta, Anand

    2011-10-14

    Changes in glycosylation have long been associated with disease. While there are many methods to detect changes in glycosylation, plant derived lectins are often used to determine changes on specific proteins or molecules of interest. One change in glycosylation that has been observed by us and by others is a disease or antigen associated increase in fucosylation on N-linked glycans. To measure this change, the fucose binding Aleuria aurantia lectin (AAL) is often utilized in plate and solution based assays. AAL is a mushroom derived lectin that contains five fucose binding sites that preferentially bind fucose linked (α-1,3, α-1,2, α-,4, and α-1,6) to N-acetyllactosamine related structures. Recently, several reports by us and by others have indicated that specific fucose linkages found on certain serum biomarker glycoprotein's are more associated with disease than others. Taking a site-directed mutagenesis approach, we have created a set of recombinant AAL proteins that display altered binding affinities to different analytes containing various fucose linkages. Copyright © 2011 Elsevier Inc. All rights reserved.

  19. Binding of angiogenesis inhibitor kringle 5 to its specific ligands by frontal affinity chromatography.

    Science.gov (United States)

    Bian, Liujiao; Li, Qian; Ji, Xu

    2015-07-03

    The interactions between angiogenesis inhibitor Kringle 5 and its five specific ligands were investigated by frontal affinity chromatography in combination with fluorescence spectra and site-directed molecular docking. The binding constants of trans-4-(aminomethyl) cyclohexane carboxylic acid (AMCHA), epsilon-aminocaproic acid (EACA), benzylamine, 7-aminoheptanoic acid (7-AHA) and L-lysine to Kringle 5 were 19.0×10(3), 7.97×10(3), 6.45×10(3), 6.07×10(3) and 4.04×10(3) L/mol, respectively. The five ligands bound to Kringle 5 on the lysine binding site in equimolar amounts, which was pushed mainly by hydrogen bond and Van der Waals force. This binding affinity was believed to be dependent on the functional group and flexible feature in ligands. This study will provide an important insight into the binding mechanism of angiogenesis inhibitor Kringle 5 to its specific ligands. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Analysis of sequence variation underlying tissue-specific transcription factor binding and gene expression.

    Science.gov (United States)

    Lower, Karen M; De Gobbi, Marco; Hughes, Jim R; Derry, Christopher J; Ayyub, Helena; Sloane-Stanley, Jacqueline A; Vernimmen, Douglas; Garrick, David; Gibbons, Richard J; Higgs, Douglas R

    2013-08-01

    Although mutations causing monogenic disorders most frequently lie within the affected gene, sequence variation in complex disorders is more commonly found in noncoding regions. Furthermore, recent genome- wide studies have shown that common DNA sequence variants in noncoding regions are associated with "normal" variation in gene expression resulting in cell-specific and/or allele-specific differences. The mechanism by which such sequence variation causes changes in gene expression is largely unknown. We have addressed this by studying natural variation in the binding of key transcription factors (TFs) in the well-defined, purified cell system of erythropoiesis. We have shown that common polymorphisms frequently directly perturb the binding sites of key TFs, and detailed analysis shows how this causes considerable (~10-fold) changes in expression from a single allele in a tissue-specific manner. We also show how a SNP, located at some distance from the recognized TF binding site, may affect the recruitment of a large multiprotein complex and alter the associated chromatin modification of the variant regulatory element. This study illustrates the principles by which common sequence variation may cause changes in tissue-specific gene expression, and suggests that such variation may underlie an individual's propensity to develop complex human genetic diseases. © 2013 WILEY PERIODICALS, INC.

  1. Displacement of specific serotonin and lysergic acid diethylamide binding by Ergalgin, a new antiserotonin drug.

    Science.gov (United States)

    Oelszner, W

    1980-01-01

    [3H]-serotonin and [3H]-lysergic acid diethylamide (LSD) bind with a high affinity, KD = 12 nM and 6 nM, respectively, to distinct receptors of rat caudate membranes in vitro. Displacement experiments with unlabeled serotonin and LSD support the hypothesis of serotonin receptors existing in an agonist and antagonist state. Methysergide and Ergalgin display quite similar potencies in displacing [3H]-serotonin and [3H]-LSD from their specific binding sites (Ki = 46,7 and 53,4 nM; 22,3 and 36,5 nM, respectively). Contrary to pharmacological findings these binding results are in favour of mixed agonist/antagonist properties of these compounds.

  2. Human Ku70 protein binds hairpin RNA and double stranded DNA through two different sites.

    Science.gov (United States)

    Anisenko, Andrey N; Knyazhanskaya, Ekaterina S; Zatsepin, Timofey S; Gottikh, Marina B

    2017-01-01

    Human protein Ku usually functions in the cell as a complex of two subunits, Ku70 and Ku80. The Ku heterodimer plays a key role in the non-homologous end joining DNA repair pathway by specifically recognizing the DNA ends at the site of the lesion. The binding of the Ku heterodimer to DNA has been well-studied, and its interactions with RNA have been also described. However, Ku70 subunit is known to have independent DNA binding capability, which is less characterized. RNA binding properties of Ku70 have not been yet specially studied. We have prepared recombinant full-length Ku70 and a set of its truncated mutants in E. coli, and studied their interactions with nucleic acids of various structures: linear single- and double-stranded DNA and RNA, as well as closed circular DNA and hairpin RNA. Ku70 has demonstrated a high affinity binding to double stranded DNA and hairpin RNA with a certain structure only. Interestingly, in contrast to the Ku heterodimer, Ku70 is found to interact with closed circular DNA. We also show for the first time that Ku70 employs two different sites for DNA and RNA binding. The double-stranded DNA is recognized by the C-terminal part of Ku70 including SAP domain as it has been earlier demonstrated, whereas hairpin RNA binding is provided by amino acids 251-438. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  3. The serotonin transporter in rhesus monkey brain: comparison of DASB and citalopram binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Zeng Zhizhen [Imaging Department, Merck Research Laboratories, West Point, PA 19486 (United States)]. E-mail: zhizhen_zeng@merck.com; Chen, T.-B. [Imaging Department, Merck Research Laboratories, West Point, PA 19486 (United States); Miller, Patricia J. [Imaging Department, Merck Research Laboratories, West Point, PA 19486 (United States); Dean, Dennis [Labeled Compound Synthesis Group, Drug Metabolism, Merck Research Laboratories, Rahway, NJ 07065-0900 (United States); Tang, Y.S. [Labeled Compound Synthesis Group, Drug Metabolism, Merck Research Laboratories, Rahway, NJ 07065-0900 (United States); Sur, Cyrille [Imaging Department, Merck Research Laboratories, West Point, PA 19486 (United States); Williams, David L. [Imaging Department, Merck Research Laboratories, West Point, PA 19486 (United States)

    2006-05-15

    We have characterized the interaction of the serotonin transporter ligand [{sup 3}H]-N,N-dimethyl-2-(2-amino-4-cyanophenylthio)-benzylamine (DASB) with rhesus monkey brain in vitro using tissue homogenate binding and autoradiographic mapping. [{sup 3}H]-DASB, a tritiated version of the widely used [{sup 11}C] positron emission tomography tracer, was found to selectively bind to a single population of sites with high affinity (K {sub d}=0.20{+-}0.04 nM). The serotonin transporter density (B {sub max}) obtained for rhesus frontal cortex was found to be 66{+-}8 fmol/mg protein using [{sup 3}H]-DASB, similar to the B {sub max} value obtained using the reference radioligand [{sup 3}H]-citalopram, a well-characterized and highly selective serotonin reuptake inhibitor (83{+-}22 fmol/mg protein). Specific binding sites of both [{sup 3}H]-DASB and [{sup 3}H]-citalopram were similarly and nonuniformly distributed throughout the rhesus central nervous system, in a pattern consistent with serotonin transporter localization reported for human brain. Regional serotonin transporter densities, estimated from optical densities of the autoradiographic images, were well correlated between the two radioligands. Finally, DASB and fluoxetine showed dose-dependent full inhibition of [{sup 3}H]-citalopram binding in a competition autoradiographic study, with K {sub i} values in close agreement with those obtained from rhesus brain homogenates. This side-by-side comparison of [{sup 3}H]-DASB and [{sup 3}H]-citalopram binding sites in rhesus tissue homogenates and in adjacent rhesus brain slices provides additional support for the use of [{sup 11}C]-DASB to assess the availability and distribution of serotonin transporters in nonhuman primates.

  4. Every Site Counts: Submitting Transcription Factor-Binding Site Information through the CollecTF Portal.

    Science.gov (United States)

    Erill, Ivan

    2015-08-01

    Experimentally verified transcription factor-binding sites represent an information-rich and highly applicable data type that aptly summarizes the results of time-consuming experiments and inference processes. Currently, there is no centralized repository for this type of data, which is routinely embedded in articles and extremely hard to mine. CollecTF provides the first standardized resource for submission and deposition of these data into the NCBI RefSeq database, maximizing its accessibility and prompting the community to adopt direct submission policies. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  5. Quantitative modeling of gene expression using DNA shape features of binding sites.

    Science.gov (United States)

    Peng, Pei-Chen; Sinha, Saurabh

    2016-07-27

    Prediction of gene expression levels driven by regulatory sequences is pivotal in genomic biology. A major focus in transcriptional regulation is sequence-to-expression modeling, which interprets the enhancer sequence based on transcription factor concentrations and DNA binding specificities and predicts precise gene expression levels in varying cellular contexts. Such models largely rely on the position weight matrix (PWM) model for DNA binding, and the effect of alternative models based on DNA shape remains unexplored. Here, we propose a statistical thermodynamics model of gene expression using DNA shape features of binding sites. We used rigorous methods to evaluate the fits of expression readouts of 37 enhancers regulating spatial gene expression patterns in Drosophila embryo, and show that DNA shape-based models perform arguably better than PWM-based models. We also observed DNA shape captures information complimentary to the PWM, in a way that is useful for expression modeling. Furthermore, we tested if combining shape and PWM-based features provides better predictions than using either binding model alone. Our work demonstrates that the increasingly popular DNA-binding models based on local DNA shape can be useful in sequence-to-expression modeling. It also provides a framework for future studies to predict gene expression better than with PWM models alone. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. 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

    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...

  7. Specific binding of lactoferrin to Escherichia coli isolated from human intestinal infections

    Energy Technology Data Exchange (ETDEWEB)

    Naidu, S.S.; Erdei, J.; Forsgren, A.; Naidu, A.S. (Departments of Medical Microbiology, Malmoe General Hospital (Sweden)); Czirok, E.; Gado, I. (National Institute of Hygiene, Budapest (Hungary)); Kalfas, S. (School of Dentistry, University of Lund, Malmoe (Sweden)); Thoren, A. (Infectious Diseases, Malmoe General Hospital (Sweden))

    1991-01-01

    The degrees of human lactoferrin (HLf) and bovine lactoferrin (BLf) binding in 169 Escherichia coli strains isolated from human intestinal infections, and in an additional 68 strains isolated from healthy individuals, were examined in a {sup 125}I-labelled protein binding assay. The binding was expressed as a percentage calculated from the total labelled ligand added to bacteria. The HLf and BLf binding to E. coli was in the range 3.7 to 73.4% and 4.8 to 61.6%, respectively. Enterotoxigenic strains demonstrated a significantly higher HLf binding (median = 19%) than enteropathogenic, enteroinvasive, enterohaemorrhagic strains or normal intestinal E. coli isolates (medians 6 to 9). Enteropathogenic strains belonging to serotypes O44 and O127 demonstrated significantly higher HLf binding compared to O26, O55, O111, O119 and O126. No significant differences in the degree of HLf or BLf binding were found between aerobactin-producing and non-producing strains. The interaction was further characterized in a high Lf-binging EPEC strain, E34663 (serotype O127). The binding was stable in the pH range 4.0 to 7.5, did not dissociate in the presence of 2M NaCl or 2M urea, and reached saturation within two h. Unlabelled HLf and BLf displaced the {sup 125}I-HLf binding to E34663 in a dose-dependent manner. Apo- and iron-saturated forms of Lf demonstrated similar binding to E34663. Among various unlabelled subephithelial matrix proteins and carbohydrates tested (in 10{sup 4}-fold excess) only fibronectin and fibrinogen caused a moderate inhibition of {sup 125}I-HLf binding. According to Scatchard plot analysis, 5,400 HLf-binding sites/cell, with an affinity constant (K{sub a}) of 1.4 x 10{sup -7} M, were estimated in strain E34663. These data establish the presence of a specific Lf-binding mechanism in E. coli. (au).

  8. Griffonia simplicifolia lectins bind specifically to endothelial cells and some epithelial cells in mouse tissues.

    Science.gov (United States)

    Laitinen, L

    1987-04-01

    The binding of Griffonia simplicifolia agglutinin-I (GSA-I) and the isolectins GSA-I-AB3 and GSA-I-B4, having affinity for some alpha-D-galactosyl and N-acetyl galactosaminyl residues was studied in different mouse tissues. In brain, cardiac muscle and skeletal muscle, the GSA-I-lectin conjugates showed prominent binding only to blood vessel endothelia. Similarly, in the liver and kidney cortex the GSA-I-conjugates selectively reacted with endothelial cells of the sinusoids and with intertubular and glomerular capillaries, respectively. However, a strong reactivity with the GSA-I-conjugates was additionally seen in the acinar cells of the pancreas, in the stratified squamous epithelia of skin and tongue, and in transitional epithelium. SDS-PAGE electrophoresis combined with the lectin-blotting technique indicated that a similar set of glycoproteins are responsible for the GSA-I binding, even in different tissues. Another lectin with specificity for alpha-D-galactose, the Maclura pomifera agglutinin, displayed a distinctly different distribution of binding sites, mainly in the basement membranes, of all mouse tissues studied. The results suggest that some alpha-D-galactosyl residues, recognized by the binding of GSA-I lectins, are preferentially expressed in endothelial cells of mouse tissues, and also provide further evidence that endothelial cells can present a highly specific surface glycosylation pattern.

  9. Myelin 2',3'-cyclic nucleotide 3'-phosphodiesterase: active-site ligand binding and molecular conformation.

    Directory of Open Access Journals (Sweden)

    Matti Myllykoski

    Full Text Available The 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase is a highly abundant membrane-associated enzyme in the myelin sheath of the vertebrate nervous system. CNPase is a member of the 2H phosphoesterase family and catalyzes the formation of 2'-nucleotide products from 2',3'-cyclic substrates; however, its physiological substrate and function remain unknown. It is likely that CNPase participates in RNA metabolism in the myelinating cell. We solved crystal structures of the phosphodiesterase domain of mouse CNPase, showing the binding mode of nucleotide ligands in the active site. The binding mode of the product 2'-AMP provides a detailed view of the reaction mechanism. Comparisons of CNPase crystal structures highlight flexible loops, which could play roles in substrate recognition; large differences in the active-site vicinity are observed when comparing more distant members of the 2H family. We also studied the full-length CNPase, showing its N-terminal domain is involved in RNA binding and dimerization. Our results provide a detailed picture of the CNPase active site during its catalytic cycle, and suggest a specific function for the previously uncharacterized N-terminal domain.

  10. Predicting DNA-binding sites of proteins from amino acid sequence

    Directory of Open Access Journals (Sweden)

    Wu Feihong

    2006-05-01

    Full Text Available Abstract Background Understanding the molecular details of protein-DNA interactions is critical for deciphering the mechanisms of gene regulation. We present a machine learning approach for the identification of amino acid residues involved in protein-DNA interactions. Results We start with a Naïve Bayes classifier trained to predict whether a given amino acid residue is a DNA-binding residue based on its identity and the identities of its sequence neighbors. The input to the classifier consists of the identities of the target residue and 4 sequence neighbors on each side of the target residue. The classifier is trained and evaluated (using leave-one-out cross-validation on a non-redundant set of 171 proteins. Our results indicate the feasibility of identifying interface residues based on local sequence information. The classifier achieves 71% overall accuracy with a correlation coefficient of 0.24, 35% specificity and 53% sensitivity in identifying interface residues as evaluated by leave-one-out cross-validation. We show that the performance of the classifier is improved by using sequence entropy of the target residue (the entropy of the corresponding column in multiple alignment obtained by aligning the target sequence with its sequence homologs as additional input. The classifier achieves 78% overall accuracy with a correlation coefficient of 0.28, 44% specificity and 41% sensitivity in identifying interface residues. Examination of the predictions in the context of 3-dimensional structures of proteins demonstrates the effectiveness of this method in identifying DNA-binding sites from sequence information. In 33% (56 out of 171 of the proteins, the classifier identifies the interaction sites by correctly recognizing at least half of the interface residues. In 87% (149 out of 171 of the proteins, the classifier correctly identifies at least 20% of the interface residues. This suggests the possibility of using such classifiers to identify

  11. Cerebral decortication reverses the effect of amphetamine on striatal D2 dopamine binding site density.

    Science.gov (United States)

    Robertson, H A

    1986-12-23

    Unilateral ablation of the cerebral cortex produces a decrease of approximately 38% in striatal D2 dopamine binding site density. Chronic d-amphetamine treatment also produces a decrease of about 22% in D2 binding site density. Paradoxically, when rats were hemidecorticated and treated chronically with d-amphetamine, the density of striatal D2 dopamine binding sites on the decorticated side was increased (by about 50%) rather than decreased. Evidently d-amphetamine-induced down-regulation of D2 dopamine binding sites depends on an intact corticostriatal pathway.

  12. Binding site distribution of nuclear transport receptors and transport complexes in single nuclear pore complexes.

    Science.gov (United States)

    Kahms, Martin; Lehrich, Philipp; Hüve, Jana; Sanetra, Nils; Peters, Reiner

    2009-09-01

    Transport through the nuclear pore complex (NPC) involves a large channel and an abundance of binding sites for nuclear transport receptors (NTRs). However, the mechanistically important distribution of NTR-binding sites along the channel is vividly debated. In this study, we visualized binding site distributions directly by two complementary optical super-resolution methods, single-molecule microscopy and 4Pi microscopy. First, we analyzed the distribution of RanGDP because this important nuclear transport substrate has two types of binding sites at the NPC, direct and indirect, NTR-mediated sites. We found that the direct binding sites had a maximum at approximately -30 nm with regard to the NPC center, whereas the indirect transport-relevant binding sites peaked at approximately -10 nm. The 20 nm-shift could be only resolved by 4Pi microscopy because of a two to threefold improved localization precision as compared with single-molecule microscopy. Then we analyzed the distribution of the NTR Kapbeta1 and a Kapbeta1-based transport complex and found them to have also binding maxima at approximately -10 nm. These observations support transport models in which NTR binding sites are distributed all along the transport channel and argue against models in which the cytoplasmic entrance of the channel is surrounded by a large cloud of binding sites.

