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Sample records for cation specific binding

  1. The specificity of protection against cationic antimicrobial peptides by lactoferrin binding protein B.

    Science.gov (United States)

    Morgenthau, Ari; Partha, Sarathy K; Adamiak, Paul; Schryvers, Anthony B

    2014-10-01

    A variety of Gram-negative pathogens possess host-specific lactoferrin (Lf) receptors that mediate the acquisition of iron from host Lf. The integral membrane protein component of the receptor, lactoferrin binding protein A specifically binds host Lf and is required for acquisition of iron from Lf. In contrast, the role of the bi-lobed surface lipoprotein, lactoferrin binding protein B (LbpB), in Lf binding and iron acquisition is uncertain. A common feature of LbpBs from most species is the presence of clusters of negatively charged amino acids in the protein's C-terminal lobe. Recently it has been shown that the negatively charged regions from the Neisseria meningitidis LbpB are responsible for protecting against an 11 amino acid cationic antimicrobial peptide (CAP), lactoferricin (Lfcin), derived from human Lf. In this study we investigated whether the LbpB confers resistance to other CAPs since N. meningitidis is likely to encounter other CAPs from the host. LbpB provided protection against the cathelicidin derived peptide, cathelicidin related antimicrobial peptide (mCRAMP), but did not confer protection against Tritrp 1 or LL37 under our experimental conditions. When tested against a range of rationally designed synthetic peptides, LbpB was shown to protect against IDR-1002 and IDR-0018 but not against HH-2 or HHC10. PMID:25038734

  2. Effects of Divalent Cations and Disulfide Bond Reducing Agents on Specific Binding of Growth Hormone to Liver Membrane Receptors from Snakehead Fish (Ophiocephalus argus, Cantor).

    Science.gov (United States)

    Sun, Xun; Zhang, Xin-Na; Zhu, Shang-Quan; Zheng, Han-Qi

    2000-01-01

    Divalent cations, Ca(2 ), Mg(2 ) and Mn(2 ) enhance the binding of bream growth hormone (brGH) to snakehead fish liver membrane, and their optimum concentration was found to be 8 12 mmol/L, at which Ca(2 ), Mg(2 ) and Mn(2 ) could increase, respectively, the specific binding to 230%, 180%, and 200%, compared with the binding in the absence of ions. The Eadie-Scatchard plot was used for the dynamic analysis of the Ca(2 ) binding site. A low affinity Ca(2 ) binding site was found in the GH-receptor complex with K(m)=0.384 mmol/L, and the affinity constant (K(a)) was increased from 1.045x10(9) L.mol(-1) to 1.295x10(9) L.mol(-1) by the addition of 10 mmol/L CaCl(2). The effects of disulfide bond reducing agents, DTT and ME, on (125)I-brGH binding to growth hormone receptor (GHR) on snakehead fish liver memebrane were also analyzed. The addition of 0.1 20 mmol/L DTT or 0.01% 1% ME to the radioreceptor assay system caused a significant dose dependent increase in the specific binding for (125)I-brGH. In the presence of 0.8 mmol/L DTT or 0.08% ME, the specific binding of (125)I-brGH was increased from 10.2% to 15.5% and 13.2% respectively, and the affinity constant was also increased from 1.265x10(9) L.mol(-1) to 2.185x10(9) L.mol(-1) and 1.625x10(9) L.mol(-1), respectively but no changes in the binding capacity were observed. Further studies showed that the effects of reductants on the specific binding of brGH were due in part to the ligand itself and in part to GHR. In addition, it was observed that one of the three disulfide bonds of brGH could be reduced by 0.8 mmol/L DTT.

  3. Predictive model of cationic surfactant binding to humic substances

    NARCIS (Netherlands)

    Ishiguro, M.; Koopal, L.K.

    2011-01-01

    The humic substances (HS) have a high reactivity with other components in the natural environment. An important factor for the reactivity of HS is their negative charge. Cationic surfactants bind strongly to HS by electrostatic and specific interaction. Therefore, a surfactant binding model is devel

  4. Binding of cationic surfactants to humic substances

    NARCIS (Netherlands)

    Ishiguro, M.; Tan, W.; Koopal, L.K.

    2007-01-01

    Commercial surfactants are introduced into the environment either through waste products or site-specific contamination. The amphiphilic nature of both surfactants and humic substances (HS) leads to their mutual attraction especially when surfactant and HS are oppositely charged. Binding of the cati

  5. Cations bind only weakly to amides in aqueous solutions.

    Science.gov (United States)

    Okur, Halil I; Kherb, Jaibir; Cremer, Paul S

    2013-04-01

    We investigated salt interactions with butyramide as a simple mimic of cation interactions with protein backbones. The experiments were performed in aqueous metal chloride solutions using two spectroscopic techniques. In the first, which provided information about contact pair formation, the response of the amide I band to the nature and concentration of salt was monitored in bulk aqueous solutions via attenuated total reflection Fourier transform infrared spectroscopy. It was found that molar concentrations of well-hydrated metal cations (Ca(2+), Mg(2+), Li(+)) led to the rise of a peak assigned to metal cation-bound amides (1645 cm(-1)) and a decrease in the peak associated with purely water-bound amides (1620 cm(-1)). In a complementary set of experiments, the effect of cation identity and concentration was investigated at the air/butyramide/water interface via vibrational sum frequency spectroscopy. In these studies, metal ion-amide binding led to the ordering of the adjacent water layer. Such experiments were sensitive to the interfacial partitioning of cations in either a contact pair with the amide or as a solvent separated pair. In both experiments, the ordering of the interactions of the cations was: Ca(2+) > Mg(2+) > Li(+) > Na(+) ≈ K(+). This is a direct cationic Hofmeister series. Even for Ca(2+), however, the apparent equilibrium dissociation constant of the cation with the amide carbonyl oxygen was no tighter than ∼8.5 M. For Na(+) and K(+), no evidence was found for any binding. As such, the interactions of metal cations with amides are far weaker than the analogous binding of weakly hydrated anions.

  6. Binding of alkaline cations to the double-helical form of gramicidin.

    OpenAIRE

    Chen, Y; Wallace, B.A.

    1996-01-01

    Gramicidin is a polypeptide antibiotic that forms monovalent cation-specific channels in membrane environments. In organic solvents and in lipids containing unsaturated fatty acid chains, it forms a double-helical "pore" structure, in which two monomers are intertwined. This form of gramicidin can bind two cations inside its lumen, and the crystal structures of both an ion complex and an ion-free form have been determined. In this study, we have used circular dichroism (CD) spectroscopy to ex...

  7. Alkali cation specific adsorption onto fcc(111) transition metal electrodes.

    Science.gov (United States)

    Mills, J N; McCrum, I T; Janik, M J

    2014-07-21

    The presence of alkali cations in electrolyte solutions is known to impact the rate of electrocatalytic reactions, though the mechanism of such impact is not conclusively determined. We use density functional theory (DFT) to examine the specific adsorption of alkali cations to fcc(111) electrode surfaces, as specific adsorption may block catalyst sites or otherwise impact surface catalytic chemistry. Solvation of the cation-metal surface structure was investigated using explicit water models. Computed equilibrium potentials for alkali cation adsorption suggest that alkali and alkaline earth cations will specifically adsorb onto Pt(111) and Pd(111) surfaces in the potential range of hydrogen oxidation and hydrogen evolution catalysis in alkaline solutions.

  8. Somatostatin binding in human gastrointestinal tissues: effect of cations and somatostatin analogues.

    OpenAIRE

    Miller, G. V.; Preston, S R; Woodhouse, L. F.; Farmery, S M; Primrose, J N

    1993-01-01

    This study characterises the somatostatin binding site in human gastrointestinal cancer and mucosa in terms of cationic specificity and relative affinity for three somatostatin analogues. Competitive displacement assays were performed on plasma membranes from human gastric and colonic tissues using radiolabelled somatostatin-14 as ligand. Comparison was made with the somatostatin binding site in rat cerebral cortex. In gastrointestinal tissue, magnesium decreased and sodium increased specific...

  9. Strong and specific effects of cations on lysozyme chloride solubility.

    Science.gov (United States)

    Bénas, Philippe; Legrand, Laurent; Riès-Kautt, Madeleine

    2002-10-01

    The influence of salt nature and concentration on tetragonal lysozyme chloride crystal solubility is presented for a set of mono-, di- and trivalent cations (Cs(+), Rb(+), Mn(2+), Co(2+) and Yb(3+)). The results show that cations have as strong an effect on protein solubility as anions and that they present their own particular effects as co-ions. Indeed, after decreasing at low ionic strength, lysozyme solubility increases with high concentration of polyvalent cations, probably due to co-ion binding and therefore the concomitant increase of the net charge of the protein-salt complex. These new results are discussed in order to progress in the understanding of the crystallisation process at the atomic level. PMID:12351866

  10. Influence of 3‧-3‧ inversion of polarity site within d(TGGGGT) on inter quartet cation binding

    Science.gov (United States)

    Šket, Primož; Korbar, Tjaša; Plavec, Janez

    2014-10-01

    Stability, dynamics and function of nucleic acids are affected by nature of cations that are involved in interaction with specific functionalities. Introduction of inversion of polarity sites represents a very useful and chemically accessible backbone modification, which can alter the binding affinity of cations. NMR study on cation binding between G-quartets in tetramolecular G-quadruplex adopted by d(5‧TGG3‧-3‧GGT5‧) with 3‧-3‧ inversion of polarity sites in the middle of G-tract showed existence of two different G-quadruplex forms with all strands in parallel orientation, where all guanine residues adopt anti conformation around glycosidic bonds in the presence of 15NH4+ ions. In one of the forms all three binding sites are equally populated, while in the second form the binding site next to the inversion of polarity site is not fully populated by 15NH4+ ions.

  11. Cationic lipid membranes-specific interactions with counter-ions

    International Nuclear Information System (INIS)

    Lipids bearing net electric charges in their hydrophilic headgroups are ubiquitous in biological membranes. Recently, the interest in cationic lipids has surged because of their potential as non-viral transfection vectors. In order to utilize cationic lipids in transfer of nucleic acids and to elucidate the role of charged lipids in cellular membranes in general, their complex interactions within the membrane and with the molecules in the surrounding media need to be thoroughly characterized. Yet, even interactions between monovalent counter-ions and charged lipids are inadequately understood. We studied the interactions of the cationic gemini surfactant (2R,3R)-2,3-dimethoxy-1,4- bis(N-hexadecyl-N,N-dimethylammonium)butane dibromide (RR-1) with chloride, bromide, fluoride, and iodide as counter-ions by differential scanning calorimetry and Langmuir balance. Chloride interacts avidly with RR-1, efficiently condensing the monolayer, decreasing the collapse pressure, and elevating the main transition temperature. With bromide and iodide clearly different behaviour was observed, indicating specific interactions between RR-1 and these counter-ions. Moreover, with fluoride as a counter-ion and in pure water identical results were obtained, demonstrating inefficient electrostatic screening of the headgroups of RR-1 and suggesting fluoride being depleted on the surface of RR-1 membranes

  12. Cationic lipid membranes-specific interactions with counter-ions

    Energy Technology Data Exchange (ETDEWEB)

    Ryhaenen, Samppa J; Saeily, V Matti J; Kinnunen, Paavo K J [Helsinki Biophysics and Biomembrane Group, Institute of Biomedicine, Biomedicum, University of Helsinki, PO Box 63 (Haartmaninkatu 8), Helsinki FIN-00014 (Finland)

    2006-07-19

    Lipids bearing net electric charges in their hydrophilic headgroups are ubiquitous in biological membranes. Recently, the interest in cationic lipids has surged because of their potential as non-viral transfection vectors. In order to utilize cationic lipids in transfer of nucleic acids and to elucidate the role of charged lipids in cellular membranes in general, their complex interactions within the membrane and with the molecules in the surrounding media need to be thoroughly characterized. Yet, even interactions between monovalent counter-ions and charged lipids are inadequately understood. We studied the interactions of the cationic gemini surfactant (2R,3R)-2,3-dimethoxy-1,4- bis(N-hexadecyl-N,N-dimethylammonium)butane dibromide (RR-1) with chloride, bromide, fluoride, and iodide as counter-ions by differential scanning calorimetry and Langmuir balance. Chloride interacts avidly with RR-1, efficiently condensing the monolayer, decreasing the collapse pressure, and elevating the main transition temperature. With bromide and iodide clearly different behaviour was observed, indicating specific interactions between RR-1 and these counter-ions. Moreover, with fluoride as a counter-ion and in pure water identical results were obtained, demonstrating inefficient electrostatic screening of the headgroups of RR-1 and suggesting fluoride being depleted on the surface of RR-1 membranes.