  13. Capture-C reveals preformed chromatin interactions between HIF-binding sites and distant promoters.

    Science.gov (United States)

    Platt, James L; Salama, Rafik; Smythies, James; Choudhry, Hani; Davies, James Oj; Hughes, Jim R; Ratcliffe, Peter J; Mole, David R

    2016-10-01

    Hypoxia-inducible factor (HIF) directs an extensive transcriptional cascade that transduces numerous adaptive responses to hypoxia. Pan-genomic analyses, using chromatin immunoprecipitation and transcript profiling, have revealed large numbers of HIF-binding sites that are generally associated with hypoxia-inducible transcripts, even over long chromosomal distances. However, these studies do not define the specific targets of HIF-binding sites and do not reveal how induction of HIF affects chromatin conformation over distantly connected functional elements. To address these questions, we deployed a recently developed chromosome conformation assay that enables simultaneous high-resolution analyses from multiple viewpoints. These assays defined specific long-range interactions between intergenic HIF-binding regions and one or more promoters of hypoxia-inducible genes, revealing the existence of multiple enhancer-promoter, promoter-enhancer, and enhancer-enhancer interactions. However, neither short-term activation of HIF by hypoxia, nor long-term stabilization of HIF in von Hippel-Lindau (VHL)-defective cells greatly alters these interactions, indicating that at least under these conditions, HIF can operate on preexisting patterns of chromatin-chromatin interactions that define potential transcriptional targets and permit rapid gene activation by hypoxic stress. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  14. The dialectic role of progesterone.

    Science.gov (United States)

    Huber, Johannes C; Ott, Johannes

    2009-04-20

    Progesterone is known to be metabolized into various metabolites exerting various effects, predominantly into 5alpha-pregnanes and 4-pregnenes. Studies on uterine tissues showed numerous progesterone-converting enzymes such as 5alpha-reductase (5alphaR), 5beta-reductase, 3alpha-, 3beta-, 20alpha-, and 20beta-hydroxysteroid oxidoreductases and others. The main progesterone-metabolizing enzymes in human breast tissues are 5alphaR, 3alpha-HSO 3beta-HSO, and 20alpha-HSO. Tumor genesis in the breast has been shown to be enhanced by high 5alphaR activity and suppression of 3alpha-HSO and 20alpha-HSO. A major determinant of 5alphaR, the breast's gate-keeping enzyme activity is the genetic variation in the enzyme's gene. Two polymorphisms within the steroid 5alphaR type 2 gene, Ala>Thr at codon 49 and Val>Leu at codon 89 have been reported to strongly affect the enzyme's activity, even in regard to breast cancer risk. As steroid hormones are known to be converted into many other steroids occupying different receptors and thereby exerting various different effects, progesterone receptors are important factors when mediating the hormone's effects. The progesterone receptor (PR) gene is transcribed from one gene by two alternative promoters and translated into PR-B, a potent transcriptional activator, and PR-A, the shorter isoform, necessary to oppose the effects of PR-B. In addition, endocrine reactions are modulated by epigenetics. The expression of progesterone receptors has been shown to be up- and downregulated by various epigenetic mechanisms. Many factors must be also taken into account in hormonal (replacement) therapy. Thus natural steroids should not be disparaged as treatment options for gender-specific diseases. An update on endocrinological knowledge and experience is rather mandatory for gynaecologists.

  15. Identification of critical residues in loop E in the 5-HT3ASR binding site

    Directory of Open Access Journals (Sweden)

    Muthalagi Mani

    2002-06-01

    Full Text Available Abstract Background The serotonin type 3 receptor (5-HT3R is a member of a superfamily of ligand gated ion channels. All members of this family share a large degree of sequence homology and presumably significant structural similarity. A large number of studies have explored the structure-function relationships of members of this family, particularly the nicotinic and GABA receptors. This information can be utilized to gain additional insights into specific structural and functional features of other receptors in this family. Results Thirteen amino acids in the mouse 5-HT3ASR that correspond to the putative E binding loop of the nicotinic α7 receptor were chosen for mutagenesis. Due to the presence of a highly conserved glycine in this region, it has been suggested that this binding loop is comprised of a hairpin turn and may form a portion of the ligand-binding site in this ion channel family. Mutation of the conserved glycine (G147 to alanine eliminated binding of the 5-HT3R antagonist [3H]granisetron. Three tyrosine residues (Y140, Y142 and Y152 also significantly altered the binding of 5-HT3R ligands. Mutations in neighboring residues had little or no effect on binding of these ligands to the 5-HT3ASR. Conclusion Our data supports a role for the putative E-loop region of the 5-HT3R in the binding of 5-HT, mCPBG, d-tc and lerisetron. 5-HT and mCPBG interact with Y142, d-tc with Y140 and lerisetron with both Y142 and Y152. Our data also provides support for the hypothesis that this region of the receptor is present in a loop structure.

  16. Two distinctive β subunits are separately involved in two binding sites of imidacloprid with different affinities in Locusta migratoria manilensis.

    Science.gov (United States)

    Bao, Haibo; Liu, Yang; Zhang, Yixi; Liu, Zewen

    2017-08-01

    Due to great diversity of nicotinic acetylcholine receptor (nAChR) subtypes in insects, one β subunit may be contained in numerous nAChR subtypes. In the locust Locusta migratoria, a model insect species with agricultural importance, the third β subunits (Locβ3) was identified in this study, which reveals at least three β subunits in this insect species. Imidacloprid was found to bind nAChRs in L. migratoria central nervous system at two sites with different affinities, with Kd values of 0.16 and 10.31nM. The specific antisera (L1-1, L2-1 and L3-1) were raised against fusion proteins at the large cytoplasmic loop of Locβ1, Locβ2 and Locβ3 respectively. Specific immunodepletion of Locβ1 with antiserum L1-1 resulted in the selective loss of the low affinity binding site for imidacloprid, whereas the immunodepletion of Locβ3 with L3-1 caused the selective loss of the high affinity site. Dual immunodepletion with L1-1 and L3-1 could completely abolish imidacloprid binding. In contrast, the immunodepletion of Locβ2 had no significant effect on the specific [3H]imidacloprid binding. Taken together, these data indicated that Locβ1 and Locβ3 were respectively contained in the low- and high-affinity binding sites for imidacloprid in L. migratoria, which is different to the previous finding in Nilaparvata lugens that Nlβ1 was in two binding sites for imidacloprid. The involvement of two β subunits separately in two binding sites may decrease the risk of imidacloprid resistance due to putative point mutations in β subunits in L. migratoria. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Evolving Transcription Factor Binding Site Models From Protein Binding Microarray Data

    KAUST Repository

    Wong, Ka-Chun

    2016-02-02

    Protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner. In this paper, we describe the PBM motif model building problem. We apply several evolutionary computation methods and compare their performance with the interior point method, demonstrating their performance advantages. In addition, given the PBM domain knowledge, we propose and describe a novel method called kmerGA which makes domain-specific assumptions to exploit PBM data properties to build more accurate models than the other models built. The effectiveness and robustness of kmerGA is supported by comprehensive performance benchmarking on more than 200 datasets, time complexity analysis, convergence analysis, parameter analysis, and case studies. To demonstrate its utility further, kmerGA is applied to two real world applications: 1) PBM rotation testing and 2) ChIP-Seq peak sequence prediction. The results support the biological relevance of the models learned by kmerGA, and thus its real world applicability.

  18. Split tasks of asymmetric nucleotide-binding sites in the heterodimeric ABC exporter EfrCD.

    Science.gov (United States)

    Hürlimann, Lea M; Hohl, Michael; Seeger, Markus A

    2017-06-01

    Many heterodimeric ATP-binding cassette (ABC) exporters evolved asymmetric ATP-binding sites containing a degenerate site incapable of ATP hydrolysis due to noncanonical substitutions in conserved sequence motifs. Recent studies revealed that nucleotide binding to the degenerate site stabilizes contacts between the nucleotide-binding domains (NBDs) of the inward-facing transporter and regulates ATP hydrolysis at the consensus site via allosteric coupling mediated by the D-loops. However, it is unclear whether nucleotide binding to the degenerate site is strictly required for substrate transport. In this study, we examined the functional consequences of a systematic set of mutations introduced at the degenerate and consensus site of the multidrug efflux pump EfrCD of Enterococcus faecalis. Mutating motifs which differ among the two ATP-binding sites (Walker B, switch loop, and ABC signature) or which are involved in interdomain communication (D-loop and Q-loop) led to asymmetric results in the functional assays and were better tolerated at the degenerate site. This highlights the importance of the degenerate site to allosterically regulate the events at the consensus site. Mutating invariant motifs involved in ATP binding and NBD closure (A-loop and Walker A) resulted in equally reduced transport activities, regardless at which ATP-binding site they were introduced. In contrast to previously investigated heterodimeric ABC exporters, mutation of the degenerate site Walker A lysine completely inactivated ATPase activity and substrate transport, indicating that ATP binding to the degenerate site is essential for EfrCD. This study provides novel insights into the split tasks of asymmetric ATP-binding sites of heterodimeric ABC exporters. © 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

  19. Characterization of a human coagulation factor Xa-binding site on Viperidae snake venom phospholipases A2 by affinity binding studies and molecular bioinformatics

    Directory of Open Access Journals (Sweden)

    Gowda Veerabasappa T

    2007-12-01

    Full Text Available Abstract Background The snake venom group IIA secreted phospholipases A2 (SVPLA2, present in the Viperidae snake family exhibit a wide range of toxic and pharmacological effects. They exert their different functions by catalyzing the hydrolysis of phospholipids (PL at the membrane/water interface and by highly specific direct binding to: (i presynaptic membrane-bound or intracellular receptors; (ii natural PLA2-inhibitors from snake serum; and (iii coagulation factors present in human blood. Results Using surface plasmon resonance (SPR protein-protein interaction measurements and an in vitro biological test of inhibition of prothrombinase activity, we identify a number of Viperidae venom SVPLA2s that inhibit blood coagulation through direct binding to human blood coagulation factor Xa (FXa via a non-catalytic, PL-independent mechanism. We classify the SVPLA2s in four groups, depending on the strength of their binding. Molecular electrostatic potentials calculated at the surface of 3D homology-modeling models show a correlation with inhibition of prothrombinase activity. In addition, molecular docking simulations between SVPLA2 and FXa guided by the experimental data identify the potential FXa binding site on the SVPLA2s. This site is composed of the following regions: helices A and B, the Ca2+ loop, the helix C-β-wing loop, and the C-terminal fragment. Some of the SVPLA2 binding site residues belong also to the interfacial binding site (IBS. The interface in FXa involves both, the light and heavy chains. Conclusion We have experimentally identified several strong FXa-binding SVPLA2s that disrupt the function of the coagulation cascade by interacting with FXa by the non-catalytic PL-independent mechanism. By theoretical methods we mapped the interaction sites on both, the SVPLA2s and FXa. Our findings may lead to the design of novel, non-competitive FXa inhibitors.

  20. Characterization of a human coagulation factor Xa-binding site on Viperidae snake venom phospholipases A2 by affinity binding studies and molecular bioinformatics.

    Science.gov (United States)

    Faure, Grazyna; Gowda, Veerabasappa T; Maroun, Rachid C

    2007-12-06

    The snake venom group IIA secreted phospholipases A2 (SVPLA2), present in the Viperidae snake family exhibit a wide range of toxic and pharmacological effects. They exert their different functions by catalyzing the hydrolysis of phospholipids (PL) at the membrane/water interface and by highly specific direct binding to: (i) presynaptic membrane-bound or intracellular receptors; (ii) natural PLA2-inhibitors from snake serum; and (iii) coagulation factors present in human blood. Using surface plasmon resonance (SPR) protein-protein interaction measurements and an in vitro biological test of inhibition of prothrombinase activity, we identify a number of Viperidae venom SVPLA2s that inhibit blood coagulation through direct binding to human blood coagulation factor Xa (FXa) via a non-catalytic, PL-independent mechanism. We classify the SVPLA2s in four groups, depending on the strength of their binding. Molecular electrostatic potentials calculated at the surface of 3D homology-modeling models show a correlation with inhibition of prothrombinase activity. In addition, molecular docking simulations between SVPLA2 and FXa guided by the experimental data identify the potential FXa binding site on the SVPLA2s. This site is composed of the following regions: helices A and B, the Ca2+ loop, the helix C-beta-wing loop, and the C-terminal fragment. Some of the SVPLA2 binding site residues belong also to the interfacial binding site (IBS). The interface in FXa involves both, the light and heavy chains. We have experimentally identified several strong FXa-binding SVPLA2s that disrupt the function of the coagulation cascade by interacting with FXa by the non-catalytic PL-independent mechanism. By theoretical methods we mapped the interaction sites on both, the SVPLA2s and FXa. Our findings may lead to the design of novel, non-competitive FXa inhibitors.

  1. Effect of sodium molybdate on the thyroxine-binding affinity of transport cytosol proteins.

    Science.gov (United States)

    Svobodová, V; Kraml, J; Schreiber, V

    1985-01-01

    The presence of sodium molybdate during tissue homogenization is known to increase the number of cytosol binding sites for glucocorticoids, progesterone, androgens and oestrogens. We wondered whether a phenomenon similar to this stabilization of steroid receptors would also occur in thyroxine-binding cytosol protein. We found that the presence of sodium molybdate (10 mmol/l) in rat adenohypophyseal cytosol increased its thyroxine-binding capacity by up to 96%. In the case of binding protein cytosol minus molybdate, Ka = 5.5 X 10(9) l.mol-1, whereas for cytosol plus molybdate Ka(1) = 6.0 X 10(9) l.mol-1 and Ka(2) = 3.0 X 10(10) l.mol-1. Cytosol prepared without molybdate did not contain a binding protein class with a higher Ka. The effect is stereo-specific and the LT4 bond is not displaced by DT4.

  2. Characterization of 6-mercaptopurine binding to bovine serum albumin and its displacement from the binding sites by quercetin and rutin

    Energy Technology Data Exchange (ETDEWEB)

    Ehteshami, Mehdi [Nutrition Research Center, School of Health and Nutrition, Tabriz University of Medical Sciences, Tabriz 51644-14766 (Iran, Islamic Republic of); Rasoulzadeh, Farzaneh [Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz 51644-14766 (Iran, Islamic Republic of); Mahboob, Soltanali [Nutrition Research Center, School of Health and Nutrition, Tabriz University of Medical Sciences, Tabriz 51644-14766 (Iran, Islamic Republic of); Rashidi, Mohammad-Reza, E-mail: rashidi@tbzmed.ac.ir [Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz 51644-14766 (Iran, Islamic Republic of)

    2013-03-15

    Binding of a drug to the serum albumins as major serum transport proteins can be influenced by other ligands leading to alteration of its pharmacological properties. In the present study, binding characteristics of 6-mercaptopurine (6-MP) with bovine serum albumin (BSA) together with its displacement from its binding site by quercetin and rutin have been investigated by the spectroscopic method. According to the binding parameters, a static quenching component in overall dynamic quenching process is operative in the interaction between 6-MP and BSA. The binding of 6-MP to BSA occurred spontaneously due to entropy-driven hydrophobic interactions. The synchronous fluorescence spectroscopy study revealed that the secondary structure of BSA is changed in the presence of 6-MP and both Tyr and Trp residues participate in the interaction between 6-MP and BSA with the later one being more dominant. The binding constant value of 6-MP-BSA in the presence of quercetin and rutin increased. 6-MP was displaced by ibuprofen indicating that the binding site of 6-MP on albumin is site II. Therefore, the change of the pharmacokinetic and pharmacodynamic properties of 6-MP by quercetin and rutin through alteration of binding capacity of 6-MP to the serum albumin cannot be ruled out. In addition, the displacement study showed that 6-MP is located in site II of BSA. - Highlights: Black-Right-Pointing-Pointer Participation of both Tyr and particularly Trp residues in the interaction between 6-MP and BSA. Black-Right-Pointing-Pointer Involvement of a static quenching component in an overall dynamic quenching process. Black-Right-Pointing-Pointer Ability of quercetin and rutin to change the binding constants of 6-MP-BSA complex. Black-Right-Pointing-Pointer Binding of 6-MP to BSA through entropy-driven hydrophobic interactions.

  3. Mutational analysis of the IFNAR1 binding site on IFNalpha2 reveals the architecture of a weak ligand-receptor binding-site.

    Science.gov (United States)

    Roisman, Laila C; Jaitin, Diego A; Baker, Darren P; Schreiber, Gideon

    2005-10-21

    Type I interferons activate cellular responses by forming a ternary complex with two receptor components, IFNAR1 and IFNAR2. While the binding of the IFNAR2 receptor to interferon is of high affinity and well characterized, the binding to IFNAR1 is weak, transient, and poorly understood. Here, we mapped the complete binding region of IFNAR1 on IFNalpha2 by creating a panel of 21 single alanine mutant proteins, and determined their binding affinities. The IFNAR1 binding site on IFNalpha2 maps to the center of the B and C helices, opposite to the binding site for IFNAR2. No hot spots for binding were found in the interface, with individual mutations having an up to fivefold effect on binding. Of the nine residues that affected binding, three adjacent conserved residues, located on the B helix, conferred an increase in the binding affinity to IFNAR1, as well as an increase in the biological activity of the interferon mutant. This suggests that binding of alpha interferons to the IFNAR1 receptor is sub-optimal. A correlation between binding affinity and biological activity was found, albeit not across the whole range of affinities. In WISH cells, but not DAUDI cells, the anti-proliferative activity was markedly affected by fluctuations in the IFNalpha2 affinity towards the IFNAR1 receptor. On the other hand, the antiviral activity of interferons on WISH cells seems to change in accordance to the binding affinity towards IFNAR1 only as long as the binding affinity is not beyond twofold of the wild-type. In accordance, the biological roles of the two interferon-receptor subunits are discussed.

  4. Spatial Analysis and Quantification of the Thermodynamic Driving Forces in Protein-Ligand Binding: Binding Site Variability

    Science.gov (United States)

    Raman, E. Prabhu; MacKerell, Alexander D.

    2015-01-01

    The thermodynamic driving forces behind small molecule-protein binding are still not well understood, including the variability of those forces associated with different types of ligands in different binding pockets. To better understand these phenomena we calculate spatially resolved thermodynamic contributions of the different molecular degrees of freedom for the binding of propane and methanol to multiple pockets on the proteins Factor Xa and p38 MAP kinase. Binding thermodynamics are computed using a statistical thermodynamics based end-point method applied on a canonical ensemble comprising the protein-ligand complexes and the corresponding free states in an explicit solvent environment. Energetic and entropic contributions of water and ligand degrees of freedom computed from the configurational ensemble provides an unprecedented level of detail into the mechanisms of binding. Direct protein-ligand interaction energies play a significant role in both non-polar and polar binding, which is comparable to water reorganization energy. Loss of interactions with water upon binding strongly compensates these contributions leading to relatively small binding enthalpies. For both solutes, the entropy of water reorganization is found to favor binding in agreement with the classical view of the “hydrophobic effect”. Depending on the specifics of the binding pocket, both energy-entropy compensation and reinforcement mechanisms are observed. Notable is the ability to visualize the spatial distribution of the thermodynamic contributions to binding at atomic resolution showing significant differences in the thermodynamic contributions of water to the binding of propane versus methanol. PMID:25625202

  5. A tool for calculating binding-site residues on proteins from PDB structures

    Directory of Open Access Journals (Sweden)

    Hu Jing

    2009-08-01

    Full Text Available Abstract Background In the research on protein functional sites, researchers often need to identify binding-site residues on a protein. A commonly used strategy is to find a complex structure from the Protein Data Bank (PDB that consists of the protein of interest and its interacting partner(s and calculate binding-site residues based on the complex structure. However, since a protein may participate in multiple interactions, the binding-site residues calculated based on one complex structure usually do not reveal all binding sites on a protein. Thus, this requires researchers to find all PDB complexes that contain the protein of interest and combine the binding-site information gleaned from them. This process is very time-consuming. Especially, combing binding-site information obtained from different PDB structures requires tedious work to align protein sequences. The process becomes overwhelmingly difficult when researchers have a large set of proteins to analyze, which is usually the case in practice. Results In this study, we have developed a tool for calculating binding-site residues on proteins, TCBRP http://yanbioinformatics.cs.usu.edu:8080/ppbindingsubmit. For an input protein, TCBRP can quickly find all binding-site residues on the protein by automatically combining the information obtained from all PDB structures that consist of the protein of interest. Additionally, TCBRP presents the binding-site residues in different categories according to the interaction type. TCBRP also allows researchers to set the definition of binding-site residues. Conclusion The developed tool is very useful for the research on protein binding site analysis and prediction.

  6. A tool for calculating binding-site residues on proteins from PDB structures.

    Science.gov (United States)

    Hu, Jing; Yan, Changhui

    2009-08-03

    In the research on protein functional sites, researchers often need to identify binding-site residues on a protein. A commonly used strategy is to find a complex structure from the Protein Data Bank (PDB) that consists of the protein of interest and its interacting partner(s) and calculate binding-site residues based on the complex structure. However, since a protein may participate in multiple interactions, the binding-site residues calculated based on one complex structure usually do not reveal all binding sites on a protein. Thus, this requires researchers to find all PDB complexes that contain the protein of interest and combine the binding-site information gleaned from them. This process is very time-consuming. Especially, combing binding-site information obtained from different PDB structures requires tedious work to align protein sequences. The process becomes overwhelmingly difficult when researchers have a large set of proteins to analyze, which is usually the case in practice. In this study, we have developed a tool for calculating binding-site residues on proteins, TCBRP http://yanbioinformatics.cs.usu.edu:8080/ppbindingsubmit. For an input protein, TCBRP can quickly find all binding-site residues on the protein by automatically combining the information obtained from all PDB structures that consist of the protein of interest. Additionally, TCBRP presents the binding-site residues in different categories according to the interaction type. TCBRP also allows researchers to set the definition of binding-site residues. The developed tool is very useful for the research on protein binding site analysis and prediction.

  7. 29 CFR 1926.752 - Site layout, site-specific erection plan and construction sequence.

    Science.gov (United States)

    2010-07-01

    ... 29 Labor 8 2010-07-01 2010-07-01 false Site layout, site-specific erection plan and construction sequence. 1926.752 Section 1926.752 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND... Steel Erection § 1926.752 Site layout, site-specific erection plan and construction sequence. (a...

  8. Shapes of antibody binding sites: qualitative and quantitative analyses based on a geomorphic classification scheme.

    Science.gov (United States)

    Lee, Michelle; Lloyd, Peter; Zhang, Xiyun; Schallhorn, Julie M; Sugimoto, Keiki; Leach, Andrew G; Sapiro, Guillermo; Houk, K N

    2006-07-07

    The topography of antibody binding sites has been classified into five types that evoke familiar geomorphic features of the Earth. The 229 antibody crystal structures from the Protein Data Bank were analyzed and classified into these classes. Relationships to previous topography classifications by Rees et al., who defined three classes, and Thornton et al., who defined four classes, are identified. An algorithm was developed to identify the antibody binding site class automatically based on the definition and the shape of the binding site. A three-dimensional convex hull was formed around the complementarity determining regions (CDRs) of the antibody. The convex hull was then "trimmed" to fit the binding site by using distance criteria and morphological techniques. Once the program identified the binding site shape, a statistical and distance based analysis was performed to classify automatically the antibody into one of the five geomorphic classes. The five antibody topography classes are as follows: cave (mostly hapten binders), crater (mostly protein and peptide/carbohydrate/nucleic acid binders), canyon, valley, and plain (mostly protein binders). Comparisons of the binding sites of empty and of complexed antibody binding sites gave an indication of how the shape of the binding site is influenced by binding of the antigen.

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

    Directory of Open Access Journals (Sweden)

    David Paramelle

    Full Text Available 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

  10. Site-directed alkylation of multiple opioid receptors. I. Binding selectivity

    Energy Technology Data Exchange (ETDEWEB)

    James, I.F.; Goldstein, A.

    1984-05-01

    A method for measuring and expressing the binding selectivity of ligands for mu, delta, and kappa opioid binding sites is reported. Radioligands are used that are partially selective for these sites in combination with membrane preparations enriched in each site. Enrichment was obtained by treatment of membranes with the alkylating agent beta-chlornaltrexamine in the presence of appropriate protecting ligands. After enrichment for mu receptors, (/sup 3/H) dihydromorphine bound to a single type of site as judged by the slope of competition binding curves. After enrichment for delta or kappa receptors, binding sites for (/sup 3/H) (D-Ala2, D-Leu5)enkephalin and (3H)ethylketocyclazocine, respectively, were still not homogeneous. There were residual mu sites in delta-enriched membranes but no evidence for residual mu or delta sites in kappa-enriched membranes were found. This method was used to identify ligands that are highly selective for each of the three types of sites.

  11. [125I]RTI-55 binding to cocaine-sensitive dopaminergic and serotonergic uptake sites in the human brain.

    Science.gov (United States)

    Little, K Y; Kirkman, J A; Carroll, F I; Breese, G R; Duncan, G E

    1993-12-01

    [125I]RTI-55 is a newly synthesized cocaine congener that may offer advantages over other ligands previously used to examine cocaine binding sites. However, the in vitro pharmacological and anatomical characterization of [125I]RTI-55 binding sites has not been previously performed in human brain. To determine the specificity, stability, and feasibility of [125I]RTI-55 for use in radioligand binding assays in postmortem human tissue, a series of experiments were performed characterizing [125I]RTI-55 binding sites in human brain using homogenized membrane preparations and quantitative autoradiography. Analysis of the association, dissociation, and saturation data favored two-phase processes. A curve-fitting analysis of the data derived in saturation experiments found a high-affinity site with KD = 66 +/- 35 pM and Bmax = 13.2 +/- 10.1 pmol/g of tissue and a low-affinity site with KD = 1.52 +/- 0.55 nM and Bmax of 47.5 +/- 11.2 pmol/g of tissue. Competition by ligands known to bind to the dopamine transporter showed a rank order of RTI-55 > GBR-12909 > maxindol > WIN 35428 > = methylphenidate > (-)-cocaine > buproprion > (+)-amphetamine. Binding to serotonergic sites was evaluated in the midbrain. Results of the saturation experiment performed autoradiographically in the midbrain showed a single site with KD = 370 +/- 84 pM. It appears that [125I]RTI-55 should be useful in further studies of the regulation of cocaine binding sites using postmortem human specimens.

  12. Estrogen and progesterone receptor-binding sites on the chicken vitellogenin II gene: synergism of steroid hormone action.

    OpenAIRE

    Cato, A C; Heitlinger, E; Ponta, H; Klein-Hitpass, L; Ryffel, G U; Bailly, A; Rauch, C; Milgrom, E

    1988-01-01

    The chicken vitellogenin II gene is transcriptionally activated by estrogens. In transient transfection experiments in human T47D cells that contain receptors for various steroids, we showed estradiol, progestin, and androgen responses of a chimeric chicken vitellogenin II construct. This construct consists of DNA sequences from -626 to -590 upstream of the start of transcription of the chicken vitellogenin gene linked to the herpes simplex virus thymidine kinase promoter driving the transcri...

  13. Hydrolysis at One of the Two Nucleotide-binding Sites Drives the Dissociation of ATP-binding Cassette Nucleotide-binding Domain Dimers*

    Science.gov (United States)

    Zoghbi, Maria E.; Altenberg, Guillermo A.

    2013-01-01

    The functional unit of ATP-binding cassette (ABC) transporters consists of two transmembrane domains and two nucleotide-binding domains (NBDs). ATP binding elicits association of the two NBDs, forming a dimer in a head-to-tail arrangement, with two nucleotides “sandwiched” at the dimer interface. Each of the two nucleotide-binding sites is formed by residues from the two NBDs. We recently found that the prototypical NBD MJ0796 from Methanocaldococcus jannaschii dimerizes in response to ATP binding and dissociates completely following ATP hydrolysis. However, it is still unknown whether dissociation of NBD dimers follows ATP hydrolysis at one or both nucleotide-binding sites. Here, we used luminescence resonance energy transfer to study heterodimers formed by one active (donor-labeled) and one catalytically defective (acceptor-labeled) NBD. Rapid mixing experiments in a stop-flow chamber showed that NBD heterodimers with one functional and one inactive site dissociated at a rate indistinguishable from that of dimers with two hydrolysis-competent sites. Comparison of the rates of NBD dimer dissociation and ATP hydrolysis indicated that dissociation followed hydrolysis of one ATP. We conclude that ATP hydrolysis at one nucleotide-binding site drives NBD dimer dissociation. PMID:24129575

  14. EO-199, a specific antagonist of antiarrhythmic drugs: Assessment by binding experiments and in vivo studies

    Energy Technology Data Exchange (ETDEWEB)

    Oppenheimer, E.; Harel, G.; Lipinsky, D.; Sarne, Y. (Tel-Aviv Univ. (Israel))

    1991-01-01

    EO-199, a demethylated analog of the novel class I antiarrhythmic drug EO-122 was found to antagonize the antiarrhythmic activity of EO-122 and that of procainamide (Class I{sub A}). EO-199 did not block significantly the activity of a class I{sub B} antiarrhythmic agent, lidocaine. EO-199 also displaced the specific binding of ({sup 3}H)EO-122 to rate heart membranes similarly to procainamide whereas lidocaine did not. The correlation between binding experiments and pharmacological effects points to a possible subclassification of these drugs; the two chemical analogs EO-199 and EO-122, as well as procainamide (I{sub A}) but not lidocaine (I{sub B}), compete at the same site or the same state of the sodium channel. The availability of a specific antagonist might be useful for studying the mechanism of action of antiarrhythmic drugs as well as an antidote in cases of antiarrhythmics overdose intoxication.

  15. DNA binding specificity and cleavage activity of Pacmmar transposase.

    Science.gov (United States)

    Delaurière, Laurence; Chénais, Benoît; Pradier, Elisabeth; Hardivillier, Yann; Renault, Sylvaine; Casse, Nathalie

    2009-08-04

    Mariner-like elements (MLEs) are members of the Tc1/mariner superfamily of transposable elements which transpose by a "cut and paste" mechanism. Most of the MLEs characterized to date are transpositionally inactive due to the accumulation of mutations in their transposase gene. Here, we report the biochemical study of two copies of the Pacmmar element (Pacmmar1.1 and Pacmmar1.2), isolated from the coastal crab Pachygrapsus marmoratus. These two copies present an open reading frame encoding a putative active transposase. Using an in vitro transposition assay, we show that Pacmmar transposases are unable to perform by themselves the transposition reaction. However, we demonstrate by an electrophoretic mobility shift assay that both transposases bind specifically to the inverted terminal repeat of the Pacmmar element. Moreover, an in vitro cleavage assay showed that both transposases have the capacity to cleave the transposon. The in vitro cleavage activity of Pacmmar transposases appears imprecise, suggesting the requirement of specific host factors or the presence of mutations which have modified the cleavage specificity of the enzyme.

  16. Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission

    DEFF Research Database (Denmark)

    Maric, Hans Michael; Hausrat, Torben Johann; Neubert, Franziska

    2017-01-01

    γ-Aminobutyric acid type A and glycine receptors are the major mediators of fast synaptic inhibition in the human central nervous system and are established drug targets. However, all drugs targeting these receptors bind to the extracellular ligand-binding domain of the receptors, which inherentl...

  17. Screening Mixtures of Small Molecules for Binding to Multiple Sites on the Surface Tetanus Toxin C Fragment by Bioaffinity NMR

    Energy Technology Data Exchange (ETDEWEB)

    Cosman, M; Zeller, L; Lightstone, F C; Krishnan, V V; Balhorn, R

    2002-01-01

    The clostridial neurotoxins include the closely related tetanus (TeNT) and botulinum (BoNT) toxins. Botulinum toxin is used to treat severe muscle disorders and as a cosmetic wrinkle reducer. Large quantities of botulinum toxin have also been produced by terrorists for use as a biological weapon. Because there are no known antidotes for these toxins, they thus pose a potential threat to human health whether by an accidental overdose or by a hostile deployment. Thus, the discovery of high specificity and affinity compounds that can inhibit their binding to neural cells can be used as antidotes or in the design of chemical detectors. Using the crystal structure of the C fragment of the tetanus toxin (TetC), which is the cell recognition and cell surface binding domain, and the computational program DOCK, sets of small molecules have been predicted to bind to two different sites located on the surface of this protein. While Site-1 is common to the TeNT and BoNTs, Site-2 is unique to TeNT. Pairs of these molecules from each site can then be linked together synthetically to thereby increase the specificity and affinity for this toxin. Electrospray ionization mass spectroscopy was used to experimentally screen each compound for binding. Mixtures containing binders were further screened for activity under biologically relevant conditions using nuclear magnetic resonance (NMR) methods. The screening of mixtures of compounds offers increased efficiency and throughput as compared to testing single compounds and can also evaluate how possible structural changes induced by the binding of one ligand can influence the binding of the second ligand. In addition, competitive binding experiments with mixtures containing ligands predicted to bind the same site could identify the best binder for that site. NMR transfer nuclear Overhauser effect (trNOE) confirm that TetC binds doxorubicin but that this molecule is displaced by N-acetylneuraminic acid (sialic acid) in a mixture that

  18. Influence of menstrual cycle on platelet serotonin uptake site and serotonin2A receptor binding.

    Science.gov (United States)

    Wihlbäck, Anna-Carin; Sundström Poromaa, Inger; Bixo, Marie; Allard, Per; Mjörndal, Tom; Spigset, Olav

    2004-07-01

    Depression and anxiety are common health problems affecting women, particularly during the reproductive years. Major depression is two to three times as common in women than in men. Neuroendocrine factors are likely to contribute to this overall increased risk for developing mood disorders in women, and the neuroendocrine influence is most obviously seen in women with premenstrual dysphoric disorder (PMDD) as these women experience depressed mood and anxiety premenstrually only during ovulatory cycles. Moreover, dysfunction of serotonergic transmission has been regarded as an important mechanism in several psychiatric disorders and ovarian steroids have been shown to profoundly influence the activity of the serotonergic system. Given these facts, the purpose of this study was to examine whether binding of [3H]paroxetine to the platelet serotonin transporter or binding of [3H]lysergic acid diethylamide ([3H]LSD) to the platelet 5-HT2A receptor are influenced by the cyclical changes in circulating estradiol and progesterone that occur during the menstrual cycle. We examined 28 healthy women, without oral contraceptives and with regular menstrual cycles. In the late follicular phase, Bmax for [3H]paroxetine binding was significantly higher than in the ovulatory (pLSD binding was significantly higher in the early follicular phase and the early luteal phase compared to the mid-luteal phase (pLSD binding were found (p<0.05, respectively).

  19. The chromosomal high-affinity binding sites for the Drosophila dosage compensation complex.

    Directory of Open Access Journals (Sweden)

    Tobias Straub

    2008-12-01

    Full Text Available Dosage compensation in male Drosophila relies on the X chromosome-specific recruitment of a chromatin-modifying machinery, the dosage compensation complex (DCC. The principles that assure selective targeting of the DCC are unknown. According to a prevalent model, X chromosome targeting is initiated by recruitment of the DCC core components, MSL1 and MSL2, to a limited number of so-called "high-affinity sites" (HAS. Only very few such sites are known at the DNA sequence level, which has precluded the definition of DCC targeting principles. Combining RNA interference against DCC subunits, limited crosslinking, and chromatin immunoprecipitation coupled to probing high-resolution DNA microarrays, we identified a set of 131 HAS for MSL1 and MSL2 and confirmed their properties by various means. The HAS sites are distributed all over the X chromosome and are functionally important, since the extent of dosage compensation of a given gene and its proximity to a HAS are positively correlated. The sites are mainly located on non-coding parts of genes and predominantly map to regions that are devoid of nucleosomes. In contrast, the bulk of DCC binding is in coding regions and is marked by histone H3K36 methylation. Within the HAS, repetitive DNA sequences mainly based on GA and CA dinucleotides are enriched. Interestingly, DCC subcomplexes bind a small number of autosomal locations with similar features.