  13. 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...

  14. Membrane potential and surface potential in mitochondria: uptake and binding of lipophilic cations.

    Science.gov (United States)

    Rottenberg, H

    1984-01-01

    The uptake and binding of the lipophilic cations ethidium+, tetraphenylphosphonium+ (TPP+), triphenylmethylphosphonium+ (TPMP+), and tetraphenylarsonium+ (TPA+) in rat liver mitochondria and submitochondrial particles were investigated. The effects of membrane potential, surface potentials and cation concentration on the uptake and binding were elucidated. The accumulation of these cations by mitochondria is described by an uptake and binding to the matrix face of the inner membrane in addition to the binding to the cytosolic face of the inner membrane. The apparent partition coefficients between the external medium and the cytosolic surface of the inner membrane (K'o) and the internal matrix volume and matrix face of the inner membrane (K'i) were determined and were utilized to estimate the membrane potential delta psi from the cation accumulation factor Rc according to the relation delta psi = RT/ZF ln [(RcVo - K'o)/(Vi + K'i)] where Vo and Vi are the volume of the external medium and the mitochondrial matrix, respectively, and Rc is the ratio of the cation content of the mitochondria and the medium. The values of delta psi estimated from this equation are in remarkably good agreement with those estimated from the distribution of 86Rb in the presence of valinomycin. The results are discussed in relation to studies in which the membrane potential in mitochondria and bacterial cells was estimated from the distribution of lipophilic cations. PMID:6492133

  15. Regulation by divalent cations of 3H-baclofen binding to GABA/sub B/ sites in rat cerebellar membranes

    International Nuclear Information System (INIS)

    When investigating the effects of divalent cations (Mg2+, Ca2+, Sr2+, Ba2+, Mn2+ and Ni2+) on 3H-baclofen binding to rat cerebellar synaptic membranes, we found that the specific binding of 3H-baclofen was not only dependent on divalent cations, but was increased dose-dependently in the presence of these cations. The effects were in the following order of potency: Mn2+ approx. = Ni2+ > Mg2+ > Ca2+ > Sr2+ > Ba2+. Scatchard analysis of the binding data revealed a single component of the binding sites in the presence of 2.5 mM MgCl2, 2.5 mM CaCl2 or 0.3 mM MnCl2 whereas two components appeared in the presence of 2.5 mM MnCl2 or 1 mM NiCl2. In the former, divalent cations altered the apparent affinity (K/sub d/) without affecting density of the binding sites (B/sub max/). In the latter, the high-affinity sites showed a higher affinity and lower density of the binding sites than did the single component of the former. As the maximal effects of four cations (Mg2+, Ca2+, Mn2+, and Ni2+) were not additive, there are probably common sites of action of these divalent cations. Among the ligands for GABA/sub B/ sites, the affinity for (-), (+) and (+/-)baclofen, GABA and β-phenyl GABA increased 2 - 6 fold in the presence of 2.5 mM MnCl2, in comparison with that in HEPES-buffered Krebs solution (containing 2.5 mM CaCl2 and 1.2 mM MgSO4), whereas that for muscimol was decreased to one-fifth. Thus, the affinity of GABA/sub B/ sites for its ligands is probably regulated by divalent cations, through common sites of action

  16. Study of Ion Specific Interactions of Alkali Cations with Dicarboxylate Dianions

    Energy Technology Data Exchange (ETDEWEB)

    Murdachaew, Garold; Valiev, Marat; Kathmann, Shawn M.; Wang, Xue B.

    2012-02-10

    Alkali metal cations often show pronounced ion specific interactions and selectivity with macromolecules in biological processes, colloids, and interfacial sciences, but a fundamental understanding about the underlying microscopic mechanism is still very limited. Here we report a direct probe of interactions between alkali metal cations (M{sup +}) and dicarboxylate dianions, O{sub 2}C(CH{sub 2})nCO{sub 2} (D{sub n}{sup 2-}) in the gas phase by combined photoelectron spectroscopy (PES) and ab initio electronic structure calculation on nine M{sup +}-D{sub n}{sup 2-} complexes (M = Li, Na, K; n = 2, 4, 6). PES spectra show that the electron binding energy (EBE) decreases from Li{sup +} to Na{sup +} to K{sup +} for complexes of M{sup +}-D{sub 2}{sup 2-}, whereas the order is Li{sup +} binding environments are found to depend upon dicarboxylate size n and the specific cation M{sup +}. The observed variance of EBEs reflects how well a specific dicarboxylate dianion accommodates each M{sup +}. This work demonstrates the delicate interplay among several factors (electrostatic interaction, size matching, and strain energy) that likely play critical roles in determining the structures and energetics of gaseous clusters as well as ion specificity and selectivity in solutions and biological systems.

  17. Dissecting electrostatic screening, specific ion binding, and ligand binding in an energetic model for glycine riboswitch folding

    Energy Technology Data Exchange (ETDEWEB)

    Lipfert, Jan; Sim, Adelene Y.L.; Herschlag, Daniel; Doniach, Sebastian (Stanford)

    2010-09-17

    Riboswitches are gene-regulating RNAs that are usually found in the 5{prime}-untranslated regions of messenger RNA. As the sugar-phosphate backbone of RNA is highly negatively charged, the folding and ligand-binding interactions of riboswitches are strongly dependent on the presence of cations. Using small angle X-ray scattering (SAXS) and hydroxyl radical footprinting, we examined the cation dependence of the different folding stages of the glycine-binding riboswitch from Vibrio cholerae. We found that the partial folding of the tandem aptamer of this riboswitch in the absence of glycine is supported by all tested mono- and divalent ions, suggesting that this transition is mediated by nonspecific electrostatic screening. Poisson-Boltzmann calculations using SAXS-derived low-resolution structural models allowed us to perform an energetic dissection of this process. The results showed that a model with a constant favorable contribution to folding that is opposed by an unfavorable electrostatic term that varies with ion concentration and valency provides a reasonable quantitative description of the observed folding behavior. Glycine binding, on the other hand, requires specific divalent ions binding based on the observation that Mg{sup 2+}, Ca{sup 2+}, and Mn{sup 2+} facilitated glycine binding, whereas other divalent cations did not. The results provide a case study of how ion-dependent electrostatic relaxation, specific ion binding, and ligand binding can be coupled to shape the energetic landscape of a riboswitch and can begin to be quantitatively dissected.

  18. Impact of the associated cation on chloride binding of Portland cement paste

    Energy Technology Data Exchange (ETDEWEB)

    De Weerdt, K., E-mail: klaartje.d.weerdt@ntnu.no [Department of Structural Engineering, Norwegian University of Science and Technology (Norway); Department of Engineering and Applied Sciences, University of Bergamo (Italy); Colombo, A. [Department of Structural Engineering, Norwegian University of Science and Technology (Norway); Department of Engineering and Applied Sciences, University of Bergamo (Italy); Coppola, L. [Department of Engineering and Applied Sciences, University of Bergamo (Italy); Justnes, H. [SINTEF Building and Infrastructure, Trondheim (Norway); Geiker, M.R. [Department of Structural Engineering, Norwegian University of Science and Technology (Norway)

    2015-02-15

    Well hydrated cement paste was exposed to MgCl{sub 2}, CaCl{sub 2} and NaCl solutions at 20 °C. The chloride binding isotherms for free chloride concentrations ranging up to 1.5 mol/l were determined experimentally. More chlorides were found to be bound when the associated cation was Mg{sup 2} {sup +} or Ca{sup 2} {sup +} compared to Na{sup +}. The chloride binding capacity of the paste appeared to be related to the pH of the exposure solution. In order to explain the cation dependency of the chloride binding a selection of samples was investigated in detail using experimental techniques such as TG, XRD and SEM–EDS to identify the phases binding the chlorides. The experimentally obtained data were compared with the calculations of a thermodynamic model, GEMS. It was concluded that the measured change in chloride binding depending on the cation was mainly governed by the pH of the exposure solution and thereby the binding capacity of the C-S-H.

  19. Impact of the associated cation on chloride binding of Portland cement paste

    International Nuclear Information System (INIS)

    Well hydrated cement paste was exposed to MgCl2, CaCl2 and NaCl solutions at 20 °C. The chloride binding isotherms for free chloride concentrations ranging up to 1.5 mol/l were determined experimentally. More chlorides were found to be bound when the associated cation was Mg2 + or Ca2 + compared to Na+. The chloride binding capacity of the paste appeared to be related to the pH of the exposure solution. In order to explain the cation dependency of the chloride binding a selection of samples was investigated in detail using experimental techniques such as TG, XRD and SEM–EDS to identify the phases binding the chlorides. The experimentally obtained data were compared with the calculations of a thermodynamic model, GEMS. It was concluded that the measured change in chloride binding depending on the cation was mainly governed by the pH of the exposure solution and thereby the binding capacity of the C-S-H

  20. Experimental and theoretical characterization of the high-affinity cation binding site of the purple membrane

    OpenAIRE

    Pardo, Leonardo; Sepulcre Sánchez, Francesc; Cladera Cerdà, Josep Bartomeu; Duñach, Mireia; Labarta, A.; Tejada, J.; Padrós Morell, Esteve

    1998-01-01

    Binding of Mn2+ or Mg2+ to the high-affinity site of the purple membrane from Halobacterium salinarium has been studied by superconducting quantum interference device magnetometry or by ab initio quantum mechanical calculations, respectively. The binding of Mn2+ cation, in a low-spin state, to the high-affinity site occurs through a major octahedral local symmetry character with a minor rhombic distortion and a coordination number of six. A molecular model of this binding site in the Schiff b...

  1. Binding properties of oxacalix[4]arenes derivatives toward metal cations; Interactions entre cations metalliques et derives des oxacalix[4]arenes

    Energy Technology Data Exchange (ETDEWEB)

    Mellah, B

    2006-11-15

    The objective of this work was to establish the binding properties of oxacalix[4]arene derivatives with different numbers of the oxa bridges, functional groups (ketones, pyridine, ester, amide and methoxy) and conformations. Their interactions with alkali and alkaline-earth, heavy and transition metal cations have been evaluated according to different approaches: (i) extraction of corresponding picrates from an aqueous phase into dichloromethane; (ii) determination of the thermodynamic parameters of complexation in methanol and/or acetonitrile by UV-spectrophotometry and micro-calorimetry; (iii) determination of the stoichiometry of the complexes by ESI-MS; (iv) {sup 1}H-NMR titrations allowing to localize the metal ions in the ligand cavity. In a first part dealing on homo-oxacalix[4]arenes, selectivities for Na{sup +}, K{sup +}, Ca{sup 2+}, Pb{sup 2+} and Mn{sup 2+} of ketones derivatives was shown. The presence of oxa bridge in these derivatives increases their efficiency while decreasing their selectivity with respect to related calixarenes. The pyridine derivative prefers transition and heavy metal cations, in agreement with the presence of the soft nitrogen atoms. In the second part, di-oxacalix[4]arene ester and secondary amide derivatives were shown to be less effective than tertiary amide counterparts but to present high selectivities for Li{sup +}, Ba{sup 2+}, Zn{sup 2+} and Hg{sup 2+}. A third part devoted to the octa-homo-tetra-oxacalix[4]arene tetra-methoxy shows that the 1:1 metal complexes formed are generally more stable than those of calixarenes, suggesting the participation of the oxygen atoms of the bridge in the complexation. Selectivity for Cs{sup +}, Ba{sup 2+}, Cu{sup 2+} and Hg{sup 2+} were noted. (author)

  2. Specific cationic emission of cisplatin following ionization by swift protons

    Science.gov (United States)

    Moretto-Capelle, Patrick; Champeaux, Jean-Philippe; Deville, Charlotte; Sence, Martine; Cafarelli, Pierre

    2016-05-01

    We have investigated collision-induced ionization and fragmentation by 100 keV protons of the radio sensitizing molecule cisplatin, which is used in cancer treatments. A large emission of HCl+ and NH2+ is observed, but surprisingly, no cationic fragments containing platinum are detected, in contrast to ionization-dissociation induced by electronic collision. Theoretical investigations show that the ionization processes take place on platinum and on chlorine atoms. We propose new ionization potentials for cisplatin. Dissociation limits corresponding to the measured fragmentation mass spectrum have been evaluated and the theoretical results show that the non-observed cationic fragments containing platinum are mostly associated with low dissociation energies. We have also investigated the reaction path for the hydrogen transfer from the NH3 group to the Cl atom, as well as the corresponding dissociation limits from this tautomeric form. Here again the cations containing platinum correspond to lower dissociation limits. Thus, the experimental results suggest that excited states, probably formed via inner-shell ionization of the platinum atom of the molecule, correlated to higher dissociation limits are favored.

  3. Binding of Divalent Cations to Polygalacturonate: A Mechanism Driven by the Hydration Water.

    Science.gov (United States)

    Huynh, Uyen T D; Lerbret, Adrien; Neiers, Fabrice; Chambin, Odile; Assifaoui, Ali

    2016-02-11

    We have investigated the interactions between polygalacturonate (polyGal) and four divalent cations (M(2+) = Ba(2+), Ca(2+), Mg(2+), Zn(2+)) that differ in size and affinity for water. Our results evidence that M(2+)-polyGal interactions are intimately linked to the affinity of M(2+) for water. Mg(2+) interacts so strongly with water that it remains weakly bound to polyGal (polycondensation) by sharing water molecules from its first coordination shell with the carboxylate groups of polyGal. In contrast, the other cations form transient ionic pairs with polyGal by releasing preferentially one water molecule (for Zn(2+)) or two (for Ca(2+) and Ba(2+)), which corresponds to monodentate and bidentate binding modes with carboxylates, respectively. The mechanism for the binding of these three divalent cations to polyGal can be described by two steps: (i) monocomplexation and formation of point-like cross-links between polyGal chains (at low M(2+)/Gal molar ratios, R) and (ii) dimerization (at higher R). The threshold molar ratio, R*, between these two steps depends on the nature of divalent cations and is lower for calcium ions (R* barium ions (R* > 0.3). This difference may be explained by the intermediate affinity of Ca(2+) for water with respect to those of Zn(2+) and Ba(2+), which may induce the formation of cross-links of intermediate flexibility. By comparison, the lower and higher flexibilities of the cross-links formed by Zn(2+) and Ba(2+), respectively, may shift the formation of dimers to higher molar ratios (R*). PMID:26771109

  4. Cationic polymers for DNA origami coating - examining their binding efficiency and tuning the enzymatic reaction rates.

    Science.gov (United States)

    Kiviaho, Jenny K; Linko, Veikko; Ora, Ari; Tiainen, Tony; Järvihaavisto, Erika; Mikkilä, Joona; Tenhu, Heikki; Nonappa; Kostiainen, Mauri A

    2016-06-01

    DNA origamis are fully tailored, programmable, biocompatible and readily functionalizable nanostructures that provide an excellent foundation for the development of sophisticated drug-delivery systems. However, the DNA origami objects suffer from certain drawbacks such as low cell-transfection rates and low stability. A great deal of studies on polymer-based transfection agents, mainly focusing on polyplex formation and toxicity, exists. In this study, the electrostatic binding between a brick-like DNA origami and cationic block-copolymers was explored. The effect of the polymer structure on the binding was investigated and the toxicity of the polymer-origami complexes evaluated. The study shows that all of the analyzed polymers had a suitable binding efficiency irrespective of the block structure. It was also observed that the toxicity of polymer-origami complexes was insignificant at the biologically relevant concentration levels. Besides brick-like DNA origamis, tubular origami carriers equipped with enzymes were also coated with the polymers. By adjusting the amount of cationic polymers that cover the DNA structures, we showed that it is possible to control the enzyme kinetics of the complexes. This work gives a starting point for further development of biocompatible and effective polycation-based block copolymers that can be used in coating different DNA origami nanostructures for various bioapplications. PMID:27219684

  5. Tuning the ion selectivity of tetrameric cation channels by changing the number of ion binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Derebe, Mehabaw G.; Sauer, David B.; Zeng, Weizhong; Alam, Amer; Shi, Ning; Jiang, Youxing (UTSMC); (ETH Zurich)

    2015-11-30

    Selective ion conduction across ion channel pores is central to cellular physiology. To understand the underlying principles of ion selectivity in tetrameric cation channels, we engineered a set of cation channel pores based on the nonselective NaK channel and determined their structures to high resolution. These structures showcase an ensemble of selectivity filters with a various number of contiguous ion binding sites ranging from 2 to 4, with each individual site maintaining a geometry and ligand environment virtually identical to that of equivalent sites in K{sup +} channel selectivity filters. Combined with single channel electrophysiology, we show that only the channel with four ion binding sites is K{sup +} selective, whereas those with two or three are nonselective and permeate Na{sup +} and K{sup +} equally well. These observations strongly suggest that the number of contiguous ion binding sites in a single file is the key determinant of the channel's selectivity properties and the presence of four sites in K{sup +} channels is essential for highly selective and efficient permeation of K{sup +} ions.

  6. A cation-pi interaction in the binding site of the glycine receptor is mediated by a phenylalanine residue

    DEFF Research Database (Denmark)

    Pless, Stephan Alexander; Millen, Kat S; Hanek, Ariele P;

    2008-01-01

    Cys-loop receptor binding sites characteristically contain many aromatic amino acids. In nicotinic ACh and 5-HT3 receptors, a Trp residue forms a cation-pi interaction with the agonist, whereas in GABA(A) receptors, a Tyr performs this role. The glycine receptor binding site, however, contains pr...

  7. Binding of cationic surfactants to DNA, protein and DNA-protein mixtures.

    Science.gov (United States)

    Gani, S A; Chattoraj, D K; Mukherjee, D C

    1999-06-01

    Extent of binding (gamma 2(1)) of cationic surfactants cetyltrimethyl ammonium bromide (CTAB), myristyltrimethyl ammonium bromide (MTAB) and dodecyl trimethyl ammonium bromide (DTAB) to calf-thymus DNA, bovine serum albumin (BSA) and to their binary mixture respectively have been measured as function of bulk concentration of the surfactant by using equilibrium dialysis technique. Binding of CTAB has been studied at different pH, ionic strength (mu), temperature and biopolymer composition and with native and denatured states of the biopolymers. The chain-length of different long chain amines plays a significant role in the extent of binding under identical solution condition. The binding ratios for CTAB to collagen, gelatin, DNA-collagen and DNA-gelatin mixtures respectively have also been determined. The conformational structures of different biopolymers are observed to play significant role in macromolecular interactions between protein and DNA in the presence of CTAB. From the experimental values of the maximum binding ratio (gamma 2m) at the saturation level for each individual biopolymer, ideal values (gamma 2m)id have been theoretically calculated for binary mixtures of biopolymers using additivity rule. The protein-DNA-CTAB interaction in mixture has been explained in terms of the deviation (delta) of (gamma 2m) from (gamma 2m)id in the presence of a surfactant in bulk. The binding of surfactants to biopolymers and to their binary mixtures are compared more precisely in terms of the Gibbs' free energy decrease (-delta G degree) for the saturation of the binding sites in the biopolymers or biopolymer mixtures with the change of the bulk surfactant activity from zero to unity in the rational mole fraction scale. PMID:10650715

  8. A specific fluorescent chemosensor for copper(Ⅱ) cation recognition

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The fluorescent spectra of 2, 6-bis(benzimidazol-2-yl) pyridine (compound 2) and its N-substituted compound (1) are studied. The fluorescent characteristics of these compounds and the complexes formed from these compounds with different metal ions have also been investigated. The results show that compound 1 possesses a specific ability to form complex with Cu2+ ions, but compound 2 has not such a property. It is proposed that the specific recognition ability of compound 1 to Cu2+ may attribute to the cyclic configuration of this compound in polar solvent.

  9. Specific receptor binding of staphylococcal enterotoxins by murine splenic lymphocytes.

    OpenAIRE

    Buxser, S; Bonventre, P F; Archer, D L

    1981-01-01

    We describe a reliable assay to measure the specific binding of 125I-labeled staphylococcal enterotoxin A (SEA) by murine spleen cells. Toxin binding by lymphocytes was specific in that it was inhibited by unlabeled SEA but not by unrelated proteins. The biological activity of SEA (T-lymphocyte mitogenesis) correlated with toxin binding to splenic lymphocytes. In the presence of high concentrations of [125I]SEA, specific binding increased rapidly and approached saturation after 2 h. Toxin bin...

  10. Regulation by divalent cations of /sup 3/H-baclofen binding to GABA/sub B/ sites in rat cerebellar membranes

    Energy Technology Data Exchange (ETDEWEB)

    Kato, K.; Goto, M.; Fukuda, H.

    1983-02-21

    When investigating the effects of divalent cations (Mg/sup 2 +/, Ca/sup 2 +/, Sr/sup 2 +/, Ba/sup 2 +/, Mn/sup 2 +/ and Ni/sup 2 +/) on /sup 3/H-baclofen binding to rat cerebellar synaptic membranes, we found that the specific binding of /sup 3/H-baclofen was not only dependent on divalent cations, but was increased dose-dependently in the presence of these cations. The effects were in the following order of potency: Mn/sup 2 +/ approx. = Ni/sup 2 +/ > Mg/sup 2 +/ > Ca/sup 2 +/ > Sr/sup 2 +/ > Ba/sup 2 +/. Scatchard analysis of the binding data revealed a single component of the binding sites in the presence of 2.5 mM MgCl/sub 2/, 2.5 mM CaCl/sub 2/ or 0.3 mM MnCl/sub 2/ whereas two components appeared in the presence of 2.5 mM MnCl/sub 2/ or 1 mM NiCl/sub 2/. In the former, divalent cations altered the apparent affinity (K/sub d/) without affecting density of the binding sites (B/sub max/). In the latter, the high-affinity sites showed a higher affinity and lower density of the binding sites than did the single component of the former. As the maximal effects of four cations (Mg/sup 2 +/, Ca/sup 2 +/, Mn/sup 2 +/, and Ni/sup 2 +/) were not additive, there are probably common sites of action of these divalent cations. Among the ligands for GABA/sub B/ sites, the affinity for (-), (+) and (+/-)baclofen, GABA and ..beta..-phenyl GABA increased 2 - 6 fold in the presence of 2.5 mM MnCl/sub 2/, in comparison with that in HEPES-buffered Krebs solution (containing 2.5 mM CaCl/sub 2/ and 1.2 mM MgSO/sub 4/), whereas that for muscimol was decreased to one-fifth. Thus, the affinity of GABA/sub B/ sites for its ligands is probably regulated by divalent cations, through common sites of action.

  11. Cationic polymers for DNA origami coating - examining their binding efficiency and tuning the enzymatic reaction rates

    Science.gov (United States)

    Kiviaho, Jenny K.; Linko, Veikko; Ora, Ari; Tiainen, Tony; Järvihaavisto, Erika; Mikkilä, Joona; Tenhu, Heikki; Nonappa, Affc; Kostiainen, Mauri A.

    2016-06-01

    DNA origamis are fully tailored, programmable, biocompatible and readily functionalizable nanostructures that provide an excellent foundation for the development of sophisticated drug-delivery systems. However, the DNA origami objects suffer from certain drawbacks such as low cell-transfection rates and low stability. A great deal of studies on polymer-based transfection agents, mainly focusing on polyplex formation and toxicity, exists. In this study, the electrostatic binding between a brick-like DNA origami and cationic block-copolymers was explored. The effect of the polymer structure on the binding was investigated and the toxicity of the polymer-origami complexes evaluated. The study shows that all of the analyzed polymers had a suitable binding efficiency irrespective of the block structure. It was also observed that the toxicity of polymer-origami complexes was insignificant at the biologically relevant concentration levels. Besides brick-like DNA origamis, tubular origami carriers equipped with enzymes were also coated with the polymers. By adjusting the amount of cationic polymers that cover the DNA structures, we showed that it is possible to control the enzyme kinetics of the complexes. This work gives a starting point for further development of biocompatible and effective polycation-based block copolymers that can be used in coating different DNA origami nanostructures for various bioapplications.DNA origamis are fully tailored, programmable, biocompatible and readily functionalizable nanostructures that provide an excellent foundation for the development of sophisticated drug-delivery systems. However, the DNA origami objects suffer from certain drawbacks such as low cell-transfection rates and low stability. A great deal of studies on polymer-based transfection agents, mainly focusing on polyplex formation and toxicity, exists. In this study, the electrostatic binding between a brick-like DNA origami and cationic block-copolymers was explored. The

  12. RNA Binding Specificity of Drosophila Muscleblind†

    OpenAIRE

    Goers, Emily S.; Voelker, Rodger B.; Gates, Devika P.; Berglund, J. Andrew

    2008-01-01

    Members of the muscleblind family of RNA binding proteins found in Drosophila and mammals are key players in both the human disease myotonic dystrophy and the regulation of alternative splicing. Recently, the mammalian muscleblind-like protein, MBNL1, has been shown to have interesting RNA binding properties with both endogenous and disease-related RNA targets. Here we report the characterization of RNA binding properties of the Drosophila muscleblind protein Mbl. Mutagenesis of double-strand...