  20. CRISPR Outsourcing: Commissioning IHF for Site-Specific Integration of Foreign DNA at the CRISPR Array.

    Science.gov (United States)

    Wei, Yunzhou; Terns, Michael P

    2016-06-16

    In this issue of Molecular Cell, Nuñez et al. (2016) report that site-specific integration of foreign DNA into CRISPR loci by the Cas1-Cas2 integrase complex is promoted by a host factor, IHF (integration host factor), that binds and bends CRISPR leader DNA. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Downregulation of (3H)Ro5-4864 binding sites after exposure to peripheral-type benzodiazepines in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, M.D.; Wang, J.K.; Morgan, J.I.; Spector, S.

    1986-09-01

    Peripheral-type benzodiazepine (BZD) binding sites undergo a rapid and pronounced downregulation after exposure to these compounds in vitro. Friend erythroleukemia cells were incubated with micromolar concentrations of BZD after which they were washed thoroughly and the binding of the specific peripheral-type BZD radioligand (/sup 3/H)Ro5-4864 was determined. Exposure to the peripheral-type BZD Ro7-3351 decreased the number of (/sup 3/H)Ro5-4864 binding sites from 324 to 41 fmol/10(6) cells with no change in affinity. Downregulation appears to require active cellular processes because it is blocked when exposure to BZD is at 4/sup 0/C rather than at 37/sup 0/C. Furthermore, whereas (/sup 3/H)Ro5-4864 binding is decreased substantially in membrane preparations made from downregulated cells, it is not altered when membrane preparations from control cells are exposed to BZD. The time course of downregulation is quite rapid, as it occurs within minutes. In contrast, the return of sites requires days and there is a close relationship between return of sites and growth of new cells. The ability of BZDs to downregulate correlates more closely with affinity for the peripheral-type site than with biological activity. The ability to undergo downregulation is characteristic of receptors and its occurrence suggests that peripheral-type BZD binding sites are functional receptors.

  2. A molecular model of the folate binding site of Pneumocystis carinii dihydrofolate reductase

    Science.gov (United States)

    Southerland, William M.

    1994-04-01

    The inhibition of Pneumocystis carinii dihydrofolate reductase (DHFR) continues to be the major treatment strategy for P. carinii pneumonia (PCP). The design of new anti-pneumocystis agents would be significantly enhanced by the availability of a 3D model of the methotrexate (MTX) binding site of the P. carinii DHFR. However, an X-ray crystal structure of the P. carinii DHFR is not yet available. Alignment of the amino acid sequences of P. carinii and Lactobacillus casei DHFRs indicates that the two proteins show approximately 80% homology among MTX binding-site residues. This high level of homology suggests that the L. casei DHFR MTX binding-site structure could serve as a structural template in developing a model of the P. carinii DHFR MTX binding site. Therefore, the X-ray crystal structure of L. casei DHFR was used to develop a 3D model of the methotrexate binding site of P. carinii DHFR. The molecular modeling and dynamics software QUANTA/CHARMm was used. Amino acid residue mutations and deletions were performed using QUANTA and macromolecular minimizations were achieved with CHARMm. The MTX binding-site residues of L. casei DHFR were mutated to the corresponding residues of the P. carinii DHFR sequence. The resulting structure was extensively minimized. The resulting P. carinii MTX binding-site model showed significant differences in hydrogen-bonding patterns from the L. casei MTX binding site. Also, the P. carinii site is more hydrophobic than the corresponding L. casei site. Analysis of atom-to-atom close contacts between methotrexate and protein binding-site residues indicates that the P. carinii MTX binding-site complex is primarily stabilized by hydrophobic interactions, while the L. casei complex is mostly stabilized by electrostatic interactions. The model is consistent with the observed increased sensitivity of P. carinii DHFR to lipid-soluble inhibitors and provides a rational basis for the design of new anti-pneumocystis agents.

  3. Development of site-specific earthquake response spectra for eastern US sites

    Energy Technology Data Exchange (ETDEWEB)

    Beavers, J.E.; Brock, W.R.; Hunt, R.J.; Shaffer, K.E.

    1993-08-01

    Site-specific earthquake, uniform-hazard response spectra have been defined for the Department of Energy Oak Ridge, Tennessee, and Portsmouth, Ohio, sites for use in evaluating existing facilities and designing new facilities. The site-specific response spectra were defined from probabilistic and deterministic seismic hazard studies following the requirements in DOE-STD-1024-92, ``Guidelines for Probabilistic Seismic Hazard Curves at DOE Sites.` For these two sites, the results show that site-specific uniform-hazard response spectra are slightly higher in the high-frequency range and considerably lower in the low-frequency range compared with response spectra defined for these sites in the past.

  4. Quantitative analysis of pheromone-binding protein specificity

    OpenAIRE

    Katti, S.; Lokhande, N.; González, D.; Cassill, A.; Renthal, R.

    2012-01-01

    Many pheromones have very low water solubility, posing experimental difficulties for quantitative binding measurements. A new method is presented for determining thermodynamically valid dissociation constants for ligands binding to pheromone-binding proteins (OBPs), using β-cyclodextrin as a solubilizer and transfer agent. The method is applied to LUSH, a Drosophila OBP that binds the pheromone 11-cis vaccenyl acetate (cVA). Refolding of LUSH expressed in E. coli was assessed by measuring N-p...

  5. Composite organization of the cobalamin binding and cubilin recognition sites of intrinsic factor

    DEFF Research Database (Denmark)

    Fedosov, Sergey N; Fedosova, Natalya U; Berglund, Lars

    2005-01-01

    of the ligand. Each isolated fragment of IF was tested for the binding to the specific receptor cubilin in the presence or absence of Cbl. Neither apo nor holo forms of IF(20) and IF(30) were recognized by the receptor. When two fragments were mixed and incubated with Cbl, they associated into a stable complex......, IF(30+20).Cbl, which bound to cubilin as well as the noncleaved IF(50).Cbl complex. We suggest that formation of the cubilin recognition site on IF is caused by assembly of two distant domains, which allows the saturated protein to be recognized by the receptor. The obtained parameters for ligand...

  6. A role for the weak DnaA binding sites in bacterial replication origins

    DEFF Research Database (Denmark)

    Charbon, Godefroid; Løbner-Olesen, Anders

    2011-01-01

    DnaA initiates the chromosomal DNA replication in nearly all bacteria, and replication origins are characterized by binding sites for the DnaA protein (DnaA-boxes) along with an ‘AT-rich’ region. However, great variation in number, spatial organization and specificity of DnaA-boxes is observed...... between species. In the study by Taylor et al. (2011), new and unexpectedly weak DnaA-boxes were identified within the Caulobacter crescentus origin of replication (Cori). The position of weak and stronger DnaA-boxes follows a pattern seen in Escherichia coli oriC. This raises the possibility...

  7. Genome-wide redistribution of BRD4 binding sites in transformation resistant cells

    Directory of Open Access Journals (Sweden)

    Han Si

    2015-03-01

    Full Text Available Hutchinson–Gilford progeria syndrome (HGPS patients do not develop cancer despite a significant accumulation of DNA damage in their cells. We have recently reported that HGPS cells are refractory to experimental oncogenic transformation and we identified the bromodomain-containing 4 protein (BRD4 as a mediator of the transformation resistance. ChIP-sequencing experiments revealed distinct genome-wide binding patterns for BRD4 in HGPS cells when compared to control wild type cells. Here we provide a detailed description of the ChIP-seq dataset (NCBI GEO accession number GSE61325, the specific and common BRD4 binding sites between HGPS and control cells, and the data analysis procedure associated with the publication by Fernandez et al., 2014 in Cell Reports 9, 248-260 [1].

  8. GFI1B controls its own expression binding to multiple sites.

    Science.gov (United States)

    Anguita, Eduardo; Villegas, Ana; Iborra, Francisco; Hernández, Aurora

    2010-01-01

    Transcription factors play essential roles in both normal and malignant hematopoiesis. This is the case for the growth factor independent 1b (GFI1B) transcription factor, which is required for erythroid and megakaryocytic differentiation and over-expressed in leukemic patients and cell lines. To investigate GFI1B regulation, we searched for multispecies conserved non-coding elements between GFI1B and neighboring genes. We used a formaldehyde-assisted isolation of regulatory elements (FAIRE) assay and DNase1 hypersensitivity to assess the chromatin conformation of these sites. Next, we analyzed transcription factor binding and histone modifications at the GFI1B locus including the conserved non-coding elements by a chromatin immunoprecipitation assay. Finally, we studied the interaction of the GFI1B promoter and the conserved non-coding elements with the chromatin conformation capture technique and used immunofluorescence to evaluate GFI1B levels in individual cells. We localized several conserved non-coding elements containing multiple erythroid specific transcription factor binding sites at the GFI1B locus. In GFI1B-expressing cells a subset of these conserved non-coding elements and the promoter adopt a close spatial conformation, localize with open chromatin sites, harbor chromatin modifications associated with gene activation and bind multiple transcription factors and co-repressors. Conclusions Our findings indicate that GFI1B regulatory elements behave as activators and repressors. Different protein levels within a cell population suggest that cells must activate and repress GFI1B continuously to control its final level. These data are consistent with a model of GFI1B regulation in which GFI1B binds to its own promoter and to the conserved non-coding elements as its levels rise. This would attract repressor complexes that progressively down-regulate the gene. GFI1B expression would decrease until a stage at which the activating complexes predominate and

  9. MotifMap-RNA: a genome-wide map of RBP binding sites.

    Science.gov (United States)

    Liu, Yu; Sun, Sha; Bredy, Timothy; Wood, Marcelo; Spitale, Robert C; Baldi, Pierre

    2017-07-01

    RNA plays a critical role in gene expression and its regulation. RNA binding proteins (RBPs), in turn, are important regulators of RNA. Thanks to the availability of large scale data for RBP binding motifs and in vivo binding sites results in the form of eCLIP experiments, it is now possible to computationally predict RBP binding sites across the whole genome. We describe MotifMap-RNA, an extension of MotifMap which predicts binding sites for RBP motifs across human and mouse genomes and allows large scale querying of predicted binding sites. The data and corresponding web server are available from: http://motifmap-rna.ics.uci.edu/ as part of the MotifMap web portal. rspitale@uci.edu or pfbaldi@uci.edu. Supplementary data are available at Bioinformatics online.

  10. Computational identification of combinatorial regulation and transcription factor binding sites.

    Science.gov (United States)

    Ryu, Taewoo; Kim, Younghoon; Kim, Dae-Won; Lee, Doheon

    2007-08-15

    A number of computational methods have been used to unravel the core mechanisms governing the regulation of gene expression, but these techniques examine only portions of the genetic regulatory mechanism. For example, some studies have failed to include the combined action of multiple transcription factors (TFs) or the importance of TF binding constraints (i.e., the binding position and orientation), while others have examined combinations of only 2 or 3 TFs. Thus, we sought to develop a new method for identifying regulatory modules in yeast, using an algorithm that includes all combinations of TFs plus a number of binding constraints when identifying target genes. We successfully developed a computational method for using microarray and TF-DNA interaction data to identify regulatory modules. All possible combinations of yeast TFs and various binding constraints were tested to identify regulatory modules. Within the identified modules, target genes were found to have common binding constraints such as fixed binding regions and orientations for each TF. Moreover, targets showed similar mRNA expression profiles and high functional coherence. Our novel approach, which accounts for both combined actions of TFs and their binding constraints, can be used to identify target genes and reliably predict regulatory modules over a broad range of functional categories. Complete results and additional information are available online at http://bisl. kaist.ac.kr/~dhlee/comModule/index.html. (c) 2007 Wiley Periodicals, Inc.

  11. Novel approach for selecting the best predictor for identifying the binding sites in DNA binding proteins.

    Science.gov (United States)

    Nagarajan, R; Ahmad, Shandar; Gromiha, M Michael

    2013-09-01

    Protein-DNA complexes play vital roles in many cellular processes by the interactions of amino acids with DNA. Several computational methods have been developed for predicting the interacting residues in DNA-binding proteins using sequence and/or structural information. These methods showed different levels of accuracies, which may depend on the choice of data sets used in training, the feature sets selected for developing a predictive model, the ability of the models to capture information useful for prediction or a combination of these factors. In many cases, different methods are likely to produce similar results, whereas in others, the predictors may return contradictory predictions. In this situation, a priori estimates of prediction performance applicable to the system being investigated would be helpful for biologists to choose the best method for designing their experiments. In this work, we have constructed unbiased, stringent and diverse data sets for DNA-binding proteins based on various biologically relevant considerations: (i) seven structural classes, (ii) 86 folds, (iii) 106 superfamilies, (iv) 194 families, (v) 15 binding motifs, (vi) single/double-stranded DNA, (vii) DNA conformation (A, B, Z, etc.), (viii) three functions and (ix) disordered regions. These data sets were culled as non-redundant with sequence identities of 25 and 40% and used to evaluate the performance of 11 different methods in which online services or standalone programs are available. We observed that the best performing methods for each of the data sets showed significant biases toward the data sets selected for their benchmark. Our analysis revealed important data set features, which could be used to estimate these context-specific biases and hence suggest the best method to be used for a given problem. We have developed a web server, which considers these features on demand and displays the best method that the investigator should use. The web server is freely available at

  12. Site-specific Auger electron spectra of ethyl trifluoroacelate molecules studied by magnetic bottle electron spectrometer

    Science.gov (United States)

    Iwayama, Hiroshi; Shigemasa, Eiji; Hikosaka, Yasumasa; Nakano, Motoyoshi; Ito, Kenji; Lablanquie, Pascal; Penet, Francis; Andric, Lidija; Selles, Patricia

    2012-11-01

    We performed multielectron coincidence measurements for inner-shell photoionizations of ethyl trifluoroacelate molecules (C4H5F3O2) using a magnetic bottle electron spectrometer. From a two dimensional coincidence map between a photoelectron and Auger electron for C 1s ionizations, we extracted site-specific Auger electron spectra for each carbon site and corresponding binding energy of doubly charged states.

  13. Progesterone, Estradiol and their Receptors in Leiomyomata and the

    African Journals Online (AJOL)

    Estradiol and progesterone binding in uterine leiomyomata and in normal uterine tissues. Obstetrics and. Gynaecology. 1980; 55: 4-20. 4. Jorge RP, Edgrad C, Jacques G,. Christine V, Bernard S and Albert N. Effect of Decapeptyl, an agonist analog of GnRH on estrogens, estrogen sulfates, and progesterone receptors in.

  14. Structure of Dioclea virgata lectin: relations between carbohydrate binding site and nitric oxide production

    Energy Technology Data Exchange (ETDEWEB)

    Delatorre, P.; Gadelha, C.A.A.; Santi-Gadelha, T. [Universidade Federal da Paraiba (UFPB), Joao Pessoa, PB (Brazil); Nobrega, R.B.; Rocha, B.A.M.; Nascimento, K.S.; Naganao, C.S.; Sampaio, A.H.; Cavada, B.S. [Universidade Federal do Ceara (UFC), Fortaleza, CE (Brazil); Pires, A.F.; Assreuy, A.M.S. [Universidade Estadual do Ceara (UECE), Fortaleza, CE (Brazil)

    2012-07-01

    Full text: Lectins are proteins/glycoproteins with at least one noncatalytic domain binding reversibly to specific monosaccharides or oligosaccharides. By binding to carbohydrate moieties on the cell surface, lectins participate in a range of cellular processes without changing the properties of the carbohydrates involved. The lectin of Dioclea virgata (DvirL), both native and complexed with X-man, was submitted to X-ray diffraction analysis and the crystal structure was compared to that of other Diocleinae lectins in order to better understand differences in biological proper- ties, especially with regard to the ability of lectins to induce nitric oxide (NO) production. The DvirL diffraction analysis revealed that both the native crystal and the X-Man-complexed form are orthorhombic and belong to space group I222. The cell parameters were: a=65.4 , b=86.6 and c=90.2 (native structure), and a=61.89 , b=87.67 and c=88.78 (X-Man-complexed structure). An association was observed between the volume of the carbohydrate recognition domain (CRD), the ability to induce NO production and the relative positions of Tyr12, Arg228 and Leu99. Thus, differences in biological activity induced by Diocleinae lectins are related to the configuration of amino acid residues in the carbohydrate binding site and to the structural conformation of subsequent regions capable of influencing site-ligand interactions. In conclusion, the ability of Diocleinae lectins to induce NO production depends on CRD configuration. (author)

  15. Interaction of mono- and dianions with cyanase: evidence for apparent half-site binding.

    Science.gov (United States)

    Anderson, P M; Johnson, W V; Endrizzi, J A; Little, R M; Korte, J J

    1987-06-30

    Cyanase is an inducible enzyme in Escherichia coli that catalyzes bicarbonate-dependent hydrolysis of cyanate. The dianions oxalate, oxalacetate, and malonate are slow-binding inhibitors of cyanase, and some monoanions such as azide and chloride also inhibit cyanase activity [Anderson, P. M., & Little, R. M. (1986) Biochemistry 25, 1621-1626]. The purpose of this study was to investigate the interaction of selected dianions and monoanions by kinetic and equilibrium dialysis binding studies in an effort to obtain information about the active site and catalytic mechanism. Measurement of the effectiveness of 30 different dianions as inhibitors of cyanase showed a significant degree of structural and/or isomeric specificity and considerable variation with respect to the slow-binding nature of the inhibition. Oxalate and oxalacetate both show extreme slow-binding inhibition at very low concentrations. Kinetic studies of the rate of inhibition of cyanase by oxalate showed that the reaction is pseudo first order with respect to oxalate concentration and the results are consistent with a pathway in which oxalate forms a complex with the enzyme in a rapid initial reversible step followed by a slow isomerization step leading to a complex with a very low dissociation constant. The rate of inhibition is significantly reduced by the presence of relatively low concentrations of either azide (analogue of cyanate) or bicarbonate. Equilibrium dialysis binding studies showed that the stoichiometry of binding at saturation for oxalate, malonate, chloride, and bicarbonate is about 0.5 mol of ligand bound/mol of subunit for each compound.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. In vitro selection of zinc fingers with altered DNA-binding specificity.