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

    DEFF Research Database (Denmark)

    1998-01-01

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

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

    DEFF Research Database (Denmark)

    Laursen, Mette; Yatime, Laure; Nissen, Poul;

    2013-01-01

    The Na+,K+-ATPase maintains electrochemical gradients for Na+ and K+ that are critical for animal cells. Cardiotonic steroids (CTSs), widely used in the clinic and recently assigned a role as endogenous regulators of intracellular processes, are highly specific inhibitors of the Na+,K+-ATPase. Here......325 toward the cation site and thereby hindering deep ouabain binding. Thus, the structural data establish a basis for the interpretation of the biochemical evidence pointing at direct K+–Mg2+ competition and explain the well-known antagonistic effect of K+ on CTS binding....

  15. Thermodynamic stability and retinol binding property of {beta}-lactoglobulin in the presence of cationic surfactants

    Energy Technology Data Exchange (ETDEWEB)

    Sahihi, M. [Department of Chemistry, Isfahan University of Technology, Isfahan 84156-83111 (Iran, Islamic Republic of); Bordbar, A.K., E-mail: bordbar@chem.ui.ac.ir [Department of Chemistry, University of Isfahan, Isfahan 81746-73441 (Iran, Islamic Republic of); Ghayeb, Y. [Department of Chemistry, Isfahan University of Technology, Isfahan 84156-83111 (Iran, Islamic Republic of)

    2011-08-15

    Highlights: > The stability parameters of {beta}-lactoglobulin, BLG, in the presence of C{sub n}TAB have been evaluated. > Rising in hydrocarbon chain length increases the denaturating power of surfactants. > C{sub n}TAB enhances the retinol binding affinity of BLG in all of its concentration range. - Abstract: In this work the stability parameters of bovine {beta}-lactoglobulin, variant A (BLG-A), with regard to their transition curves induced by dodecyltrimethylammonium bromide (C{sub 12}TAB), tetradecyltrimethylammonium bromide (C{sub 14}TAB) and hexadecyltrimethylammonium bromide (C{sub 16}TAB) as cationic surfactants, were determined at 298 K. For each transition curve, the conventional method of analysis which assumes a linear concentration dependence of the pre- and post-transition base lines, gave the most realistic values for {Delta}G{sub D}(H{sub 2}O). The results represent the increase in the denaturating power of surfactants with an increase in hydrocarbon chain length. The value of about 22.27 kJ . mol{sup -1} was obtained for {Delta}G{sub D}(H{sub 2}O) from transition curves. Subsequently, the retinol binding property of BLG as its functional indicator was investigated in the presence of these surfactants using the spectrofluorimeter titration method. The results represent the substantial enhancement of retinol binding affinity of BLG in the presence of these surfactants.

  16. An insight into the binding between ester-functionalized cationic Gemini surfactant and lysozyme

    Energy Technology Data Exchange (ETDEWEB)

    Patel, Rajan, E-mail: rpatel@jmi.ac.in; Maurya, Jitendra Kumar; Mir, Muzaffar Ul Hassan; Kumari, Meena; Maurya, Neha

    2014-10-15

    The interactions of cationic dodecyl betainate gemini (DBG) surfactant with lysozyme was studied by fluorescence, time resolved fluorescence, UV–visible, circular dichroism, and molecular docking methods. The results showed that the DBG quenched the fluorescence of lysozyme through static quenching mechanism as confirmed by time resolved spectroscopy. The Stern–Volmer quenching constant (K{sub sv}) and relevant thermodynamic parameters such as enthalpy change (ΔH), Gibbs free energy change (ΔG) and entropy change (ΔS) for interaction system were calculated at different temperatures. The results revealed that hydrophobic forces played a major role in the interactions process. The results of synchronous fluorescence, UV–visible and CD spectra demonstrated that the binding of DBG with lysozyme induces conformational changes in lysozyme. Moreover, the molecular modeling results shows the possible binding sites in the interaction system. - Highlights: • DBG quenches the fluorescence intensity of lysozyme and changes its conformation. • Quenching follows the static mechanism. • DBG are first used to investigate their effects on lysozyme. • Hydrophobic interactions played a major role in the binding process.

  17. Ceruloplasmin revisited: structural and functional roles of various metal cation-binding sites

    International Nuclear Information System (INIS)

    The three-dimensional molecular structure of human serum ceruloplasmin has been reinvestigated using X-ray synchrotron data collected at 100 K from a crystal frozen to liquid-nitrogen temperature. The three-dimensional molecular structure of human serum ceruloplasmin has been reinvestigated using X-ray synchrotron data collected at 100 K from a crystal frozen to liquid-nitrogen temperature. The resulting model, with an increase in resolution from 3.1 to 2.8 Å, gives an overall improvement of the molecular structure, in particular the side chains. In addition, it enables the clear definition of previously unidentified Ca2+-binding and Na+-binding sites. The Ca2+ cation is located in domain 1 in a configuration very similar to that found in the activated bovine factor Va. The Na+ sites appear to play a structural role in providing rigidity to the three protuberances on the top surface of the molecule. These features probably help to steer substrates towards the mononuclear copper sites prior to their oxidation and to restrict the size of the approaching substrate. The trinuclear copper centre appears to differ from the room-temperature structure in that a dioxygen moiety is bound in a similar way to that found in the endospore coat protein CotA from Bacillus subtilis

  18. Interaction of Sodium Hyaluronate with a Biocompatible Cationic Surfactant from Lysine: A Binding Study.

    Science.gov (United States)

    Bračič, Matej; Hansson, Per; Pérez, Lourdes; Zemljič, Lidija F; Kogej, Ksenija

    2015-11-10

    Mixtures of natural and biodegradable surfactants and ionic polysaccharides have attracted considerable research interest in recent years because they prosper as antimicrobial materials for medical applications. In the present work, interactions between the lysine-derived biocompatible cationic surfactant N(ε)-myristoyl-lysine methyl ester, abbreviated as MKM, and the sodium salt of hyaluronic acid (NaHA) are investigated in aqueous media by potentiometric titrations using the surfactant-sensitive electrode and pyrene-based fluorescence spectroscopy. The critical micelle concentration in pure surfactant solutions and the critical association concentration in the presence of NaHA are determined based on their dependence on the added electrolyte (NaCl) concentration. The equilibrium between the protonated (charged) and deprotonated (neutral) forms of MKM is proposed to explain the anomalous binding isotherms observed in the presence of the polyelectrolyte. The explanation is supported by theoretical model calculations of the mixed-micelle equilibrium and the competitive binding of the two MKM forms to the surface of the electrode membrane. It is suggested that the presence of even small amounts of the deprotonated form can strongly influence the measured electrode response. Such ionic-nonionic surfactant mixtures are a special case of mixed surfactant systems where the amount of the nonionic component cannot be varied independently as was the case for some of the earlier studies.

  19. A cation-π interaction at a phenylalanine residue in the glycine receptor binding site is conserved for different agonists

    DEFF Research Database (Denmark)

    Pless, Stephan Alexander; Hanek, Ariele P; Price, Kerry L;

    2011-01-01

    Cation-π interactions have been demonstrated to play a major role in agonist-binding in Cys-loop receptors. However, neither the aromatic amino acid contributing to this interaction nor its location is conserved among Cys-loop receptors. Likewise, it is not clear how many different agonists of a ...

  20. A Low Protein Binding Cationic Poly(2-oxazoline) as Non-Viral Vector

    KAUST Repository

    He, Zhijian

    2015-04-02

    © 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. Developing safe and efficient non-viral gene delivery systems remains a major challenge. We present a new cationic poly(2-oxazoline) (CPOx) block copolymer for gene therapy that was synthesized by sequential polymerization of non-ionic 2-methyl-2-oxazoline and a new 2-oxazoline monomer, 2-(N-methyl, N-Boc-amino)-methyl-2-oxazoline, followed by deprotection of the pendant secondary amine groups. Upon mixing with plasmid DNA (pDNA), CPOx forms small (diameter ≈80 nm) and narrowly dispersed polyplexes (PDI <0.2), which are stable upon dilution in saline and against thermal challenge. These polyplexes exhibited low plasma protein binding and very low cytotoxicity in vitro compared to the polyplexes of pDNA and poly(ethylene glycol)-b-poly(L-lysine) (PEG-b-PLL). CPOx/pDNA polyplexes at N/P = 5 bound considerably less plasma protein compared to polyplexes of PEG-b-PLL at the same N/P ratio. This is a unique aspect of the developed polyplexes emphasizing their potential for systemic delivery in vivo. The transfection efficiency of the polyplexes in B16 murine melanoma cells was low after 4 h, but increased significantly for 10 h exposure time, indicative of slow internalization of polyplexes. Addition of Pluronic P85 boosted the transfection using CPOx/pDNA polyplexes considerably. The low protein binding of CPOx/pDNA polyplexes is particularly interesting for the future development of targeted gene delivery.

  1. Cationic oligonucleotides can mediate specific inhibition of gene expression in Xenopus oocytes.

    OpenAIRE

    Bailey, C P; Dagle, J M; Weeks, D L

    1998-01-01

    Base-specific hydrogen bonding between an oligonucleotide and the purines in the major groove of a DNA duplex provide an approach to selective inhibition of gene expression. Oligonucleotide-mediated triplex formation in vivo may be enhanced by a number of different chemical modifications. We have previously described an in vitro analysis of triplex formation using oligonucleotides containing internucleoside phosphate linkages modified with the cation N , N -diethyl-ethylenediamine (DEED). Whe...

  2. Specific uranyl binding by macrocyclic ligands attached to resins

    International Nuclear Information System (INIS)

    Macrocyclic polydentates have attracted enormous attention from chemists because of their unique and significant characteristics of the strong and selective binding of a variety of metal ions. The metal binding is governed mostly by the size of the macroring and the nature of heteroatoms involved. The most important role of the macrocyclic structure is, in general, the so-called macrocyclic effect - to increase (making less negative) a large negative entropy change involved in the polydentate chelation. Basic strategy of uranium binding, is to design a ligand of very strong metal binding to take advantage of this macrocyclic effect, where number of chelating heteroatoms and their spatial arrangement is designed to be most appropriate for uranyl (UO22+) binding, since in natural sea water uranium is dissolved mostly in a form of uranyl carbonate. The following macrocylic ligands, hexamine, hexaketone, hexacarboxylic acid, were prepared and tested. The macrocyclic hexacarboxylic ligand was the most promising. The addition of hexacarboxylic acid to a uranyl tricarbonate solution gave a change of visible absorption due to the competitive formation of the uranyl complex. From this competitive binding, a relative formation constant was estimated to be 10-5, giving a log K/sub f/ value of 16.4 at 250C for the uranyl complex. This value is the largest among the hosts ever reported to bind uranyl ion.The selectivity of the macrocyclic hexacarboxylic ligand was also ascertained by testing with other metal cations. Results indicate that uranyl ions can be extracted efficiently from sea water using the hexacarboxylic acid ligands which are attached to a polymer insoluble in water

  3. Sunshine and specific binding of serotonin transporters in Finnish man

    International Nuclear Information System (INIS)

    Aim: Visible light (400-700 nm) exposure decreases melatonin, norepinephrine, and acetylcholine whereas cortisol, serotonin, CABA, and dopamine levels increase. Light could be of particular relevance in the pathophysiology of neuropsychiatric disorders such as winter type affective disorder. The aim of the present study was to measure seasonal variation of specific binding of serotonin transporters (SERT) in man. Material and Methods: Thirty six white Caucasian males were studied. Their mean age was 38 years (range: 19-64 years). All subjects were medically health. A dose of 185 MBq of [123I]nor-b-CIT (supplied by MAP Medical Technologies Oy, Tikkakoski, Finland) was intravenously injected. SPECT scans were performed on a triple-head Siemens Multi SPECT 3 gamma camera equipped with fan-beam collimators. Regions of interest were drawn onto the midbrain (free + non-specific + specific binding) and onto the cerebellum (free + non-specific binding). The specific binding of the midbrain was calculated as (midbrain-cerebellum)/cerebellum. The findings of the study subjects were grouped onto the 6 sub-groups (six subjects per sub-group: January, March, May, July, September and November). In addition, blood platelets content was followed up for 12 months in 18 healthy males. The maximal binding potential (Bmax: fmol/mg protein) of platelets was determined. Results: Dependence of the specific binding of SERT in the midbrain and Bmax of human blood platelets on daily sunshine is presented. The data suggest lower specific binding of SERT in summer than in winter although this difference did not reach a statistical significance due to a small number of study subjects. Conclusion: Visible light exposure can alter specific binding of SERT in Finnish healthy males. The findings of in vivo molecular imaging support seasonal variation of human blood platelets content

  4. 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.

  5. TAL Effector DNA-Binding Principles and Specificity.

    Science.gov (United States)

    Richter, Annekatrin; Streubel, Jana; Boch, Jens

    2016-01-01

    Transcription activator-like effectors (TALEs) are proteins with a unique DNA-binding domain that confers both a predictable and programmable specificity. The DNA-binding domain consists typically of 34-amino acid near-identical repeats. The repeats form a right-handed superhelical structure that wraps around the DNA double helix and exposes the variable amino acids at position 13 of each repeat to the sense strand DNA bases. Each repeat binds one base in a highly specific, non-overlapping, and comma-free fashion. Although TALE specificities are encoded in a simple way, sophisticated rules can be taken into account to build highly efficient DNA-binding modules for biotechnological use. PMID:26443210

  6. Xanthurenic acid binds to neuronal G-protein-coupled receptors that secondarily activate cationic channels in the cell line NCB-20.

    Science.gov (United States)

    Taleb, Omar; Maammar, Mohammed; Brumaru, Daniel; Bourguignon, Jean-Jacques; Schmitt, Martine; Klein, Christian; Kemmel, Véronique; Maitre, Michel; Mensah-Nyagan, Ayikoe Guy

    2012-01-01

    Xanthurenic acid (XA) is a metabolite of the tryptophan oxidation pathway through kynurenine and 3-hydroxykynurenine. XA was until now considered as a detoxification compound and dead-end product reducing accumulation of reactive radical species. Apart from a specific role for XA in the signaling cascade resulting in gamete maturation in mosquitoes, nothing was known about its functions in other species including mammals. Based upon XA distribution, transport, accumulation and release in the rat brain, we have recently suggested that XA may potentially be involved in neurotransmission/neuromodulation, assuming that neurons presumably express specific XA receptors. Recently, it has been shown that XA could act as a positive allosteric ligand for class II metabotropic glutamate receptors. This finding reinforces the proposed signaling role of XA in brain. Our present results provide several lines of evidence in favor of the existence of specific receptors for XA in the brain. First, binding experiments combined with autoradiography and time-course analysis led to the characterization of XA binding sites in the rat brain. Second, specific kinetic and pharmacological properties exhibited by these binding sites are in favor of G-protein-coupled receptors (GPCR). Finally, in patch-clamp and calcium imaging experiments using NCB-20 cells that do not express glutamate-induced calcium signals, XA elicited specific responses involving activation of cationic channels and increases in intracellular Ca(2+) concentration. Altogether, these results suggest that XA, acting through a GPCR-induced cationic channel modulatory mechanism, may exert excitatory functions in various brain neuronal pathways. PMID:23139790

  7. Xanthurenic acid binds to neuronal G-protein-coupled receptors that secondarily activate cationic channels in the cell line NCB-20.

    Directory of Open Access Journals (Sweden)

    Omar Taleb

    Full Text Available Xanthurenic acid (XA is a metabolite of the tryptophan oxidation pathway through kynurenine and 3-hydroxykynurenine. XA was until now considered as a detoxification compound and dead-end product reducing accumulation of reactive radical species. Apart from a specific role for XA in the signaling cascade resulting in gamete maturation in mosquitoes, nothing was known about its functions in other species including mammals. Based upon XA distribution, transport, accumulation and release in the rat brain, we have recently suggested that XA may potentially be involved in neurotransmission/neuromodulation, assuming that neurons presumably express specific XA receptors. Recently, it has been shown that XA could act as a positive allosteric ligand for class II metabotropic glutamate receptors. This finding reinforces the proposed signaling role of XA in brain. Our present results provide several lines of evidence in favor of the existence of specific receptors for XA in the brain. First, binding experiments combined with autoradiography and time-course analysis led to the characterization of XA binding sites in the rat brain. Second, specific kinetic and pharmacological properties exhibited by these binding sites are in favor of G-protein-coupled receptors (GPCR. Finally, in patch-clamp and calcium imaging experiments using NCB-20 cells that do not express glutamate-induced calcium signals, XA elicited specific responses involving activation of cationic channels and increases in intracellular Ca(2+ concentration. Altogether, these results suggest that XA, acting through a GPCR-induced cationic channel modulatory mechanism, may exert excitatory functions in various brain neuronal pathways.

  8. Pulse radiolysis and spectrophotometric studies on the binding of organic cations with heparin

    Science.gov (United States)

    Jakubowska, Małgorzata; Adamus, Jan; Gębicki, Jerzy; Marcinek, Andrzej; Sikora, Adam

    2014-06-01

    Here we present the spectroscopic and pulse radiolysis studies of the interactions of heparin and some organic cations:methylene blue (MB), 1-methylnicotinamide (MNA+), and its dimer 1,3-bis(1-methylnicotinamide)propane (bis(MNA+)).

  9. Crystal Structure of an Integron Gene Cassette-Associated Protein from Vibrio cholerae Identifies a Cationic Drug-Binding Module

    Energy Technology Data Exchange (ETDEWEB)

    Deshpande, Chandrika N.; Harrop, Stephen J.; Boucher, Yan; Hassan, Karl A.; Di Leo, Rosa; Xu, Xiaohui; Cui, Hong; Savchenko, Alexei; Chang, Changsoo; Labbate, Maurizio; Paulsen, Ian T.; Stokes, H.W.; Curmi, Paul M.G.; Mabbutt, Bridget C. (MIT); (UT-Australia); (Macquarie); (Toronto); (New South)

    2012-02-15

    The direct isolation of integron gene cassettes from cultivated and environmental microbial sources allows an assessment of the impact of the integron/gene cassette system on the emergence of new phenotypes, such as drug resistance or virulence. A structural approach is being exploited to investigate the modularity and function of novel integron gene cassettes. We report the 1.8 {angstrom} crystal structure of Cass2, an integron-associated protein derived from an environmental V. cholerae. The structure defines a monomeric beta-barrel protein with a fold related to the effector-binding portion of AraC/XylS transcription activators. The closest homologs of Cass2 are multi-drug binding proteins, such as BmrR. Consistent with this, a binding pocket made up of hydrophobic residues and a single glutamate side chain is evident in Cass2, occupied in the crystal form by polyethylene glycol. Fluorescence assays demonstrate that Cass2 is capable of binding cationic drug compounds with submicromolar affinity. The Cass2 module possesses a protein interaction surface proximal to its drug-binding cavity with features homologous to those seen in multi-domain transcriptional regulators. Genetic analysis identifies Cass2 to be representative of a larger family of independent effector-binding proteins associated with lateral gene transfer within Vibrio and closely-related species. We propose that the Cass2 family not only has capacity to form functional transcription regulator complexes, but represents possible evolutionary precursors to multi-domain regulators associated with cationic drug compounds.

  10. Crystal structure of an integron gene cassette-associated protein from Vibrio cholerae identifies a cationic drug-binding module.

    Directory of Open Access Journals (Sweden)

    Chandrika N Deshpande

    Full Text Available The direct isolation of integron gene cassettes from cultivated and environmental microbial sources allows an assessment of the impact of the integron/gene cassette system on the emergence of new phenotypes, such as drug resistance or virulence. A structural approach is being exploited to investigate the modularity and function of novel integron gene cassettes.We report the 1.8 Å crystal structure of Cass2, an integron-associated protein derived from an environmental V. cholerae. The structure defines a monomeric beta-barrel protein with a fold related to the effector-binding portion of AraC/XylS transcription activators. The closest homologs of Cass2 are multi-drug binding proteins, such as BmrR. Consistent with this, a binding pocket made up of hydrophobic residues and a single glutamate side chain is evident in Cass2, occupied in the crystal form by polyethylene glycol. Fluorescence assays demonstrate that Cass2 is capable of binding cationic drug compounds with submicromolar affinity. The Cass2 module possesses a protein interaction surface proximal to its drug-binding cavity with features homologous to those seen in multi-domain transcriptional regulators.Genetic analysis identifies Cass2 to be representative of a larger family of independent effector-binding proteins associated with lateral gene transfer within Vibrio and closely-related species. We propose that the Cass2 family not only has capacity to form functional transcription regulator complexes, but represents possible evolutionary precursors to multi-domain regulators associated with cationic drug compounds.

  11. Ancestral Protein Reconstruction Yields Insights into Adaptive Evolution of Binding Specificity in Solute-Binding Proteins.

    Science.gov (United States)

    Clifton, Ben E; Jackson, Colin J

    2016-02-18

    The promiscuous functions of proteins are an important reservoir of functional novelty in protein evolution, but the molecular basis for binding promiscuity remains elusive. We used ancestral protein reconstruction to experimentally characterize evolutionary intermediates in the functional expansion of the polar amino acid-binding protein family, which has evolved to bind a variety of amino acids with high affinity and specificity. High-resolution crystal structures of an ancestral arginine-binding protein in complex with l-arginine and l-glutamine show that the promiscuous binding of l-glutamine is enabled by multi-scale conformational plasticity, water-mediated interactions, and selection of an alternative conformational substate productive for l-glutamine binding. Evolution of specialized glutamine-binding proteins from this ancestral protein was achieved by displacement of water molecules from the protein-ligand interface, reducing the entropic penalty associated with the promiscuous interaction. These results provide a structural and thermodynamic basis for the co-option of a promiscuous interaction in the evolution of binding specificity.

  12. Human DC-SIGN binds specific human milk glycans.

    Science.gov (United States)

    Noll, Alexander J; Yu, Ying; Lasanajak, Yi; Duska-McEwen, Geralyn; Buck, Rachael H; Smith, David F; Cummings, Richard D

    2016-05-15

    Human milk glycans (HMGs) are prebiotics, pathogen receptor decoys and regulators of host physiology and immune responses. Mechanistically, human lectins (glycan-binding proteins, hGBP) expressed by dendritic cells (DCs) are of major interest, as these cells directly contact HMGs. To explore such interactions, we screened many C-type lectins and sialic acid-binding immunoglobulin-like lectins (Siglecs) expressed by DCs for glycan binding on microarrays presenting over 200 HMGs. Unexpectedly, DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) showed robust binding to many HMGs, whereas other C-type lectins failed to bind, and Siglec-5 and Siglec-9 showed weak binding to a few glycans. By contrast, most hGBP bound to multiple glycans on other microarrays lacking HMGs. An α-linked fucose residue was characteristic of HMGs bound by DC-SIGN. Binding of DC-SIGN to the simple HMGs 2'-fucosyl-lactose (2'-FL) and 3-fucosyl-lactose (3-FL) was confirmed by flow cytometry to beads conjugated with 2'-FL or 3-FL, as well as the ability of the free glycans to inhibit DC-SIGN binding. 2'-FL had an IC50 of ∼1 mM for DC-SIGN, which is within the physiological concentration of 2'-FL in human milk. These results demonstrate that DC-SIGN among the many hGBP expressed by DCs binds to α-fucosylated HMGs, and suggest that such interactions may be important in influencing immune responses in the developing infant. PMID:26976925

  13. Cationic solid-lipid nanoparticles can efficiently bind and transfect plasmid DNA

    NARCIS (Netherlands)

    Olbrich, C; Bakowsky, U; Muller, RH; Kneuer, C

    2001-01-01

    The suitability of cationically modified solid-lipid nanoparticles (SLN) as a novel transfection agent was investigated. SLN were produced by hot homogenisation using either Compritol ATO 888 or paraffin as matrix lipid, a mixture of Tween 80 and Span 85 as tenside and either EQ1 (NN-di-(beta-steaor

  14. Structural Determinants of Specific Lipid Binding to Potassium Channels

    NARCIS (Netherlands)

    Weingarth, M.H.; Prokofyev, A.; van der Cruijsen, E.A.W.; Nand, D.; Bonvin, A.M.J.J.; Pongs, O.; Baldus, M.