    Science.gov (United States)

    Jamieson, A C; Kim, S H; Wells, J A

    1994-05-17

    We have used random mutagenesis and phage display to alter the DNA-binding specificity of Zif268, a transcription factor that contains three zinc finger domains. Four residues in the helix of finger 1 of Zif268 that potentially mediate DNA binding were identified from an X-ray structure of the Zif268-DNA complex. A library was constructed in which these residues were randomly mutated and the Zif268 variants were fused to a truncated version of the gene III coat protein on the surface of M13 filamentous phage particles. The phage displayed the mutant proteins in a monovalent fashion and were sorted by repeated binding and elution from affinity matrices containing different DNA sequences. When the matrix contained the natural nine base pair operator sequence 5'-GCG-TGG-GCG-3', native-like zinc fingers were isolated. New finger 1 variants were found by sorting with two different operators in which the singly modified triplets, GTG and TCG, replaced the native finger 1 triplet, GCG. Overall, the selected finger 1 variants contained a preponderance of polar residues at the four sites. Interestingly, the net charge of the four residues in any selected finger never derived more that one unit from neutrality despite the fact that about half the variants contained three or four charged residues over the four sites. Measurements of the dissociation constants for two of these purified finger 1 variants by gel-shift assay showed their specificities to vary over a 10-fold range, with the greatest affinity being for the DNA binding site for which they were sorted.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Thermodynamic and structural investigation of the specific SDS binding of humicola insolens cutinase

    Science.gov (United States)

    Kold, David; Dauter, Zbigniew; Laustsen, Anne K; Brzozowski, Andrzej M; Turkenburg, Johan P; Nielsen, Anders D; Koldsø, Heidi; Petersen, Evamaria; Schiøtt, Birgit; De Maria, Leonardo; Wilson, Keith S; Svendsen, Allan; Wimmer, Reinhard

    2014-01-01

    The interaction of lipolytic enzymes with anionic surfactants is of great interest with respect to industrially produced detergents. Here, we report the interaction of cutinase from the thermophilic fungus Humicola insolens with the anionic surfactant SDS, and show the enzyme specifically binds a single SDS molecule under nondenaturing concentrations. Protein interaction with SDS was investigated by NMR, ITC and molecular dynamics simulations. The NMR resonances of the protein were assigned, with large stretches of the protein molecule not showing any detectable resonances. SDS is shown to specifically interact with the loops surrounding the catalytic triad with medium affinity (Ka ≈ 105 M−1). The mode of binding is closely similar to that seen previously for binding of amphiphilic molecules and substrate analogues to cutinases, and hence SDS acts as a substrate mimic. In addition, the structure of the enzyme has been solved by X-ray crystallography in its apo form and after cocrystallization with diethyl p-nitrophenyl phosphate (DNPP) leading to a complex with monoethylphosphate (MEP) esterified to the catalytically active serine. The enzyme has the same fold as reported for other cutinases but, unexpectedly, esterification of the active site serine is accompanied by the ethylation of the active site histidine which flips out from its usual position in the triad. PMID:24832484

  18. DNA methylation in the CTCF-binding site I and the expression pattern of the H19 gene

    DEFF Research Database (Denmark)

    Esteves, Leda I C V; Javaroni, Afonso C; Nishimoto, Inês N

    2005-01-01

    transcription factor whose binding to the H19-DMR is suppressed by DNA methylation. Our study included a group of 41 head and neck squamous cell carcinoma (HNSCC) samples. The imprinting status of the H19 gene was analyzed in 11 out of 35 positive cases for H19 gene expression, and only 1 of them showed loss......Loss of allele-specific expression by the imprinted genes IGF2 and H19 has been correlated with a differentially methylated region (DMR) upstream to the H19 gene. The H19-DMR contains seven potential CCCTC-binding factor (CTCF) binding sites. CTCF is a chromatin insulator and a multifunctional......2 and only 1 presented regional recurrence. These data support the hypothesis that H19 expression could be used as a prognostic marker to indicate recurrence in early stage tumors. We also examined the methylation of the CTCF binding site 1 in a subgroup of these samples. The H19 gene silencing...

  19. Savannah River Site`s Site Specific Plan. Environmental restoration and waste management, fiscal year 1992

    Energy Technology Data Exchange (ETDEWEB)

    1991-08-01

    This Site Specific Plan (SSP) has been prepared by the Savannah River Site (SRS) in order to show the Environmental Restoration and Waste Management activities that were identified during the preparation of the Department of Energy-Headquarters (DOE-HQ) Environmental Restoration and Waste Management Five-Year Plan (FYP) for FY 1992--1996. The SSP has been prepared in accordance with guidance received from DOE-HQ. DOE-SR is accountable to DOE-HQ for the implementation of this plan. The purpose of the SSP is to develop a baseline for policy, budget, and schedules for the DOE Environmental Restoration and Waste Management activities. The plan explains accomplishments since the Fiscal Year (FY) 1990 plan, demonstrates how present and future activities are prioritized, identifies currently funded activities and activities that are planned to be funded in the upcoming fiscal year, and describes future activities that SRS is considering.

  20. rVISTA for Comparative Sequence-Based Discovery of Functional Transcription Factor Binding Sites

    Energy Technology Data Exchange (ETDEWEB)

    Loots, Gabriela G.; Ovcharenko, Ivan; Pachter, Lior; Dubchak, Inna; Rubin, Edward M.

    2002-03-08

    Identifying transcriptional regulatory elements represents a significant challenge in annotating the genomes of higher vertebrates. We have developed a computational tool, rVISTA, for high-throughput discovery of cis-regulatory elements that combines transcription factor binding site prediction and the analysis of inter-species sequence conservation. Here, we illustrate the ability of rVISTA to identify true transcription factor binding sites through the analysis of AP-1 and NFAT binding sites in the 1 Mb well-annotated cytokine gene cluster1 (Hs5q31; Mm11). The exploitation of orthologous human-mouse data set resulted in the elimination of 95 percent of the 38,000 binding sites predicted upon analysis of the human sequence alone, while it identified 87 percent of the experimentally verified binding sites in this region.

  1. Biochemical and structural definition of the l-afadin- and actin-binding sites of alpha-catenin.

    Science.gov (United States)

    Pokutta, Sabine; Drees, Frauke; Takai, Yoshimi; Nelson, W James; Weis, William I

    2002-05-24

    alpha-Catenin is an integral component of adherens junctions, where it links cadherins to the actin cytoskeleton. alpha-Catenin is also required for the colocalization of the nectin/afadin/ponsin adhesion system to adherens junctions, and it specifically associates with the nectin-binding protein afadin. A proteolytic fragment of alpha-catenin, residues 385-651, contains the afadin-binding site. The three-dimensional structure of this fragment comprises two side-by-side four-helix bundles, both of which are required for afadin binding. The alpha-catenin fragment 385-651 binds afadin more strongly than the full-length protein, suggesting that the full-length protein harbors a cryptic binding site for afadin. Comparison of the alpha-catenin 385-651 structure with the recently solved structure of the alpha-catenin M-fragment (Yang, J., Dokurno, P., Tonks, N. K., and Barford, D. (2001) EMBO J. 20, 3645-3656) reveals a surprising flexibility in the orientation of the two four-helix bundles. alpha-Catenin and the actin-binding protein vinculin share sequence and most likely structural similarity within their actin-binding domains. Despite this homology, actin binding requires additional sequences adjacent to this region.

  2. Biochemical and Structural Definition of the l-Afadin- and Actin-binding Sites of α-Catenin*

    Science.gov (United States)

    Pokutta, Sabine; Drees, Frauke; Takai, Yoshimi; Nelson, W. James; Weis, William I.

    2012-01-01

    α -Catenin is an integral component of adherens junctions, where it links cadherins to the actin cytoskeleton. α-Catenin is also required for the colocalization of the nectin/afadin/ponsin adhesion system to adherens junctions, and it specifically associates with the nectin-binding protein afadin. A proteolytic fragment of α-catenin, residues 385–651, contains the afadin-binding site. The three-dimensional structure of this fragment comprises two side-by-side four-helix bundles, both of which are required for afadin binding. The α-catenin fragment 385–651 binds afadin more strongly than the full-length protein, suggesting that the full-length protein harbors a cryptic binding site for afadin. Comparison of the α-catenin 385–651 structure with the recently solved structure of the α-catenin M-fragment (Yang, J., Dokurno, P., Tonks, N. K., and Barford, D. (2001) EMBO J. 20, 3645–3656) reveals a surprising flexibility in the orientation of the two four-helix bundles. α-Catenin and the actin-binding protein vinculin share sequence and most likely structural similarity within their actin-binding domains. Despite this homology, actin binding requires additional sequences adjacent to this region. PMID:11907041

  3. Mapping the ADF/cofilin binding site on monomeric actin by competitive cross-linking and peptide array: evidence for a second binding site on monomeric actin.

    Science.gov (United States)

    Mannherz, Hans Georg; Ballweber, Edda; Galla, Marco; Villard, Sylvie; Granier, Claude; Steegborn, Clemens; Schmidtmann, Anja; Jaquet, Kornelia; Pope, Brian; Weeds, Alan G

    2007-02-23

    The binding sites for actin depolymerising factor (ADF) and cofilin on G-actin have been mapped by competitive chemical cross-linking using deoxyribonuclease I (DNase I), gelsolin segment 1 (G1), thymosin beta4 (Tbeta4), and vitamin D-binding protein (DbP). To reduce ADF/cofilin induced actin oligomerisation we used ADP-ribosylated actin. Both vitamin D-binding protein and thymosin beta4 inhibit binding by ADF or cofilin, while cofilin or ADF and DNase I bind simultaneously. Competition was observed between ADF or cofilin and G1, supporting the hypothesis that cofilin preferentially binds in the cleft between sub-domains 1 and 3, similar to or overlapping the binding site of G1. Because the affinity of G1 is much higher than that of ADF or cofilin, even at a 20-fold excess of the latter, the complexes contained predominantly G1. Nevertheless, cross-linking studies using actin:G1 complexes and ADF or cofilin showed the presence of low concentrations of ternary complexes containing both ADF or cofilin and G1. Thus, even with monomeric actin, it is shown for the first time that binding sites for both G1 and ADF or cofilin can be occupied simultaneously, confirming the existence of two separate binding sites. Employing a peptide array with overlapping sequences of actin overlaid by cofilin, we have identified five sequence stretches of actin able to bind cofilin. These sequences are located within the regions of F-actin predicted to bind cofilin in the model derived from image reconstructions of electron microscopical images of cofilin-decorated filaments. Three of the peptides map to the cleft region between sub-domains 1 and 3 of the upper actin along the two-start long-pitch helix, while the other two are in the DNase I loop corresponding to the site of the lower actin in the helix. In the absence of any crystal structures of ADF or cofilin in complex with actin, these studies provide further information about the binding sites on F-actin for these important actin

  4. Identification of Host Insulin Binding Sites on Schistosoma japonicum Insulin Receptors.

    Directory of Open Access Journals (Sweden)

    Rachel J Stephenson

    Full Text Available Schistosoma japonicum insulin receptors (SjIRs have been identified as encouraging vaccine candidates. Interrupting or blocking the binding between host insulin and the schistosome insulin receptors (IRs may result in reduced glucose uptake leading to starvation and stunting of worms with a reduction in egg output. To further understand how schistosomes are able to exploit host insulin for development and growth, and whether these parasites and their mammalian hosts compete for the same insulin source, we identified insulin binding sites on the SjIRs. Based on sequence analysis and the predicted antigenic structure of the primary sequences of the SjIRs, we designed nine and eleven peptide analogues from SjIR-1 and SjIR-2, respectively. Using the Octet RED system, we identified analogues derived from SjIR-1 (10 and SjIR-2 (20, 21 and 22 with insulin-binding sequences specific for S. japonicum. Nevertheless, the human insulin receptor (HIR may compete with the SjIRs in binding human insulin in other positions which are important for HIR binding to insulin. However, no binding occurred between insulin and parasite analogues derived from SjIR-1 (2, 7 and 8 and SjIR-2 (14, 16 and 18 at the same locations as HIR sequences which have been shown to have strong insulin binding affinities. Importantly, we found two analogues (1 and 3, derived from SjIR-1, and two analogues (13 and 15 derived from SjIR-2, were responsible for the major insulin binding affinity in S. japonicum. These peptide analogues were shown to have more than 10 times (in KD value stronger binding capacity for human insulin compared with peptides derived from the HIR in the same sequence positions. Paradoxically, analogues 1, 3, 13 and 15 do not appear to contain major antigenic determinants which resulted in poor antibody responses to native S. japonicum protein. This argues against their future development as peptide-vaccine candidates.

  5. Stiochiometry of maltodextrin-binding sites in LamB, an outer membrane protein from Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Gehring, K.; Cheng, Chaohsiung; Jap, B.K. (Lawrence Berkeley Lab., CA (United States)); Nikaido, H. (Univ. of California, Berkeley (United States))

    1991-03-01

    The authors have directly measured the stoichiometry of maltodextrin-binding sites in LamB. Scatchard plots and computer fitting of flow dialysis (rate-of-dialysis) experiments clearly establish three independent binding sites per LamB trimer, with a dissociation constant of approximately 60 {mu}M for maltoheptaose. The current model for LamB's function as a specific pore is discussed with respect to the symmetry in LamB's kinetic properties and the implications of their results.

  6. Site-specific attachment to recombinant antibodies via introduced surface cysteine residues.

    Science.gov (United States)

    Lyons, A; King, D J; Owens, R J; Yarranton, G T; Millican, A; Whittle, N R; Adair, J R

    1990-08-01

    Many diagnostic and therapeutic applications of monoclonal antibodies require the covalent linking of effector or reporter molecules to the immunoglobulin polypeptides. Existing methods generally involve the non-selective modification of amino acid side chains, producing one or more randomly distributed attachment sites. This results in heterogeneous labelling of the antibody molecules and often to a decrease in antigen-binding due to the modification of residues close to the antigen-binding site. We report a novel strategy for site-specifically labelling antibodies through surface cysteine residues. Examination of molecular structures was used to identify amino acids of the CH1 domain of the IgG heavy chain that were accessible to solvent but not to larger molecules. Site-directed mutagenesis was used to substitute cysteine residues at these positions in the heavy chain of a mouse/human chimaeric version of the tumour-binding monoclonal antibody, B72.3. Expression of the modified antibody genes in mammalian cells yielded correctly assembled proteins that had thiol groups in pre-determined positions and showed no loss of antigen-binding activity. One of the mutants was used to demonstrate the site-specific attachment of a radio-iodinated ligand to the chimaeric B72.3 antibody.

  7. Crystal structure of vinculin in complex with vinculin binding site 50 (VBS50), the integrin binding site 2 (IBS2) of talin

    Energy Technology Data Exchange (ETDEWEB)

    Yogesha, S.D.; Rangarajan, Erumbi S.; Vonrhein, Clemens; Bricogne, Gerard; Izard, Tina (Globel Phasing); (Scripps)

    2012-05-10

    The cytoskeletal protein talin activates integrin receptors by binding of its FERM domain to the cytoplasmic tail of {beta}-integrin. Talin also couples integrins to the actin cytoskeleton, largely by binding to and activating the cytoskeletal protein vinculin, which binds to F-actin through the agency of its five-helix bundle tail (Vt) domain. Talin activates vinculin by means of buried amphipathic {alpha}-helices coined vinculin binding sites (VBSs) that reside within numerous four- and five-helix bundle domains that comprise the central talin rod, which are released from their buried locales by means of mechanical tension on the integrin:talin complex. In turn, these VBSs bind to the N-terminal seven-helix bundle (Vh1) domain of vinculin, creating an entirely new helix bundle that severs its head-tail interactions. Interestingly, talin harbors a second integrin binding site coined IBS2 that consists of two five-helix bundle domains that also contain a VBS (VBS50). Here we report the crystal structure of VBS50 in complex with vinculin at 2.3 {angstrom} resolution and show that intramolecular interactions of VBS50 within IBS2 are much more extensive versus its interactions with vinculin. Indeed, the IBS2-vinculin interaction only occurs at physiological temperature and the affinity of VBS50 for vinculin is about 30 times less than other VBSs. The data support a model where integrin binding destabilizes IBS2 to allow it to bind to vinculin.

  8. HIM-8 binds to the X chromosome pairing center and mediateschromosome-specific meiotic synapsis

    Energy Technology Data Exchange (ETDEWEB)

    Phillips, Carolyn M.; Wong, Chihunt; Bhalla, Needhi; Carlton,Peter M.; Weiser, Pinky; Meneely, Philip M.; Dernburg, Abby F.

    2005-06-05

    The him-8 gene is essential for proper meiotic segregationof the X chromosomes in C. elegans. Herewe show that loss of him-8function causes profound X-chromosome-specific defects in homolog pairingand synapsis.him-8 encodes a C2H2 zinc finger protein that is expressedduring meiosis andconcentrates at a site on the X chromosome known as themeiotic Pairing Center (PC). A role for HIM-8 in PC function is supportedby genetic interactions between PC lesions and him-8 mutations.HIM-8-bound chromosome sites associate with the nuclear envelope (NE)throughout meiotic prophase. Surprisingly, a point mutation in him-8 thatretains both chromosome binding and NE localization fails to stabilizepairing or promote synapsis. These observations indicate thatstabilization of homolog pairing is an active process in which thetethering of chromosome sites to the NE may be necessary but is notsufficient.