    2013-01-01

    We have investigated specific lipid binding to the pore domain of potassium channels KcsA and chimeric KcsAKv1.3 on the structural and functional level using extensive coarse-grained and atomistic molecular dynamics simulations, solid-state NMR, and single channel measurements. We show that, while K

  15. Computational redesign of endonuclease DNA binding and cleavage specificity

    Science.gov (United States)

    Ashworth, Justin; Havranek, James J.; Duarte, Carlos M.; Sussman, Django; Monnat, Raymond J.; Stoddard, Barry L.; Baker, David

    2006-06-01

    The reprogramming of DNA-binding specificity is an important challenge for computational protein design that tests current understanding of protein-DNA recognition, and has considerable practical relevance for biotechnology and medicine. Here we describe the computational redesign of the cleavage specificity of the intron-encoded homing endonuclease I-MsoI using a physically realistic atomic-level forcefield. Using an in silico screen, we identified single base-pair substitutions predicted to disrupt binding by the wild-type enzyme, and then optimized the identities and conformations of clusters of amino acids around each of these unfavourable substitutions using Monte Carlo sampling. A redesigned enzyme that was predicted to display altered target site specificity, while maintaining wild-type binding affinity, was experimentally characterized. The redesigned enzyme binds and cleaves the redesigned recognition site ~10,000 times more effectively than does the wild-type enzyme, with a level of target discrimination comparable to the original endonuclease. Determination of the structure of the redesigned nuclease-recognition site complex by X-ray crystallography confirms the accuracy of the computationally predicted interface. These results suggest that computational protein design methods can have an important role in the creation of novel highly specific endonucleases for gene therapy and other applications.

  16. A specific binding site recognizing a fragment of angiotensin II in bovine adrenal cortex membranes.

    Science.gov (United States)

    Bernier, S G; Fournier, A; Guillemette, G

    1994-12-12

    We have characterized a specific binding site for angiotensin IV in bovine adrenal cortex membranes. Pseudo-equilibrium studies at 37 degrees C for 2 h have shown that this binding site recognizes angiotensin IV with a high affinity (Kd = 0.24 +/- 0.03 nM). The binding site is saturable and relatively abundant (maximal binding capacity around 0.5 pmol/mg protein). Non-equilibrium kinetic analyses at 37 degrees C revealed a calculated kinetic Kd of 47 pM. The binding site is pharmacologically distinct from the classic angiotensin receptors AT1 or AT2. Competitive binding studies with bovine adrenal cortex membranes demonstrated the following rank order of effectiveness: angiotensin IV (Val-Tyr-Ile-His-Pro-Phe) = angiotensin II-(3-7) (Val-Tyr-Ile-His-Pro) > angiotensin III (Arg-Val-Tyr-Ile-His-Pro-Phe) > or = angiotensin II-(4-7) (Tyr-Ile-His-Pro) > angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) > angiotensin II-(1-6) (Asp-Arg-Val-Tyr-Ile-His) > angiotensin II-(4-8) (Tyr-Ile-His-Pro-Phe) > > > angiotensin II-(3-6) (Val-Tyr-Ile-His), angiotensin II-(4-6) (Tyr-Ile-His), L-158,809 (5,7-dimethyl-2-ethyl-3-[(2'(1-H-tetrazol-5-yl)[1,1'-biphenyl]-4-y l) methyl]-3-H-imidazo[4,5-beta]pyridine H2O) and PD 123319 (1-[4-(dimethylamino)3-methylphenyl]methyl-5-(diphenylacetyl)4,5,6 ,7- tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid). The divalent cations Mg2+ and Ca2+ were shown to diminish the binding of 125I-angiotensioffn IV to bovine adrenal cortex membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. 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.

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

    Indian Academy of Sciences (India)

    Sumedha Roy; Shayoni Dutta; Kanika Khanna; Shruti Singla; Durai Sundar

    2012-07-01

    Zinc finger proteins interact via their individual fingers to three base pair subsites on the target DNA. The four key residue positions −1, 2, 3 and 6 on the alpha-helix of the zinc fingers have hydrogen bond interactions with the DNA. Mutating these key residues enables generation of a plethora of combinatorial possibilities that can bind to any DNA stretch of interest. Exploiting the binding specificity and affinity of the interaction between the zinc fingers and the respective DNA can help to generate engineered zinc fingers for therapeutic purposes involving genome targeting. Exploring the structure–function relationships of the existing zinc finger–DNA complexes can aid in predicting the probable zinc fingers that could bind to any target DNA. Computational tools ease the prediction of such engineered zinc fingers by effectively utilizing information from the available experimental data. A study of literature reveals many approaches for predicting DNA-binding specificity in zinc finger proteins. However, an alternative approach that looks into the physico-chemical properties of these complexes would do away with the difficulties of designing unbiased zinc fingers with the desired affinity and specificity. We present a physico-chemical approach that exploits the relative strengths of hydrogen bonding between the target DNA and all combinatorially possible zinc fingers to select the most optimum zinc finger protein candidate.

  19. 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.

  20. Specific Features of Motion of Cations and Anions in Electrolyte Solutions

    CERN Document Server

    Bulavin, L A; Malomuzh, M P; Pankratov, K M

    2012-01-01

    The nature of mobility of ions and water molecules in dilute aqueous solutions of electrolytes (at most fifteen water molecules per ion) is investigated. It is shown that the behavior of the mobility coefficients of water molecules and ions, as well as the self-diffusion coefficients of water molecules, are determined by the radii of their hard shells rather than by the effect of the hydrogen bond network. It is established that the influence of hydration effects on the density of the system and the self-diffusion coefficients of water molecules does not exceed several per cent. Based on microscopic concepts, it is shown that the different behaviors of a $\\rm K^{+}$ cation and an $\\rm F^{-}$ anion with equal rigid radii are in good agreement with specific features of the intermolecular interaction described by the generalized Stillinger--David potential.

  1. Maitotoxin-induced cell death cascade in bovine aortic endothelial cells: divalent cation specificity and selectivity.

    Science.gov (United States)

    Wisnoskey, Brian J; Estacion, Mark; Schilling, William P

    2004-08-01

    The maitotoxin (MTX)-induced cell death cascade in bovine aortic endothelial cells (BAECs), a model for Ca(2+) overload-induced toxicity, reflects three sequential changes in plasmalemmal permeability. MTX initially activates Ca(2+)-permeable, nonselective cation channels (CaNSC) and causes a massive increase in cytosolic free Ca(2+) concentration ([Ca(2+)](i)). This is followed by the opening of large endogenous cytolytic/oncotic pores (COP) that allow molecules ionomycin and were significantly delayed in BAPTA-loaded cells. Experiments at the single-cell level revealed that Ba(2+) not only delayed the time to cell lysis but also caused desynchronization of the lytic phase. Last, membrane blebs, which were numerous and spherical in Ca(2+)-containing solutions, were poorly defined and greatly reduced in number in the presence of Ba(2+). Taken together, these results suggest that intracellular high-affinity Ca(2+)-binding proteins are involved in the MTX-induced changes in plasmalemmal permeability that are responsible for cell demise. PMID:15044153

  2. Cations modulate the substrate specificity of bifunctional class I O-methyltransferase from Ammi majus.

    Science.gov (United States)

    Lukacin, Richard; Matern, Ulrich; Specker, Silvia; Vogt, Thomas

    2004-11-19

    Caffeoyl-coenzyme A O-methyltransferase cDNA was cloned from dark-grown Ammi majus L. (Apiaceae) cells treated with a crude fungal elicitor and the open reading frame was expressed in Escherichia coli. The translated polypeptide of 27.1-kDa shared significant identity to other members of this highly conserved class of proteins and was 98.8% identical to the corresponding O-methyltransferase from parsley. For biochemical characterization, the recombinant enzyme could be purified to apparent homogeneity by metal-affinity chromatography, although the recombinant enzyme did not contain any affinity tag. Based on sequence analysis and substrate specificity, the enzyme classifies as a cation-dependent O-methyltransferase with pronounced preference for caffeoyl coenzyme A, when assayed in the presence of Mg2+-ions. Surprisingly, however, the substrate specificity changed dramatically, when Mg2+ was replaced by Mn2+ or Co2+ in the assays. This effect could point to yet unknown functions and substrate specificities in situ and suggests promiscuous roles for the lignin specific cluster of plant O-methyltransferases.

  3. Changes in BQCA Allosteric Modulation of [(3)H]NMS Binding to Human Cortex within Schizophrenia and by Divalent Cations.

    Science.gov (United States)

    Dean, Brian; Hopper, Shaun; Conn, P Jeffrey; Scarr, Elizabeth

    2016-05-01

    Stimulation of the cortical muscarinic M1 receptor (CHRM1) is proposed as a treatment for schizophrenia, a hypothesis testable using CHRM1 allosteric modulators. Allosteric modulators have been shown to change the activity of CHRMs using cloned human CHRMs and CHRM knockout mice but not human CNS, a prerequisite for them working in humans. Here we show in vitro that BQCA, a positive allosteric CHRM1 modulator, brings about the expected change in affinity of the CHRM1 orthosteric site for acetylcholine in human cortex. Moreover, this effect of BQCA is reduced in the cortex of a subset of subjects with schizophrenia, separated into a discrete population because of a profound loss of cortical [(3)H]pirenzepine binding. Surprisingly, there was no change in [(3)H]NMS binding to the cortex from this subset or those with schizophrenia but without a marked loss of cortical CHRM1. Hence, we explored the nature of [(3)H]pirenzepine and [(3)H]NMS binding to human cortex and showed total [(3)H]pirenzepine and [(3)H]NMS binding was reduced by Zn(2+), acetylcholine displacement of [(3)H]NMS binding was enhanced by Mg(2+) and Zn(2+), acetylcholine displacement of [(3)H]pirenzepine was reduced by Mg(2+) and enhanced by Zn(2+), whereas BQCA effects on [(3)H]NMS, but not [(3)H]pirenzepine, binding was enhanced by Mg(2+) and Zn(2+). These data suggest the orthosteric and allosteric sites on CHRMs respond differently to divalent cations and the effects of allosteric modulation of the cortical CHRM1 is reduced in a subset of people with schizophrenia, a finding that may have ramifications for the use of CHRM1 allosteric modulators in the treatment of schizophrenia.

  4. Sequence-specific binding of a hormonally regulated mRNA binding protein to cytidine-rich sequences in the lutropin receptor open reading frame.

    Science.gov (United States)

    Kash, J C; Menon, K M

    1999-12-21

    In previous studies, a lutropin receptor mRNA binding protein implicated in the hormonal regulation of lutropin receptor mRNA stability was identified. This protein, termed LRBP-1, was shown by RNA gel electrophoretic mobility shift assay to specifically interact with lutropin receptor RNA sequences. The present studies have examined the specificity of lutropin receptor mRNA recognition by LRBP-1 and mapped the contact site by RNA footprinting and by site-directed mutagenesis. LRBP-1 was partially purified by cation-exchange chromatography, and the mRNA binding properties of the partially purified LRBP-1 were examined by RNA gel electrophoretic mobility shift assay and hydroxyl-radical RNA footprinting. These data showed that the LRBP-1 binding site is located between nucleotides 203 and 220 of the receptor open reading frame, and consists of the bipartite polypyrimidine sequence 5'-UCUC-X(7)-UCUCCCU-3'. Competition RNA gel electrophoretic mobility shift assays demonstrated that homoribopolymers of poly(rC) were effective RNA binding competitors, while poly(rA), poly(rG), and poly(rU) showed no effect. Mutagenesis of the cytidine residues contained within the LRBP-1 binding site demonstrated that all the cytidines in the bipartite sequence contribute to LRBP-1 binding specificity. Additionally, RNA gel electrophoretic mobility supershift analysis showed that LRBP-1 was not recognized by antibodies against two well-characterized poly(rC) RNA binding proteins, alphaCP-1 and alphaCP-2, implicated in the regulation of RNA stability of alpha-globin and tyrosine hydroxylase mRNAs. In summary, we show that partially purified LRBP-1 binds to a polypyrimidine sequence within nucleotides 203 and 220 of lutropin receptor mRNA with a high degree of specificity which is indicative of its role in posttranscriptional control of lutropin receptor expression.

  5. Conformations of Cationized Peptides. Determination of Ligand Binding Geometries by Irmpd Spectroscopy

    Science.gov (United States)

    Dunbar, Robert C.; Steill, Jeffrey; Oomens, Jos; Polfer, Nick C.

    2009-06-01

    Spectroscopic study of the conformations of metalated amino acids has mapped out in some detail the preferences for canonical (charge solvated) versus zwitterionic (salt bridge) conformations. Corresponding studies of larger peptides are now possible. Here are described results for several singly and doubly charged metal ions with dipeptides and tripeptides. Factors including ion charge, size of cation, and side chain identity and sequence are found to be conformational determinants. IRMPD spectra of the ions were acquired by irradiating the cell with infrared light from the FELIX free electron laser at wavelengths in the approximate range 500 to 1900 cm^{-1}.

  6. Specific endothelial binding and tumor uptake of radiolabeled angiostatin

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Kyung-Han; Song, Sung Hee; Paik, Jin-Young; Byun, Sang Sung; Lee, Sang-Yoon; Choe, Yearn Seong; Kim, Byung-Tae [Department of Nuclear Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwondong, Kangnamgu, Seoul (Korea)

    2003-07-01

    Angiostatin (AS) is a potent antiangiogenic agent which inhibits tumor growth through specific action on proliferating endothelial cells. Imaging of radiolabeled AS would enhance our knowledge on the pharmacokinetics of AS and might provide useful information relating to tumor neovasculature. We therefore investigated the potential of radiolabeled AS as a novel tumor imaging agent. Human angiostatin was radioiodine labeled using the lactoperoxidase method. Competition binding studies showed a dose-dependent inhibition of {sup 125}I-AS binding to endothelial cells by excess unlabeled AS, and a displacement curve demonstrated that specific binding was dose dependent and saturable, with a K{sub d} value of 169 nM. Gel analysis showed that {sup 125}I-AS remained stable in serum for up to 24 h without significant degradation. Intravenously injected {sup 125}I-AS in rats was cleared from the blood in an exponential fashion. Biodistribution data from human colon cancer-bearing Balb/C nude mice showed high uptake in the kidneys, stomach, liver, and lungs. Tumor uptake was 3.2{+-}0.7, 2.6{+-}0.2, and 1.7{+-}0.2%ID/g at 2, 4, and 9 h after injection, respectively. Tumor to muscle count ratio increased from 3.1{+-}0.5 at 2 h to 4.4{+-}0.5 at 9 h. Serial scintigraphy from 1 to 5 h after {sup 123}I-AS injection demonstrated high uptake in the kidneys and bladder, consistent with renal excretion. There was clear demarcation of tumor by 1 h, with gradual increase in contrast over time (4-h tumor to contralateral thigh ratio =4.7{+-}1.1). Thus, radioiodine-labeled angiostatin binds specifically to endothelial cells and has potential as a novel tumor imaging agent. (orig.)

  7. Expression and divalent cation binding properties of the novel chemotactic inflammatory protein psoriasin

    DEFF Research Database (Denmark)

    Vorum, H; Madsen, Peder; Rasmussen, H H;

    1996-01-01

    Psoriasin is a novel chemotactic inflammatory protein that possesses weak similarity to the S100 family members of Ca(2+)-binding proteins, and that is highly up-regulated in hyperproliferative psoriatic keratinocytes. Here we have used the psoriasin cDNA to express recombinant human (rh) psoriasin...... in Escherichia coli as a fusion protein containing a hexa His tag and a factor Xa cleavage site in the NH2-terminus. The protein was purified by affinity chromatography on Ni(2+)-nitrilotriacetic acid agarose, digested with factor Xa, further purified by ion-exchange chromatography and characterized by two...... in KCl and four in NaCl, with an affinity just below 1 x 10(4) M-1 for the first molecule. Thus psoriasin does not bind significant amounts of Zn2+ at physiological concentrations. Mg2+ and Ca2+ are bound anti-cooperatively and binding of each of the ions (Ca2+, Zn2+, or Mg2+), is accompanied...

  8. [Cationic specificity of a Ca2+-accumulating system in smooth muscle cell mitochondria].

    Science.gov (United States)

    Veklich, T O; Kosterin, S O; Shynlova, O P

    2002-01-01

    In the experiments conducted with application of an isotopic technique (45Ca2+) on the myometrium cells suspension treated by digitonin solution (0.1 mg/ml) some properties of Ca ions accumulation system in the mitochondria--cationic and substrate specificity as well as effects of Mg2+ and some other bivalent metals ions on the Ca2+ accumulation velocity have been estimated. Ca ions accumulation from the incubation medium containing 3 mM sodium succinate Na, 2 mM Pi (as potassium K(+)-phosphate buffer, pH 7.4 at 37 degrees C), 0.01 mM (40CaCl2 + 45CaCl2) and 100 nM thapsigargin--selective inhibiting agent of endoplasmatic reticulum calcium pump were demonstrated as detected just only in presence of Mg, while not Ni, Co or Cu ions. The increase of Mg2+ concentration from 1 x 10(-6) to 10(-3) M induced the ATP dependent transport activation in the myometrium mitochondria. Under [Mg2+] increase till 40 mM this cation essentially decreased Ca2+ accumulation (by 65% from the maximal value). The optimum for Ca2+ transport in the myometrium cells suspension is Mg2+ 10 mM concentration. Ka activation apparent constant along Mg2+ value (in presence 3 mM ATP and 3 mM sodium succinate) is 4.27 mM. The above listed bivalent metals decreased Mg2+, ATP-dependent accumulation of calcium, values of inhibition apparent constants for ions Co2+, Ni2+ and Cu2+ were--2.9 x 10(-4) M, 5.1 x 10(-5) M and 4.2 x 10(-6) M respectively. For Mg2+, ATP-dependent Ca2+ transport in the uterus myocytes mitocondria a high substrate specificity is a characteristic phenomenon in elation to ATP: GTP, CTP and UTP practically fail to provide for Ca accumulation process. PMID:12199098

  9. A new bile acid-derived lariat-ether: Design, synthesis and cation binding properties

    Indian Academy of Sciences (India)

    P Babu; Uday Maitra

    2003-10-01

    A new chola lariat ether (1, a 21-crown-6) was constructed from readily available precursors. The association constant of compound 1 with alkali metal picrates was measured using Cram’s extraction protocol. Evidence is presented for the involvement of the 3-methoxy group for the complexation. Energy minimised structures show that the A-ring gets slightly distorted upon metal ion binding.

  10. Radiation stability of cations in ionic liquids. 5. Task-specific ionic liquids consisting of biocompatible cations and the puzzle of radiation hypersensitivity.

    Science.gov (United States)

    Shkrob, Ilya A; Marin, Timothy W; Wishart, James F; Grills, David C

    2014-09-01

    In 1953, an accidental discovery by Melvin Calvin and co-workers provided the first example of a solid (the α-polymorph of choline chloride) showing hypersensitivity to ionizing radiation: under certain conditions, the radiolytic yield of decomposition approached 5 × 10(4) per 100 eV (which is 4 orders of magnitude greater than usual values), suggesting an uncommonly efficient radiation-induced chain reaction. Twenty years later, the still-accepted mechanism for this rare condition was suggested by Martyn Symons, but no validation for this mechanism has been supplied. Meanwhile, ionic liquids and deep eutectic mixtures that are based on choline, betainium, and other derivitized natural amino compounds are presently finding an increasing number of applications as diluents in nuclear separations, where the constituent ions are exposed to ionizing radiation that is emitted by decaying radionuclides. Thus, the systems that are compositionally similar to radiation hypersensitive solids are being considered for use in high radiation fields, where this property is particularly undesirable! In Part 5 of this series on organic cations, we revisit the phenomenon of radiation hypersensitivity and explore mechanistic aspects of radiation-induced reactions involving this class of task-specific, biocompatible, functionalized cations, both in ionic liquids and in reference crystalline compounds. We demonstrate that Symons' mechanism needs certain revisions and rethinking, and suggest its modification. Our reconsideration suggests that there cannot be conditions leading to hypersensitivity in ionic liquids.

  11. Interfacial Binding of Divalent Cations to Calixarene-Based Langmuir Monolayers

    Energy Technology Data Exchange (ETDEWEB)

    Tulli, Ludovico G.; Wang, Wenjie; Lindemann, William R.; Kuzmenko, Ivan; Meier, Wolfgang; Vaknin, David; Shahgaldian, Patrick

    2015-02-20

    The interactions of Langmuir monolayers produced through the self-assembly of an amphiphilic p-carboxycalix[4]arene (1) with a series of divalent, fourth-period transition metals, at the air-water interface, were investigated. Changes in the interfacial behavior of 1 in response to the presence of CuCl2, CoCl2, MnCl2, and NiCl2 were studied by means of Langmuir compression isotherms and Brewster angle microscopy (BAM). The measurements revealed that the self-assembly properties of 1 are significantly affected by Cu2+ ions. The interactions of 1-based monolayers with Co2+ and Cu2+ ions were further investigated by means of synchrotron radiation-based X-ray reflectivity (XRR), X-ray near-total-reflection fluorescence (XNTRF), and grazing incidence X-ray diffraction (GIXD). XNTRF and XRR analyses revealed that the monolayer of 1 binds more strongly to Cu2+ than Co2+ ions. In the presence of relatively high concentrations of Cu2+ ions in the subphase (1.4 × 10-3 M), XNTRF exhibited anomalous depth profile behavior and GIXD measurements showed considerably strong diffuse scattering. Both measurements suggest the formation of Cu2+ clusters contiguous to the monolayer of 1.

  12. Describing the Peptide Binding Specificity of HLA-C

    DEFF Research Database (Denmark)

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

    for 5 HLA-C molecules and for all, but one, molecule we find a high frequency of binders, >70%, among these peptides. To extend the examined peptide space, we use bioinformatic prediction tools to search for additional binders. Finally, we update our prediction tool, NetMHCpan, with the HLA-C affinity......Human leukocyte antigen (HLA) presents peptides to T-cells for immune scrutiny. Whereas HLA-A and -B have been described in great detail, HLA-C has received much less attention. Here, to increase the coverage of HLA-C and the accuracy of the corresponding tools, we have generated HLA-C molecules......; peptide-binding assays, data and predictors; and tetramers; representing the most prevalent HLA-C molecules. We have combined positional scanning combinatorial peptide library (PSCPL) with a homogenous high-throughput dissociation assay and generated specificity matrices for 11 different HLA-C molecules...

  13. Cationic liposomal drug delivery system for specific targeting of human cd14+ monocytes in whole blood

    DEFF Research Database (Denmark)

    2013-01-01

    This invention concerns a liposome comprising lipids and at least one active ingredient, wherein at least one of the lipids is a cationic lipid; said liposome exhibiting a net positive charge at physiological conditions at which said liposome preferentially adheres to monocytes in freshly drawn b...

  14. Tissue-specific regulation of potassium homeostasis by high doses of cationic amino acids.

    Science.gov (United States)

    Cremades, Asunción; Del Rio-Garcia, Jesús; Lambertos, Ana; López-Garcia, Carlos; Peñafiel, Rafael

    2016-01-01

    The administration of l-arginine hydrochloride has been used for testing pituitary secretion in humans, and as an experimental model for induction of acute pancreatitis in rats and mice. Whereas in the first case, the administration of the amino acid is associated with hiperkalemia, in the model of acute pancreatitis no data are available on possible changes in potassium homeostasis. The present study shows that the acute administration to mice of l-arginine hydrochloride or other cationic amino acids almost duplicate plasma potassium levels. This effect was associated to a marked decrease of tissue potassium in both pancreas and liver. No changes were found in other tissues. These changes cannot be ascribed to the large load of chloride ions, since similar effects were produced when l-ornithine aspartate was administered. The changes in potassium levels were dependent on the dose. The displacement of intracellular potassium from the liver and pancreas to the extracellular compartment appears to be dependent on the entry of the cationic amino acid, since the administration of an equivalent dose of alfa-difluoromethyl ornithine HCl (DFMO), a non physiological analog of l-ornithine, which is poorly taken by the tissues in comparison with the physiological cationic amino acids, did not produce any change in potassium levels in pancreas and liver. The analyses of the expression of cationic amino acid transporters (CAT) suggest that the CAT-2 transporter may be implicated in the potassium/cationic amino acid interchange in liver and pancreas. The possible physiological or pathological relevance of these findings is discussed. PMID:27330882

  15. Quantitative analysis of pheromone-binding protein specificity

    OpenAIRE

    Katti, S.; Lokhande, N.; D González; 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...

  16. Tissue-specific regulation of potassium homeostasis by high doses of cationic amino acids

    OpenAIRE

    Cremades, Asunción; del Rio-Garcia, Jesús; Lambertos, Ana; López-Garcia, Carlos; Peñafiel, Rafael

    2016-01-01

    The administration of l-arginine hydrochloride has been used for testing pituitary secretion in humans, and as an experimental model for induction of acute pancreatitis in rats and mice. Whereas in the first case, the administration of the amino acid is associated with hiperkalemia, in the model of acute pancreatitis no data are available on possible changes in potassium homeostasis. The present study shows that the acute administration to mice of l-arginine hydrochloride or other cationic am...