  9. 77 FR 22772 - Environmental Management Site-Specific Advisory Board

    Science.gov (United States)

    2012-04-17

    ... Environmental Management Site-Specific Advisory Board AGENCY: Office of Environmental Management, Department of... Administration, notice is hereby given that the Environmental Management Site-Specific Advisory Board (EM SSAB... recommendations to the Assistant Secretary for Environmental Management (EM) concerning issues affecting the EM...

  10. Response Element Composition Governs Correlations between Binding Site Affinity and Transcription in Glucocorticoid Receptor Feed-forward Loops*

    Science.gov (United States)

    Sasse, Sarah K.; Zuo, Zheng; Kadiyala, Vineela; Zhang, Liyang; Pufall, Miles A.; Jain, Mukesh K.; Phang, Tzu L.; Stormo, Gary D.; Gerber, Anthony N.

    2015-01-01

    Combinatorial gene regulation through feed-forward loops (FFLs) can bestow specificity and temporal control to client gene expression; however, characteristics of binding sites that mediate these effects are not established. We previously showed that the glucocorticoid receptor (GR) and KLF15 form coherent FFLs that cooperatively induce targets such as the amino acid-metabolizing enzymes AASS and PRODH and incoherent FFLs exemplified by repression of MT2A by KLF15. Here, we demonstrate that GR and KLF15 physically interact and identify low affinity GR binding sites within glucocorticoid response elements (GREs) for PRODH and AASS that contribute to combinatorial regulation with KLF15. We used deep sequencing and electrophoretic mobility shift assays to derive in vitro GR binding affinities across sequence space. We applied these data to show that AASS GRE activity correlated (r2 = 0.73) with predicted GR binding affinities across a 50-fold affinity range in transfection assays; however, the slope of the linear relationship more than doubled when KLF15 was expressed. Whereas activity of the MT2A GRE was even more strongly (r2 = 0.89) correlated with GR binding site affinity, the slope of the linear relationship was sharply reduced by KLF15, consistent with incoherent FFL logic. Thus, GRE architecture and co-regulator expression together determine the functional parameters that relate GR binding site affinity to hormone-induced transcriptional responses. Utilization of specific affinity response functions and GR binding sites by FFLs may contribute to the diversity of gene expression patterns within GR-regulated transcriptomes. PMID:26088140

  11. Response Element Composition Governs Correlations between Binding Site Affinity and Transcription in Glucocorticoid Receptor Feed-forward Loops.

    Science.gov (United States)

    Sasse, Sarah K; Zuo, Zheng; Kadiyala, Vineela; Zhang, Liyang; Pufall, Miles A; Jain, Mukesh K; Phang, Tzu L; Stormo, Gary D; Gerber, Anthony N

    2015-08-07

    Combinatorial gene regulation through feed-forward loops (FFLs) can bestow specificity and temporal control to client gene expression; however, characteristics of binding sites that mediate these effects are not established. We previously showed that the glucocorticoid receptor (GR) and KLF15 form coherent FFLs that cooperatively induce targets such as the amino acid-metabolizing enzymes AASS and PRODH and incoherent FFLs exemplified by repression of MT2A by KLF15. Here, we demonstrate that GR and KLF15 physically interact and identify low affinity GR binding sites within glucocorticoid response elements (GREs) for PRODH and AASS that contribute to combinatorial regulation with KLF15. We used deep sequencing and electrophoretic mobility shift assays to derive in vitro GR binding affinities across sequence space. We applied these data to show that AASS GRE activity correlated (r(2) = 0.73) with predicted GR binding affinities across a 50-fold affinity range in transfection assays; however, the slope of the linear relationship more than doubled when KLF15 was expressed. Whereas activity of the MT2A GRE was even more strongly (r(2) = 0.89) correlated with GR binding site affinity, the slope of the linear relationship was sharply reduced by KLF15, consistent with incoherent FFL logic. Thus, GRE architecture and co-regulator expression together determine the functional parameters that relate GR binding site affinity to hormone-induced transcriptional responses. Utilization of specific affinity response functions and GR binding sites by FFLs may contribute to the diversity of gene expression patterns within GR-regulated transcriptomes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Electrical detection of specific versus non-specific binding events in breast cancer cells

    Science.gov (United States)

    King, Benjamin C.; Clark, Michael; Burkhead, Thomas; Sethu, Palaniappan; Rai, Shesh; Kloecker, Goetz; Panchapakesan, Balaji

    2012-10-01

    Detection of circulating tumor cells (CTCs) from patient blood samples offers a desirable alternative to invasive tissue biopsies for screening of malignant carcinomas. A rigorous CTC detection method must identify CTCs from millions of other formed elements in blood and distinguish them from healthy tissue cells also present in the blood. CTCs are known to overexpress surface receptors, many of which aid them in invading other tissue, and these provide an avenue for their detection. We have developed carbon nanotube (CNT) thin film devices to specifically detect these receptors in intact cells. The CNT sidewalls are functionalized with antibodies specific to Epithelial Cell Adhesion Molecule (EpCAM), a marker overexpressed by breast and other carcinomas. Specific binding of EpCAM to anti-EpCAM antibodies causes a change in the local charge environment of the CNT surface which produces a characteristic electrical signal. Two cell lines were tested in the device: MCF7, a mammary adenocarcinoma line which overexpresses EpCAM, and MCF10A, a non-tumorigenic mammary epithelial line which does not. Introduction of MCF7s caused significant changes in the electrical conductance of the devices due to specific binding and associated charge environment change near the CNT sidewalls. Introduction of MCF10A displays a different profile due to purely nonspecific interactions. The profile of specific vs. nonspecific interaction signatures using carbon based devices will guide development of this diagnostic tool towards clinical sample volumes with wide variety of markers.

  13. Structural basis of carbohydrate recognition by lectin II from Ulex europaeus, a protein with a promiscuous carbohydrate-binding site.

    Science.gov (United States)

    Loris, R; De Greve, H; Dao-Thi, M H; Messens, J; Imberty, A; Wyns, L

    2000-08-25

    Protein-carbohydrate interactions are the language of choice for inter- cellular communication. The legume lectins form a large family of homologous proteins that exhibit a wide variety of carbohydrate specificities. The legume lectin family is therefore highly suitable as a model system to study the structural principles of protein-carbohydrate recognition. Until now, structural data are only available for two specificity families: Man/Glc and Gal/GalNAc. No structural data are available for any of the fucose or chitobiose specific lectins. The crystal structure of Ulex europaeus (UEA-II) is the first of a legume lectin belonging to the chitobiose specificity group. The complexes with N-acetylglucosamine, galactose and fucosylgalactose show a promiscuous primary binding site capable of accommodating both N-acetylglucos amine or galactose in the primary binding site. The hydrogen bonding network in these complexes can be considered suboptimal, in agreement with the low affinities of these sugars. In the complexes with chitobiose, lactose and fucosyllactose this suboptimal hydrogen bonding network is compensated by extensive hydrophobic interactions in a Glc/GlcNAc binding subsite. UEA-II thus forms the first example of a legume lectin with a promiscuous binding site and illustrates the importance of hydrophobic interactions in protein-carbohydrate complexes. Together with other known legume lectin crystal structures, it shows how different specificities can be grafted upon a conserved structural framework. Copyright 2000 Academic Press.

  14. GenProBiS: web server for mapping of sequence variants to protein binding sites.

    Science.gov (United States)

    Konc, Janez; Skrlj, Blaz; Erzen, Nika; Kunej, Tanja; Janezic, Dusanka

    2017-05-11

    Discovery of potentially deleterious sequence variants is important and has wide implications for research and generation of new hypotheses in human and veterinary medicine, and drug discovery. The GenProBiS web server maps sequence variants to protein structures from the Protein Data Bank (PDB), and further to protein-protein, protein-nucleic acid, protein-compound, and protein-metal ion binding sites. The concept of a protein-compound binding site is understood in the broadest sense, which includes glycosylation and other post-translational modification sites. Binding sites were defined by local structural comparisons of whole protein structures using the Protein Binding Sites (ProBiS) algorithm and transposition of ligands from the similar binding sites found to the query protein using the ProBiS-ligands approach with new improvements introduced in GenProBiS. Binding site surfaces were generated as three-dimensional grids encompassing the space occupied by predicted ligands. The server allows intuitive visual exploration of comprehensively mapped variants, such as human somatic mis-sense mutations related to cancer and non-synonymous single nucleotide polymorphisms from 21 species, within the predicted binding sites regions for about 80 000 PDB protein structures using fast WebGL graphics. The GenProBiS web server is open and free to all users at http://genprobis.insilab.org. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. Quantitative analysis of pheromone-binding protein specificity.

    Science.gov (United States)

    Katti, S; Lokhande, N; González, D; Cassill, A; Renthal, R

    2013-02-01

    Many pheromones have very low water solubility, posing experimental difficulties for quantitative binding measurements. A new method is presented for determining thermodynamically valid dissociation constants for ligands binding to pheromone-binding proteins, using β-cyclodextrin as a solubilizer and transfer agent. The method is applied to LUSH, a Drosophila odorant-binding protein that binds the pheromone 11-cis vaccenyl acetate (cVA). Refolding of LUSH expressed in Escherichia coli was assessed by measuring N-phenyl-1-naphthylamine (NPN) binding and Förster resonance energy transfer between LUSH tryptophan 123 (W123) and NPN. Binding of cVA was measured from quenching of W123 fluorescence as a function of cVA concentration. The equilibrium constant for transfer of cVA between β-cyclodextrin and LUSH was determined from a linked equilibria model. This constant, multiplied by the β-cyclodextrin-cVA dissociation constant, gives the LUSH-cVA dissociation constant: ∼100 nM. It was also found that other ligands quench W123 fluorescence. The LUSH-ligand dissociation constants were determined to be ∼200 nM for the silk moth pheromone bombykol and ∼90 nM for methyl oleate. The results indicate that the ligand-binding cavity of LUSH can accommodate a variety ligands with strong binding interactions. Implications of this for the Laughlin, Ha, Jones and Smith model of pheromone reception are discussed. © 2012 Royal Entomological Society.

  16. Automatic generation of 3D motifs for classification of protein binding sites

    Directory of Open Access Journals (Sweden)

    Herzyk Pawel

    2007-08-01

    Full Text Available Abstract Background Since many of the new protein structures delivered by high-throughput processes do not have any known function, there is a need for structure-based prediction of protein function. Protein 3D structures can be clustered according to their fold or secondary structures to produce classes of some functional significance. A recent alternative has been to detect specific 3D motifs which are often associated to active sites. Unfortunately, there are very few known 3D motifs, which are usually the result of a manual process, compared to the number of sequential motifs already known. In this paper, we report a method to automatically generate 3D motifs of protein structure binding sites based on consensus atom positions and evaluate it on a set of adenine based ligands. Results Our new approach was validated by generating automatically 3D patterns for the main adenine based ligands, i.e. AMP, ADP and ATP. Out of the 18 detected patterns, only one, the ADP4 pattern, is not associated with well defined structural patterns. Moreover, most of the patterns could be classified as binding site 3D motifs. Literature research revealed that the ADP4 pattern actually corresponds to structural features which show complex evolutionary links between ligases and transferases. Therefore, all of the generated patterns prove to be meaningful. Each pattern was used to query all PDB proteins which bind either purine based or guanine based ligands, in order to evaluate the classification and annotation properties of the pattern. Overall, our 3D patterns matched 31% of proteins with adenine based ligands and 95.5% of them were classified correctly. Conclusion A new metric has been introduced allowing the classification of proteins according to the similarity of atomic environment of binding sites, and a methodology has been developed to automatically produce 3D patterns from that classification. A study of proteins binding adenine based ligands showed that

  17. Site-SpecificCu Labeling of the Serine Protease, Active Site Inhibited Factor Seven Azide (FVIIai-N), Using Copper Free Click Chemistry

    DEFF Research Database (Denmark)

    Jeppesen, Troels E; Kristensen, Lotte K; Nielsen, Carsten H

    2018-01-01

    A method for site-specific radiolabeling of the serine protease active site inhibited factor seven (FVIIai) with64Cu has been applied using a biorthogonal click reaction. FVIIai binds to tissue factor (TF), a trans-membrane protein involved in hemostasis, angiogenesis, proliferation, cell migration...

  18. Progesterone for premenstrual syndrome

    NARCIS (Netherlands)

    Ford, Olive; Lethaby, Anne; Roberts, Helen; Mol, Ben Willem J.

    2009-01-01

    BACKGROUND: About 5% of women experience severe symptoms called premenstrual syndrome (PMS), only in the two weeks before their menstrual periods. Treatment with progesterone may restore a deficiency, balance menstrual hormone levels or reduce effects of falling progesterone levels on the brain or

  19. Mapping the heparin-binding site of the osteoinductive protein NELL1 by site-directed mutagenesis.

    Science.gov (United States)

    Takahashi, Kaneyoshi; Imai, Arisa; Iijima, Masumi; Yoshimoto, Nobuo; Maturana, Andrés D; Kuroda, Shun'ichi; Niimi, Tomoaki

    2015-12-21

    Neural epidermal growth factor-like (NEL)-like 1 (NELL1) is a secretory osteogenic protein comprising an N-terminal thrombospondin-1-like (TSPN) domain, four von Willebrand factor type C domains, and six epidermal growth factor-like repeats. NELL1 shows heparin-binding activity; however, the biological significance remains to be explored. In this report, we demonstrate that NELL1 binds to cell surface proteoglycans through its TSPN domain. Major heparin-binding sites were identified on the three-dimensional structural model of the TSPN domain of NELL1. Mutant analysis of the heparin-binding sites indicated that the heparin-binding activity of the TSPN domain is involved in interaction of NELL1 with cell surface proteoglycans. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  20. 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.

  1. Subfamily-specific adaptations in the structures of two penicillin-binding proteins from Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Daniil M Prigozhin

    Full Text Available Beta-lactam antibiotics target penicillin-binding proteins including several enzyme classes essential for bacterial cell-wall homeostasis. To better understand the functional and inhibitor-binding specificities of penicillin-binding proteins from the pathogen, Mycobacterium tuberculosis, we carried out structural and phylogenetic analysis of two predicted D,D-carboxypeptidases, Rv2911 and Rv3330. Optimization of Rv2911 for crystallization using directed evolution and the GFP folding reporter method yielded a soluble quadruple mutant. Structures of optimized Rv2911 bound to phenylmethylsulfonyl fluoride and Rv3330 bound to meropenem show that, in contrast to the nonspecific inhibitor, meropenem forms an extended interaction with the enzyme along a conserved surface. Phylogenetic analysis shows that Rv2911 and Rv3330 belong to different clades that emerged in Actinobacteria and are not represented in model organisms such as Escherichia coli and Bacillus subtilis. Clade-specific adaptations allow these enzymes to fulfill distinct physiological roles despite strict conservation of core catalytic residues. The characteristic differences include potential protein-protein interaction surfaces and specificity-determining residues surrounding the catalytic site. Overall, these structural insights lay the groundwork to develop improved beta-lactam therapeutics for tuberculosis.

  2. Site-Specific Chemical Labeling of Long RNA Molecules

    DEFF Research Database (Denmark)

    Jahn, Kasper; Olsen, Eva Maria; Nielsen, Morten Muhlig

    2011-01-01

    Site-specific labeling of RNA molecules is a valuable tool for studying their structure and function. Here, we describe a new site-specific RNA labeling method, which utilizes a DNA-templated chemical reaction to attach a label at a specific internal nucleotide in an RNA molecule. The method...... is nonenzymatic and based on the formation of a four-way junction, where a donor strand is chemically coupled to an acceptor strand at a specific position via an activated chemical group. A disulfide bond in the linker is subsequently cleaved under mild conditions leaving a thiol group attached to the acceptor......-RNA strand. The site-specific thiol-modified target RNA can then be chemically labeled with an optional group, here demonstrated by coupling of a maleimide-functionalized fluorophore. The method is rapid and allows site specific labeling of both in vitro and in vivo synthesized RNA with a broad range...

  3. Progesterone resistance in endometriosis

    DEFF Research Database (Denmark)

    Patel, Bansari G; Rudnicki, Martin; Yu, Jie

    2017-01-01

    Endometriosis is a common cause of pelvic pain and affects up to 10% of women of reproductive age. Aberrant progesterone signaling in the endometrium plays a significant role in impaired decidualization and establishment of ectopic endometrial implants. Eutopic endometrial cells from women...... renders infants susceptible to neonatal uterine bleeding and endometriosis. Progesterone action is crucial to decreasing inflammation in the endometrium, and deviant progesterone signaling results in a proinflammatory phenotype. Conversely, chronic inflammation can induce a progesterone resistant state...... and their targets. Environmental toxins, such as dioxin, play a possible role in the genesis of endometriosis by permitting an inflammatory milieu. A consequence of impaired progesterone action is that hormonal therapy is rendered ineffective for a subset of women with endometriosis. Synthetic progestins...

  4. Impact of Binding Site Comparisons on Medicinal Chemistry and Rational Molecular Design.

    Science.gov (United States)

    Ehrt, Christiane; Brinkjost, Tobias; Koch, Oliver

    2016-05-12

    Modern rational drug design not only deals with the search for ligands binding to interesting and promising validated targets but also aims to identify the function and ligands of yet uncharacterized proteins having impact on different diseases. Additionally, it contributes to the design of inhibitors with distinct selectivity patterns and the prediction of possible off-target effects. The identification of similarities between binding sites of various proteins is a useful approach to cope with those challenges. The main scope of this perspective is to describe applications of different protein binding site comparison approaches to outline their applicability and impact on molecular design. The article deals with various substantial application domains and provides some outstanding examples to show how various binding site comparison methods can be applied to promote in silico drug design workflows. In addition, we will also briefly introduce the fundamental principles of different protein binding site comparison methods.

  5. 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. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Oxytocin receptor binding sites in the periphery of the neonatal mouse.

    Directory of Open Access Journals (Sweden)

    Maria A Greenwood

    Full Text Available Oxytocin (OXT is a pleiotropic regulator of physiology and behavior. An emerging body of evidence demonstrates a role for OXT in the transition to postnatal life of the infant. To identify potential sites of OXT action via the OXT receptor (OXTR in the newborn mouse, we performed receptor autoradiography on 20 μm sagittal sections of whole postnatal day 0 male and female mice on a C57BL/6J background using the 125iodinated ornithine vasotocin analog ([125I]-OVTA radioligand. A competitive binding assay on both wild-type (WT and OXTR knockout (OXTR KO tissue was used to assess the selectivity of [125I]-OVTA for neonatal OXTR. Radioactive ligand (0.05 nM [125I]-OVTA was competed against concentrations of 0 nM, 10 nM, and 1000 nM excess unlabeled OXT. Autoradiographs demonstrated the high selectivity of the radioligand for infant peripheral OXTR. Specific ligand binding activity for OXTR was observed in the oronasal cavity, the eye, whisker pads, adrenal gland, and anogenital region in the neonatal OXTR WT mouse, but was absent in neonatal OXTR KO. Nonspecific binding was observed in areas with a high lipid content such as the scapular brown adipose tissue and the liver: in these regions, binding was present in both OXTR WT and KO mice, and could not be competed away with OXT in either WT or KO mice. Collectively, these data confirm novel OXT targets in the periphery of the neonate. These peripheral OXTR sites, coupled with the immaturity of the neonate's own OXT system, suggest a role for exogenous OXT in modulating peripheral physiology and development.

  7. In Silico Investigation of the Neurotensin Receptor 1 Binding Site

    DEFF Research Database (Denmark)

    Lückmann, Michael; Holst, Birgitte; Schwartz, Thue W.

    2016-01-01

    structure of NTSR1 in complex with NTS8-13 has been detd., providing novel insights into peptide ligand recognition by 7TM receptors. SR48692, a potent and selective small mol. antagonist has previously been used extensively as a tool compd. to study NTSR1 receptor signaling properties. To investigate...... the binding mode of SR48692 and other small mol. compds. to NTSR1, we applied an Automated Ligand-guided Backbone Ensemble Receptor Optimization protocol (ALiBERO), taking receptor flexibility and ligand knowledge into account. Structurally overlapping binding poses for SR48692 and NTS8-13 were obsd., despite...

  8. Thermodynamic compensation upon binding to exosite 1 and the active site of thrombin.

    Science.gov (United States)

    Treuheit, Nicholas A; Beach, Muneera A; Komives, Elizabeth A

    2011-05-31

    Several lines of experimental evidence including amide exchange and NMR suggest that ligands binding to thrombin cause reduced backbone dynamics. Binding of the covalent inhibitor dPhe-Pro-Arg chloromethyl ketone to the active site serine, as well as noncovalent binding of a fragment of the regulatory protein, thrombomodulin, to exosite 1 on the back side of the thrombin molecule both cause reduced dynamics. However, the reduced dynamics do not appear to be accompanied by significant conformational changes. In addition, binding of ligands to the active site does not change the affinity of thrombomodulin fragments binding to exosite 1; however, the thermodynamic coupling between exosite 1 and the active site has not been fully explored. We present isothermal titration calorimetry experiments that probe changes in enthalpy and entropy upon formation of binary ligand complexes. The approach relies on stringent thrombin preparation methods and on the use of dansyl-l-arginine-(3-methyl-1,5-pantanediyl)amide and a DNA aptamer as ligands with ideal thermodynamic signatures for binding to the active site and to exosite 1. Using this approach, the binding thermodynamic signatures of each ligand alone as well as the binding signatures of each ligand when the other binding site was occupied were measured. Different exosite 1 ligands with widely varied thermodynamic signatures cause a similar reduction in ΔH and a concomitantly lower entropy cost upon DAPA binding at the active site. The results suggest a general phenomenon of enthalpy-entropy compensation consistent with reduction of dynamics/increased folding of thrombin upon ligand binding to either the active site or exosite 1.

  9. Differential modulation by cations of sigma and phencyclidine binding sites in rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Schwarz, S.; Zhou, G.Z.; Katki, A.G.; Rodbard, D. (National Institute of Child Health and Human Development, Bethesda, MD (USA))

    1990-01-01

    The present investigation attempted to differentiate haloperidol-sensitive sigma sites (sigma H) from phencyclidine (PCP) binding sites in rat brain membranes. We studied the effects of several cations at physiologically relevant concentrations on the binding of radioligands selective for sigma H sites ((3H)haloperidol, (3H)(+)3-PPP**), and (3H)(+)SKF10,047, or for PCP sites ((3H)PCP and (3H)TCP). The PCP sites displayed a markedly greater sensitivity to cations than sigma H sites. This property was reflected by a greater extent of inhibition of the binding of PCP-selective relative to sigma H-selective ligands at a given cation concentration, as well as by lower IC50's and by steeper slopes of the cation dose-response curves. Divalent cations were approximately 100 times more potent than monovalent cations. All cations were inhibitory, except Sr2+ and Ba2+ which, at micromolar concentrations, enhanced PCP binding but not sigma H binding. Thus, PCP-selective sites appeared to be distinct from sigma H sites with regards to several aspects of cation modulation. This is consistent with the view that PCP and sigma H sites are distinct molecular entities. Further, the marked cation sensitivity of the PCP site is consistent with the current hypothesis according to which the PCP site is linked to the N-methyl-D-aspartate (NMDA) receptor-cation channel complex.

  10. Impact of Alu repeats on the evolution of human p53 binding sites

    Directory of Open Access Journals (Sweden)

    Sirotin Michael V

    2011-01-01

    Full Text Available Abstract Background The p53 tumor suppressor protein is involved in a complicated regulatory network, mediating expression of ~1000 human genes. Recent studies have shown that many p53 in vivo binding sites (BSs reside in transposable repeats. The relationship between these BSs and functional p53 response elements (REs remains unknown, however. We sought to understand whether the p53 REs also reside in transposable elements and particularly in the most-abundant Alu repeats. Results We have analyzed ~160 functional p53 REs identified so far and found that 24 of them occur in repeats. More than half of these repeat-associated REs reside in Alu elements. In addition, using a position weight matrix approach, we found ~400,000 potential p53 BSs in Alu elements genome-wide. Importantly, these putative BSs are located in the same regions of Alu repeats as the functional p53 REs - namely, in the vicinity of Boxes A/A' and B of the internal RNA polymerase III promoter. Earlier nucleosome-mapping experiments showed that the Boxes A/A' and B have a different chromatin environment, which is critical for the binding of p53 to DNA. Here, we compare the Alu-residing p53 sites with the corresponding Alu consensus sequences and conclude that the p53 sites likely evolved through two different mechanisms - the sites overlapping with the Boxes A/A' were generated by CG → TG mutations; the other sites apparently pre-existed in the progenitors of several Alu subfamilies, such as AluJo and AluSq. The binding affinity of p53 to the Alu-residing sites generally correlates with the age of Alu subfamilies, so that the strongest sites are embedded in the 'relatively young' Alu repeats. Conclusions The primate-specific Alu repeats play an important role in shaping the p53 regulatory network in the context of chromatin. One of the selective factors responsible for the frequent occurrence of Alu repeats in introns may be related to the p53-mediated regulation of Alu

  11. Photoaffinity labeling with cholesterol analogues precisely maps a cholesterol-binding site in voltage-dependent anion channel-1.

    Science.gov (United States)

    Budelier, Melissa M; Cheng, Wayland W L; Bergdoll, Lucie; Chen, Zi-Wei; Janetka, James W; Abramson, Jeff; Krishnan, Kathiresan; Mydock-McGrane, Laurel; Covey, Douglas F; Whitelegge, Julian P; Evers, Alex S

    2017-06-02

    Voltage-dependent anion channel-1 (VDAC1) is a highly regulated β-barrel membrane protein that mediates transport of ions and metabolites between the mitochondria and cytosol of the cell. VDAC1 co-purifies with cholesterol and is functionally regulated by cholesterol, among other endogenous lipids. Molecular modeling studies based on NMR observations have suggested five cholesterol-binding sites in VDAC1, but direct experimental evidence for these sites is lacking. Here, to determine the sites of cholesterol binding, we photolabeled purified mouse VDAC1 (mVDAC1) with photoactivatable cholesterol analogues and analyzed the photolabeled sites with both top-down mass spectrometry (MS), and bottom-up MS paired with a clickable, stable isotope-labeled tag, FLI -tag. Using cholesterol analogues with a diazirine in either the 7 position of the steroid ring (LKM38) or the aliphatic tail (KK174), we mapped a binding pocket in mVDAC1 localized to Thr 83 and Glu 73 , respectively. When Glu 73 was mutated to a glutamine, KK174 no longer photolabeled this residue, but instead labeled the nearby Tyr 62 within this same binding pocket. The combination of analytical strategies employed in this work permits detailed molecular mapping of a cholesterol-binding site in a protein, including an orientation of the sterol within the site. Our work raises the interesting possibility that cholesterol-mediated regulation of VDAC1 may be facilitated through a specific binding site at the functionally important Glu 73 residue. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Thermodynamics of sequence-specific binding of PNA to DNA

    DEFF Research Database (Denmark)

    Ratilainen, T; Holmén, A; Tuite, E

    2000-01-01

    For further characterization of the hybridization properties of peptide nucleic acids (PNAs), the thermodynamics of hybridization of mixed sequence PNA-DNA duplexes have been studied. We have characterized the binding of PNA to DNA in terms of binding affinity (perfectly matched duplexes) and seq......For further characterization of the hybridization properties of peptide nucleic acids (PNAs), the thermodynamics of hybridization of mixed sequence PNA-DNA duplexes have been studied. We have characterized the binding of PNA to DNA in terms of binding affinity (perfectly matched duplexes...... relative to that of the perfectly matched sequence with a corresponding free energy penalty of about 15 kJ mol(-1) bp(-1). The average cost of a single mismatch is therefore estimated to be on the order of or larger than the gain of two matched base pairs, resulting in an apparent binding constant of only...

  13. Mapping of SPARC/BM-40/Osteonectin-binding Sites on Fibrillar Collagens*S⃞

    OpenAIRE

    Giudici, Camilla; Raynal, Nicolas; Wiedemann, Hanna; Wayne A. Cabral; Marini, Joan C.; Timpl, Rupert; Bächinger, Hans Peter; Farndale, Richard W.; Sasaki, Takako; Tenni, Ruggero

    2008-01-01

    The 33-kDa matrix protein SPARC (BM-40, osteonectin) binds several collagen types with moderate affinity. The collagen-binding site resides in helix αA of the extracellular calcium-binding domain of SPARC and is partially masked by helix αC. Previously, we found that the removal of helix αC caused a 10-fold increase in the affinity of SPARC for collagen, and we identified amino acids crucial for binding by site-directed mutagenesis. In this study, we used rotary shadow...

  14. Characterization of saturable binding sites for circulating pancreatic polypeptide in rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Whitcomb, D.C.; Taylor, I.L.; Vigna, S.R. (Duke Univ. Medical Center, Durham, NC (USA))

    1990-10-01

    Pancreatic polypeptide (PP) inhibits pancreatic exocrine secretion by indirect mechanisms that may be centrally mediated. The central site of action of PP that results in inhibition of pancreatic secretion has not been identified. Using autoradiography to identify 125I-PP binding to frozen sections of rat brain, we have identified saturable, high-affinity PP receptors in high concentrations in the interpenduncular nucleus, area postrema (AP), nucleus tractus solitarius, and dorsal motor nucleus of the vagus. The PP receptor differs from neuropeptide Y and peptide YY receptors in its binding specificity and location. Because PP is not produced in the brain, and the blood-brain barrier (BBB) excludes circulating peptides from most areas in the brain, we employed an in vivo radioreceptor assay to determine whether circulating PP binds to areas such as the AP that has both an incomplete BBB and a high concentration of PP receptors. 125I-PP and 131I-bovine serum albumin were infused simultaneously into rats through a peripheral vein with or without excess unlabeled PP. After 10 min, rats were killed and the brains were removed and cut into eight regions based on the autoradiographic localization of PP receptors. There was a significant (P less than 0.02) increase in saturable radiolabeled PP accumulation in the region that included the AP, demonstrating that circulating PP can bind to this area of the brain in vivo. PP is released into the circulation after a meal via mechanisms that exhibit vagal and cholinergic dependence. We speculate that PP completes a feedback loop by binding to receptors in the AP and interacting with the adjacent vagal nuclei to inhibit vagal activity.

  15. SITE-DIRECTED MUTAGENESIS OF PROPOSED ACTIVE-SITE RESIDUES OF PENICILLIN-BINDING PROTEIN-5 FROM ESCHERICHIA-COLI

    NARCIS (Netherlands)

    VANDERLINDEN, MPG; DEHAAN, L; DIDEBERG, O; KECK, W

    1994-01-01

    Alignment of the amino acid sequence of penicillin-binding protein 5 (PBP5) with the sequences of other members of the family of active-site-serine penicillin-interacting enzymes predicted the residues playing a role in the catalytic mechanism of PBP5. Apart from the active-site (Ser(44)), Lys(47),

  16. PROGESTERONE EXERTS NEUROPROTECTIVE EFFECTS AFTER BRAIN INJURY

    OpenAIRE

    Stein, Donald G.

    2007-01-01

    Progesterone, although still widely considered primarily a sex hormone, is an important agent affecting many central nervous system functions. This review assesses recent, primarily in vivo, evidence that progesterone can play an important role in promoting and enhancing repair after traumatic brain injury and stroke. Although many of its specific actions on neuroplasticity remain to be discovered, there is growing evidence that this hormone may be a safe and effective treatment for traumatic...

  17. Progesterone exerts neuroprotective effects after brain injury.

    Science.gov (United States)

    Stein, Donald G

    2008-03-01

    Progesterone, although still widely considered primarily a sex hormone, is an important agent affecting many central nervous system functions. This review assesses recent, primarily in vivo, evidence that progesterone can play an important role in promoting and enhancing repair after traumatic brain injury and stroke. Although many of its specific actions on neuroplasticity remain to be discovered, there is growing evidence that this hormone may be a safe and effective treatment for traumatic brain injury and other neural disorders in humans.

  18. Binding affinity of a small molecule to an amorphous polymer in a solvent. Part 1: free energy of binding to a binding site.

    Science.gov (United States)

    Chunsrivirot, Surasak; Diao, Ying; Trout, Bernhardt L

    2011-10-18

    Crystallization is commonly used in a separation and purification process in the production of a wide range of materials in various industries. In industry, crystallization usually starts with heterogeneous nucleation on a foreign surface. The complicated mechanism of heterogeneous nucleation is not well understood; however, we hypothesize that there might be a possible correlation between binding affinity to a surface and enhancement of nucleation. Recent studies show that amorphous polymers can be used to control crystallization, selectively produce pharmaceutical polymorphs, and discover novel pharmaceutical polymorphs. To investigate the possible correlation between the binding affinity of one molecule to key binding sites (local binding) and heterogeneous nucleation activity as well as the possibility of using this binding affinity to help guide the selection of polymers that promote heterogeneous nucleation, we computed the free energy of binding of aspirin to four nonporous cross-linked polymers in an ethanol-water 38 v% mixture. These cross-linked polymers are poly(4-acryloylmorpholine) (PAM), poly(2-carboxyethyl acrylate) (PCEA), poly(4-hydroxylbutyl acrylate) (PHBA), and polystyrene (PS); all of them were cross-linked with divinylbenzene (DVB). These systems were used because their heterogeneous nucleation activities are available in literature, and the ranking is PAM > PCEA > PHBA ≈ PS. We generated three independent surfaces for each polymer and computed the free energy of binding of aspirin to the best binding site that we found on each surface. The average free energies of binding to the best sites of PAM, PCEA, PHBA, and PS are -20.4 ± 1.0, -16.7 ± 1.0, -14.4 ± 1.1, and -13.6 ± 1.1 kcal/mol, respectively. We found that the trend of the magnitudes of the average free energies of binding to the best sites is PAM > PCEA > PHBA ≈ PS. This trend is very similar to that of heterogeneous nucleation activity. Our results suggest the importance of the

  19. Ligand-binding sites in human serum amyloid P component

    DEFF Research Database (Denmark)

    Heegaard, N.H.H.; Heegaard, Peter M. H.; Roepstorff, P.

    1996-01-01

    Amyloid P component (AP) is a naturally occurring glycoprotein that is found in serum and basement membranes, AP is also a component of all types of amyloid, including that found in individuals who suffer from Alzheimer's disease and Down's syndrome. Because AP has been found to bind strongly...

  20. Structure of P-Glycoprotein Reveals a Molecular Basis for Poly-Specific Drug Binding

    Energy Technology Data Exchange (ETDEWEB)

    Aller, Stephen G.; Yu, Jodie; Ward, Andrew; Weng, Yue; Chittaboina, Srinivas; Zhuo, Rupeng; Harrell, Patina M.; Trinh, Yenphuong T.; Zhang, Qinghai; Urbatsch, Ina L.; Chang, Geoffrey; (Scripps); (TTU)

    2009-04-22

    P-glycoprotein (P-gp) detoxifies cells by exporting hundreds of chemically unrelated toxins but has been implicated in multidrug resistance (MDR) in the treatment of cancers. Substrate promiscuity is a hallmark of P-gp activity, thus a structural description of poly-specific drug-binding is important for the rational design of anticancer drugs and MDR inhibitors. The x-ray structure of apo P-gp at 3.8 angstroms reveals an internal cavity of -6000 angstroms cubed with a 30 angstrom separation of the two nucleotide-binding domains. Two additional P-gp structures with cyclic peptide inhibitors demonstrate distinct drug-binding sites in the internal cavity capable of stereoselectivity that is based on hydrophobic and aromatic interactions. Apo and drug-bound P-gp structures have portals open to the cytoplasm and the inner leaflet of the lipid bilayer for drug entry. The inward-facing conformation represents an initial stage of the transport cycle that is competent for drug binding.

  1. Role of the IE62 Consensus Binding Site in Transactivation by the Varicella-Zoster Virus IE62 Protein ▿

    Science.gov (United States)

    White, Kris; Peng, Hua; Hay, John; Ruyechan, William T.

    2010-01-01

    The varicella-zoster virus (VZV) IE62 protein is the major transcriptional activator. IE62 is capable of associating with DNA both nonspecifically and in a sequence-specific manner via a consensus binding site (5′-ATCGT-3′). However, the function of the consensus site is poorly understood, since IE62 efficiently transactivates promoter elements lacking this sequence. In the work presented here, sequence analysis of the VZV genome revealed the presence of 245 IE62 consensus sites throughout the genome. Some 54 sites were found to be present within putative VZV promoters. Electrophoretic mobility shift assay (EMSA) experiments using an IE62 fragment containing the IE62 DNA-binding domain and duplex oligonucleotides that did or did not contain the IE62 consensus binding sequence yielded KD (equilibrium dissociation constant) values in the nanomolar range. Further, the IE62 DNA binding domain was shown to have a 5-fold-increased affinity for its consensus site compared to nonconsensus sequences. The effect of consensus site presence and position on IE62-mediated activation of native VZV and model promoters was examined using site-specific mutagenesis and transfection and superinfection reporter assays. In all promoters examined, the consensus sequence functioned as a distance-dependent repressive element. Protein recruitment assays utilizing the VZV gI promoter indicated that the presence of the consensus site increased the recruitment of IE62 but not Sp1. These data suggest a model where the IE62 consensus site functions to down-modulate IE62 activation, and interaction of IE62 with this sequence may result in loss or decrease of the ability of IE62 to recruit cellular factors needed for full promoter activation. PMID:20130051

  2. Determination of the Clostridium perfringens-binding site on fibronectin.

    Science.gov (United States)

    Katayama, Seiichi; Tagomori, Mika; Morita, Naomi; Yamasaki, Tsutomu; Nariya, Hirofumi; Okada, Mariko; Watanabe, Mariko; Hitsumoto, Yasuo

    2015-08-01

    The extracellular matrix protein fibronectin (Fn) is known to bind to the surface of Clostridium perfringens cells. Fn is a disulfide-linked homodimer protein, with each Fn polypeptide consisting of three types of repeating modules: 12 type I, 2 type II, and 15-17 type III modules. To determine the epitope on Fn recognized by C. perfringens cells, anti-Fn monoclonal antibodies (mAbs) and various Fn fragments (III2-10, rIII2-4, rIII5-7, rIII8, rIII9, rIII10) were employed. Although two C. perfringens-derived Fn-binding proteins, FbpA and FbpB, have been reported, they appear not to be the bacterium's surface Fn receptor. Moreover, both FbpA and FbpB were found to bind to C. perfringens cells. To avoid confusion, a mutant C. perfringens lacking both the fbpA and fbpB genes (MW5) was prepared using an in-frame deletion system. MW5 cells bound Fn on their surface, suggesting the presence of a putative Fn receptor(s) on C. perfringens cells. Of several anti-Fn mAbs, both HB39 and MO inhibited the binding of Fn to MW5 cells. HB39 reacted strongly with III2-10 and rIII9, and weakly with rIII2-4, rIII10 and rIII5-7 in Western blotting analysis. Binding of HB39 to Fn was inhibited in the presence of either rIII9 or rIII10, but not in the presence of rIII2-4, rIII5-7, or rIII8. Binding of Fn to MW5 cells was strongly inhibited by both III2-10 and rIII9, marginally inhibited by rIII2-4, but not affected by rIII5-7, rIII8, or rIII10. Significant binding of MW5 cells to immobilized rIII9 and rIII10 as well as immobilized III2-10 was observed. The region of Fn recognized by C. perfringens was thus mapped to the region encompassed by III9 and III10. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    Rannversson, Hafsteinn; Andersen, Jacob; Sørensen, Lena; Bang-Andersen, Benny; Park, Minyoung; Huber, Thomas; Sakmar, Thomas P.; Strømgaard, Kristian

    2016-01-01

    Despite the well-established role of the human serotonin transporter (hSERT) in the treatment of depression, the molecular details of antidepressant drug binding are still not fully understood. Here we utilize amber codon suppression in a membrane-bound transporter protein to encode photocrosslinking unnatural amino acids (UAAs) into 75 different positions in hSERT. UAAs are incorporated with high specificity, and functionally active transporters have similar transport properties and pharmacological profiles compared with wild-type transporters. We employ ultraviolet-induced crosslinking with p-azido-L-phenylalanine (azF) at selected positions in hSERT to map the binding site of imipramine, a prototypical tricyclic antidepressant, and vortioxetine, a novel multimodal antidepressant. We find that the two antidepressants crosslink with azF incorporated at different positions within the central substrate-binding site of hSERT, while no crosslinking is observed at the vestibular-binding site. Taken together, our data provide direct evidence for defining the high-affinity antidepressant binding site in hSERT. PMID:27089947

  4. Quantitative autoradiographic determination of binding sites for a peripheral benzodiazepine ligand ((/sup 3/H)PK 11195) in human iris

    Energy Technology Data Exchange (ETDEWEB)

    Valtier, D.; Malgouris, C.; Uzan, A.

    1987-01-01

    Specific binding sites of peripheral-type benzodiazepines were investigated in human iris/ciliary body (8 eyes). Examination of color-coded prints and densitometric quantification of autoradiograms were performed on slides (20 ..mu..m) labelled with (/sup 3/H)PK 11195 (1 nM) at 25 deg C. Nonspecific binding was determined with PK 11211 (5 ..mu..M) or Ro 5-4864 (5 ..mu..M). Binding sites were present on all the slides, with equivalent density in the 3 regions of the preparation (ciliary body, iris and pupil margin). The numbers of binding sites in ciliary body, iris, and pupil margin, respectively were: 42.7 +- 0.2, 30.1 +- 0.5 and 37.4 +- 0.4 femtomol/mg protein. Labelling on the pupil margin seemed to coincide with the iris sphincter muscle. The presence of peripheral benzodiazepine binding sites in iris muscular tissue, and particularly in the pupil margin, suggests that the iris preparation may be a valuable tool to detect putative physiological effects of peripheral benzodiazepines on muscular motility.

  5. Nucleotide Binding Site Communication in Arabidopsis thaliana Adenosine 5;-Phosphosulfate Kinase

    Energy Technology Data Exchange (ETDEWEB)

    Ravilious, Geoffrey E.; Jez, Joseph M. (WU)

    2012-08-31

    Adenosine 5{prime}-phosphosulfate kinase (APSK) catalyzes the ATP-dependent synthesis of adenosine 3{prime}-phosphate 5{prime}-phosphosulfate (PAPS), which is an essential metabolite for sulfur assimilation in prokaryotes and eukaryotes. Using APSK from Arabidopsis thaliana, we examine the energetics of nucleotide binary and ternary complex formation and probe active site features that coordinate the order of ligand addition. Calorimetric analysis shows that binding can occur first at either nucleotide site, but that initial interaction at the ATP/ADP site was favored and enhanced affinity for APS in the second site by 50-fold. The thermodynamics of the two possible binding models (i.e. ATP first versus APS first) differs and implies that active site structural changes guide the order of nucleotide addition. The ligand binding analysis also supports an earlier suggestion of intermolecular interactions in the dimeric APSK structure. Crystallographic, site-directed mutagenesis, and energetic analyses of oxyanion recognition by the P-loop in the ATP/ADP binding site and the role of Asp136, which bridges the ATP/ADP and APS/PAPS binding sites, suggest how the ordered nucleotide binding sequence and structural changes are dynamically coordinated for catalysis.

  6. 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.

  7. Site-specific design optimization of wind turbines

    DEFF Research Database (Denmark)

    Fuglsang, P.; Bak, C.; Schepers, J.G.

    2002-01-01

    advantage of high-wind-speed sites. It was not possible to design a single wind turbine for all wind climates investigated, since the differences in the design loads were too large. Multiple-site wind turbines should be designed for generic wind conditions, which cover wind parameters encountered at flat...... terrain sites with a high mean wind speed. Site-specific wind turbines should be designed for low-mean-wind-speed sites and complex terrain. Copyright © 2002 John Wiley & Sons, Ltd....

  8. Characteristics of binding sites of intergenic, intronic and exonic ...

    African Journals Online (AJOL)

    user

    2013-03-06

    Mar 6, 2013 ... miRNAFinder 2.2 (https://sites.google.com/site/malaheenee/home) was used to find miRNA origins (intergenic, exonic or intronic). A literature review of genes coding intronic miRNAs led to 51 oncogenes (Supplementary Table 1) that encode proteins participating in gastrointestinal and breast cancer.

  9. Brevenal, a brevetoxin antagonist from Karenia brevis, binds to a previously unreported site on mammalian sodium channels.

    Science.gov (United States)

    Gold, Elena P; Jacocks, Henry M; Bourdelais, Andrea J; Baden, Daniel G

    2013-06-01

    Brevetoxins are a family of ladder-frame polyether toxins produced by the marine dinoflagellate Karenia brevis. During blooms of K. brevis, inhalation of brevetoxins aerosolized by wind and wave action can lead to asthma-like symptoms in persons at the beach. Consumption of either shellfish or finfish contaminated by K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to binding at a defined site on, and subsequent activation of, voltage-sensitive sodium channels (VSSCs) in cell membranes (site 5). In addition to brevetoxins, K. brevis produces several other ladder-frame compounds. One of these compounds, brevenal, has been shown to antagonize the effects of brevetoxin. In an effort to further characterize to effects of brevenal, a radioactive analog ([3H]-brevenol) was produced by reducing the side-chain terminal aldehyde moiety of brevenal to an alcohol using tritiated sodium borohydride. A KD of 67 nM and Bmax of 7.1 pmol/mg protein were obtained for [3H]-brevenol in rat brain synaptosomes, suggesting a 1:1 matching with VSSCs. Brevenal and brevenol competed for [3H]-brevenol binding with Ki values of 75 nM and 56 nM, respectively. However, although both brevenal and brevenol can inhibit brevetoxin binding, brevetoxin was completely ineffective at competition for [3H]-brevenol binding. After examining other site-specific compounds, it was determined that [3H]-brevenol binds to a site that is distinct from the other known sites including the brevetoxin site (site 5) although some interaction with site 5 is apparent.

  10. The effect of group X secreted phospholipase A2 on fertilization outcome is specific and not mimicked by other secreted phospholipases A2 or progesterone.

    Science.gov (United States)

    Abi Nahed, Roland; Escoffier, Jessica; Revel, Charlaine; Jeammet, Louise; Payré, Christine; Ray, Pierre F; Hennebicq, Sylviane; Lambeau, Gerard; Arnoult, Christophe

    2014-04-01

    Mouse group X sPLA2 (mGX) is an acrosomal protein playing an important role in fertilization and controlling acrosome reaction (AR) occurring during capacitation. We demonstrated previously that sperm from mGX knock-out mice had a severely impaired fertilization potential in vitro. We also showed that treatment of wild-type sperm with recombinant mGX during capacitation improved fertilization outcome. This interesting property suggests that sPLA2s could be used to improve fertilization in assisted reproductive technologies (ART). However the molecular mechanism explaining the mGX-dependent enhancing effect on fertilization outcome remains unclear so far. Interestingly, like progesterone (P4), mGX is a very potent activator of AR and the role of mGX-induced AR in fertilization outcome was not evaluated so far. To assess the role of sPLA2-induced AR in IVF, we first tested the potency of 9 mouse and 2 human sPLA2s and P4 to trigger AR of mouse sperm. We then tested the ability of 6 of these molecules (mouse Group IIA, mouse Group IID, mouse Group X, human Group V, human Group X and P4) to improve the yield of 2-cell embryos obtained by IVF in mouse. We showed that in the mouse neither P4 nor any of the other sPLA2s tested were able to mimic the IVF improvement produced by mGX-treatment. These results demonstrate that sPLA2s are not commutable in the context of mouse sperm fertility, indicating that their utilisation in other species, is subjected to the identification of probably unique species-specific active sPLA2. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  11. HOCOMOCO: A comprehensive collection of human transcription factor binding sites models

    KAUST Repository

    Kulakovskiy, Ivan V.

    2012-11-21

    Transcription factor (TF) binding site (TFBS) models are crucial for computational reconstruction of transcription regulatory networks. In existing repositories, a TF often has several models (also called binding profiles or motifs), obtained from different experimental data. Having a single TFBS model for a TF is more pragmatic for practical applications. We show that integration of TFBS data from various types of experiments into a single model typically results in the improved model quality probably due to partial correction of source specific technique bias. We present the Homo sapiens comprehensive model collection (HOCOMOCO, http://autosome.ru/HOCOMOCO/, http://cbrc.kaust.edu.sa/ hocomoco/) containing carefully hand-curated TFBS models constructed by integration of binding sequences obtained by both low- and high-throughput methods. To construct position weight matrices to represent these TFBS models, we used ChIPMunk software in four computational modes, including newly developed periodic positional prior mode associated with DNA helix pitch. We selected only one TFBS model per TF, unless there was a clear experimental evidence for two rather distinct TFBS models. We assigned a quality rating to each model. HOCOMOCO contains 426 systematically curated TFBS models for 401 human TFs, where 172 models are based on more than one data source. The Author(s) 2012.

  12. HOCOMOCO: a comprehensive collection of human transcription factor binding sites models.

    Science.gov (United States)

    Kulakovskiy, Ivan V; Medvedeva, Yulia A; Schaefer, Ulf; Kasianov, Artem S; Vorontsov, Ilya E; Bajic, Vladimir B; Makeev, Vsevolod J

    2013-01-01

    Transcription factor (TF) binding site (TFBS) models are crucial for computational reconstruction of transcription regulatory networks. In existing repositories, a TF often has several models (also called binding profiles or motifs), obtained from different experimental data. Having a single TFBS model for a TF is more pragmatic for practical applications. We show that integration of TFBS data from various types of experiments into a single model typically results in the improved model quality probably due to partial correction of source specific technique bias. We present the Homo sapiens comprehensive model collection (HOCOMOCO, http://autosome.ru/HOCOMOCO/, http://cbrc.kaust.edu.sa/hocomoco/) containing carefully hand-curated TFBS models constructed by integration of binding sequences obtained by both low- and high-throughput methods. To construct position weight matrices to represent these TFBS models, we used ChIPMunk software in four computational modes, including newly developed periodic positional prior mode associated with DNA helix pitch. We selected only one TFBS model per TF, unless there was a clear experimental evidence for two rather distinct TFBS models. We assigned a quality rating to each model. HOCOMOCO contains 426 systematically curated TFBS models for 401 human TFs, where 172 models are based on more than one data source.

  13. Halenaquinone, a chemical compound that specifically inhibits the secondary DNA binding of RAD51.

    Science.gov (United States)

    Takaku, Motoki; Kainuma, Takashi; Ishida-Takaku, Takako; Ishigami, Shintaro; Suzuki, Hidekazu; Tashiro, Satoshi; van Soest, Rob W M; Nakao, Yoichi; Kurumizaka, Hitoshi

    2011-04-01

    Mutations and single-nucleotide polymorphisms affecting RAD51 gene function have been identified in several tumors, suggesting that the inappropriate expression of RAD51 activity may cause tumorigenesis. RAD51 is an essential enzyme for the homologous recombinational repair (HRR) of DNA double-strand breaks. In the HRR pathway, RAD51 catalyzes the homologous pairing between single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), which is the central step of the HRR pathway. To identify a chemical compound that regulates the homologous-pairing activity of RAD51, in the present study, we screened crude extract fractions from marine sponges by the RAD51-mediated homologous-pairing assay. Halenaquinone was identified as an inhibitor of the RAD51 homologous-pairing activity. A surface plasmon resonance analysis indicated that halenaquinone directly bound to RAD51. Intriguingly, halenaquinone specifically inhibited dsDNA binding by RAD51 alone or the RAD51-ssDNA complex, but only weakly affected the RAD51-ssDNA binding. In vivo, halenaquinone significantly inhibited the retention of RAD51 at double-strand break sites. Therefore, halenaquinone is a novel type of RAD51 inhibitor that specifically inhibits the RAD51-dsDNA binding. © 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

  14. The Association of Myosin IB with Actin Waves in Dictyostelium Requires Both the Plasma Membrane-Binding Site and Actin-Binding Region in the Myosin Tail

    Science.gov (United States)

    Brzeska, Hanna; Pridham, Kevin; Chery, Godefroy; Titus, Margaret A.; Korn, Edward D.

    2014-01-01

    F-actin structures and their distribution are important determinants of the dynamic shapes and functions of eukaryotic cells. Actin waves are F-actin formations that move along the ventral cell membrane driven by actin polymerization. Dictyostelium myosin IB is associated with actin waves but its role in the wave is unknown. Myosin IB is a monomeric, non-filamentous myosin with a globular head that binds to F-actin and has motor activity, and a non-helical tail comprising a basic region, a glycine-proline-glutamine-rich region and an SH3-domain. The basic region binds to acidic phospholipids in the plasma membrane through a short basic-hydrophobic site and the Gly-Pro-Gln region binds F-actin. In the current work we found that both the basic-hydrophobic site in the basic region and the Gly-Pro-Gln region of the tail are required for the association of myosin IB with actin waves. This is the first evidence that the Gly-Pro-Gln region is required for localization of myosin IB to a specific actin structure in situ. The head is not required for myosin IB association with actin waves but binding of the head to F-actin strengthens the association of myosin IB with waves and stabilizes waves. Neither the SH3-domain nor motor activity is required for association of myosin IB with actin waves. We conclude that myosin IB contributes to anchoring actin waves to the plasma membranes by binding of the basic-hydrophobic site to acidic phospholipids in the plasma membrane and binding of the Gly-Pro-Gln region to F-actin in the wave. PMID:24747353

  15. Binding sites for atrial natriuretic factor (ANF) in brain: alterations in Brattleboro rats

    Energy Technology Data Exchange (ETDEWEB)

    McCarty, R.; Plunkett, L.M.

    1986-12-01

    Binding sites for atrial natriuretic factor (ANF-28) were analyzed in discrete brain areas of Brattleboro rats with hereditary diabetes insipidus and Long-Evans (LE) controls by quantitative autoradiography. The maximum binding capacity (Bmax) and affinity constant (Ka) for /sup 125/I-ANF-28 were elevated significantly in the subfornical organ of Brattleboro rats compared to matched LE controls. In contrast, values for Bmax and Ka for /sup 125/I-ANF-28