  17. Specific ion effects on the properties of cationic Gemini surfactant monolayers

    Energy Technology Data Exchange (ETDEWEB)

    Alejo, T.; Merchan, M.D.; Velazquez, M.M., E-mail: mvsal@usal.es

    2011-06-01

    The effects of some anions of the Hofmeister series and different divalent cations of alkaline earth metals on the properties of Langmuir monolayers of the cationic Gemini surfactant ethyl-bis (dimethyl octadecylammonium bromide) have been investigated. Surface pressure and potential isotherms at the air-water interface were obtained on aqueous subphases containing sodium salts with several anions of the Hofmeister series (Cl{sup -}, NO{sub 3}{sup -}, Br{sup -}, I{sup -}, ClO{sub 4}{sup -}, and SCN{sup -}). The influence of the investigated anions on the monolayer properties can be ordered according to the Hofmeister series with a change in the order between bromide and nitrate anions. On the other hand, for a given anion, the cation of the salt also influences the surface properties of the Langmuir films. The monolayers can be transferred onto mica by the Langmuir-Blodgett technique and then the Langmuir-Blodgett films were characterized by atomic force microscopy (AFM). The AFM images show that the molecules become more closely packed and nearly vertical to the surface when anions screen the electric charge of the surfactant molecules.

  18. High DNA-Binding Affinity and Gene-Transfection Efficacy of Bioreducible Cationic Nanomicelles with a Fluorinated Core.

    Science.gov (United States)

    Wang, Long-Hai; Wu, De-Cheng; Xu, Hang-Xun; You, Ye-Zi

    2016-01-11

    During the last two decades, cationic polymers have become one of the most promising synthetic vectors for gene transfection. However, the weak interactions formed between DNA and cationic polymers result in low transfection efficacy. Furthermore, the polyplexes formed between cationic polymers and DNA generally exhibit poor stability and toxicity because of the large excess of cationic polymer typically required for complete DNA condensation. Herein, we report the preparation of a novel class of bioreducible cationic nanomicelles by the use of disulfide bonds to connect the cationic shell to the fluorocarbon core. These bioreducible nanomicelles form strong interactions with DNA and completely condense DNA at an N/P ratio of 1. The resulting nanomicelle/DNA polyplexes exhibited high biocompatibility and performed very effectively as a gene-delivery system.

  19. 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...

  20. Specific binding-adsorbent assay method and test means

    International Nuclear Information System (INIS)

    A description is given of an improved specific binding assay method and test means employing a nonspecific adsorbent for the substance to be determined, particularly hepatitis B surface (HBsub(s)) antigen, in its free state or additionally in the form of its immune complex. The invention is illustrated by 1) the radioimmunoadsorbent assay for HBsub(s) antigen, 2) the radioimmunoadsorbent assay for HBsub(s) antigen in the form of immune complex with antibody, 3) a study of adsorption characteristics of various anion exchange materials for HBsub(s) antigen, 4) the use of hydrophobic adsorbents in a radioimmunoadsorbent assay for HBsub(s) antigen and 5) the radioimmunoadsorbent assay for antibody to HBsub(s) antigen. The advantages of the present method for detecting HBsub(s) antigen compared to previous methods include the manufacturing advantages of eliminating the need for insolubilised anti-HBsub(s) and the advantages of a single incubation step, fewer manipulations, storability of adsorbent materials, increased sensitivity and versatility of detecting HBsub(s) antigen in the form of its immune complex if desired. (U.K.)

  1. Structures and binding specificity of galactose- and mannose-binding lectins from champedak: differences from jackfruit lectins.

    Science.gov (United States)

    Gabrielsen, Mads; Abdul-Rahman, Puteri Shafinaz; Othman, Shatrah; Hashim, Onn H; Cogdell, Richard J

    2014-06-01

    Galactose-binding and mannose-binding lectins from the champedak fruit, which is native to South-east Asia, exhibit useful potential clinical applications. The specificity of the two lectins for their respective ligands allows the detection of potential cancer biomarkers and monitoring of the glycosylated state of proteins in human serum and/or urine. To fully understand and expand the use of these natural proteins, their complete sequences and crystal structures are presented here, together with details of sugar binding.

  2. Monoclonal antibody modification with chelate-linked high-molecular-weight polymers: major increases in polyvalent cation binding without loss of antigen binding

    Energy Technology Data Exchange (ETDEWEB)

    Torchilin, V.P.; Klibanov, A.L.; Nossiff, N.D.; Slinkin, M.A.; Strauss, H.W.; Haber, E.; Smirnov, V.N.; Khaw, B.A.

    1987-06-01

    Polyethyleneimine or polylysines of differing molecular sizes were substituted with either EDTA or DTPA and then with succinic acid groups. These polymers were then reacted with the amino groups on myosin-specific monoclonal antibody or its Fab using a water soluble carbodiimide. The polymer-antibody complexes were capable of binding up to 150 di- or trivalent ions per mole (Mn++, Gd , or /sup 111/In ) without attendant loss of antigen binding. The polylysine derivatives of the intact antibody were rapidly cleared and sequestered in the liver, whereas the polylysine 14-kilodalton (kd) derivative of Fab was cleared from the circulation with minimal hepatic and kidney sequestration. This differed from the biodistribution of intact antimyosin or its Fab labeled with /sup 111/In via direct attachment of DTPA to the epsilon amino group of the lysyl residues. Applications in magnetic resonance and nuclear imaging are envisioned.

  3. Human DC-SIGN Binds Specific Human Milk Glycans

    Science.gov (United States)

    Noll, Alexander J.; Yu, Ying; Lasanajak, Yi; Duska-McEwen, Geralyn; Buck, Rachael H.; Smith, David F.; Cummings, Richard D.

    2016-01-01

    Human milk glycans (HMGs) are prebiotics, pathogen receptor decoys, and regulators of host physiology and immune responses. Mechanistically, human lectins (glycan-binding proteins, hGBPs) expressed by dendritic cells (DC) are of major interest, as these cells directly contact HMGs. To explore such interactions, we screened many C-type lectins and Siglecs expressed by DC for glycan binding on microarrays presenting over 200 HMGs. Unexpectedly, DC-SIGN showed robust binding to many HMGs, whereas other C-type lectins failed to bind, and Siglecs-5 and -9 showed weak binding to a few glycans. By contrast, most hGBPs bound to multiple glycans on other microarrays lacking HMGs. An α-linked fucose residue was characteristic of HMGs bound by DC-SIGN. Binding of DC-SIGN to the simple HMGs 2′-fucosyllactose (2′-FL) and 3-fucosyllactose (3-FL) was confirmed by flow cytometry to beads conjugated with 2′-FL or 3-FL, as well as the ability of the free glycans to inhibit DC-SIGN binding. 2′-FL had an IC50 of ~1 mM for DC-SIGN, which is within the physiological concentration of 2′-FL in human milk. These results demonstrate that DC-SIGN among the many hGBPs expressed by DC binds to α-fucosylated HMGs, and suggest that such interactions may be important in influencing immune responses in the developing infant. PMID:26976925

  4. Homogeneous fluorescent specific PCR for the authentication of medicinal snakes using cationic conjugated polymers.

    Science.gov (United States)

    Jiang, Chao; Yuan, Yuan; Liu, Libing; Hou, Jingyi; Jin, Yan; Huang, Luqi

    2015-11-05

    A label-free, homogenous and sensitive one-step method for the molecular authentication of medicinal snakes has been developed by combining a rapid PCR technique with water-soluble cationic conjugated polyelectrolytes (CCPs). Three medicinal snake materials (Deinagkistrodon acutus, Zaocys dhumnades and Bungarus multicinctus; a total of 35 specimens) and 48 snake specimens with similar morphologies and textures were clearly distinguished by the naked eye by utilizing a CCP-based assay in a high-throughput manner. The identification of medicinal snakes in patented Chinese drugs was successfully performed using this detection system. In contrast to previous fluorescence-labeled oligonucleotide detection and direct DNA stain hybridization assays, this method does not require designing dye-labeled primers, and unfavorable dimer fluorescence is avoided in this homogenous method.

  5. Microhydrated aromatic cluster cations: Binding motifs of 4-aminobenzonitrile-(H2O)n cluster cations with n ≤ 4

    Science.gov (United States)

    Schmies, Matthias; Miyazaki, Mitsuhiko; Fujii, Masaaki; Dopfer, Otto

    2014-12-01

    Infrared photodissociation (IRPD) spectra of mass-selected 4-aminobenzonitrile-(water)n cluster cations, ABN+-(H2O)n with n ≤ 4, recorded in the N-H and O-H stretch ranges are analyzed by quantum chemical calculations at the M06-2X/aug-cc-pVTZ level to determine the evolution of the initial microhydration process of this bifunctional aromatic cation in its ground electronic state. IRPD spectra of cold clusters tagged with Ar and N2 display higher resolution and allow for a clear-cut structural assignment. The clusters are generated in an electron impact source, which generates predominantly the most stable isomers. The IRPD spectra are assigned to single isomers for n = 1-3. The preferred cluster growth begins with sequential hydration of the two acidic NH protons of the amino group (n = 1-2), which is followed by attachment of secondary H2O ligands hydrogen-bonded to the first-shell ligands (n = 3-4). These symmetric and branched structures are more stable than those with a cyclic H-bonded solvent network. Moreover, in the size range n ≤ 4 the formation of a solvent network stabilized by strong cooperative effects is favored over interior ion hydration which is destabilized by noncooperative effects. The potential of the ABN+-H2O dimer is characterized in detail and supports the cluster growth derived from the IRPD spectra. Although the N-H bonds are destabilized by stepwise microhydration, which is accompanied by increasing charge transfer from ABN+ to the solvent cluster, no proton transfer to the solvent is observed for n ≤ 4.

  6. Microhydrated aromatic cluster cations: Binding motifs of 4-aminobenzonitrile-(H{sub 2}O){sub n} cluster cations with n ≤ 4

    Energy Technology Data Exchange (ETDEWEB)

    Schmies, Matthias; Dopfer, Otto, E-mail: dopfer@physik.tu-berlin.de [Institut für Optik und Atomare Physik, Technische Universität Berlin, Hardenbergstr. 36, 10623 Berlin (Germany); Miyazaki, Mitsuhiko; Fujii, Masaaki [Chemical Resources Laboratory, Tokyo Institute of Technology, Yokohama 226-8503 (Japan)

    2014-12-07

    Infrared photodissociation (IRPD) spectra of mass-selected 4-aminobenzonitrile-(water){sub n} cluster cations, ABN{sup +}-(H{sub 2}O){sub n} with n ≤ 4, recorded in the N–H and O–H stretch ranges are analyzed by quantum chemical calculations at the M06-2X/aug-cc-pVTZ level to determine the evolution of the initial microhydration process of this bifunctional aromatic cation in its ground electronic state. IRPD spectra of cold clusters tagged with Ar and N{sub 2} display higher resolution and allow for a clear-cut structural assignment. The clusters are generated in an electron impact source, which generates predominantly the most stable isomers. The IRPD spectra are assigned to single isomers for n = 1–3. The preferred cluster growth begins with sequential hydration of the two acidic NH protons of the amino group (n = 1–2), which is followed by attachment of secondary H{sub 2}O ligands hydrogen-bonded to the first-shell ligands (n = 3–4). These symmetric and branched structures are more stable than those with a cyclic H-bonded solvent network. Moreover, in the size range n ≤ 4 the formation of a solvent network stabilized by strong cooperative effects is favored over interior ion hydration which is destabilized by noncooperative effects. The potential of the ABN{sup +}-H{sub 2}O dimer is characterized in detail and supports the cluster growth derived from the IRPD spectra. Although the N–H bonds are destabilized by stepwise microhydration, which is accompanied by increasing charge transfer from ABN{sup +} to the solvent cluster, no proton transfer to the solvent is observed for n ≤ 4.

  7. Tropomodulin isoforms utilize specific binding functions to modulate dendrite development.

    Science.gov (United States)

    Gray, Kevin T; Suchowerska, Alexandra K; Bland, Tyler; Colpan, Mert; Wayman, Gary; Fath, Thomas; Kostyukova, Alla S

    2016-06-01

    Tropomodulins (Tmods) cap F-actin pointed ends and have altered expression in the brain in neurological diseases. The function of Tmods in neurons has been poorly studied and their role in neurological diseases is entirely unknown. In this article, we show that Tmod1 and Tmod2, but not Tmod3, are positive regulators of dendritic complexity and dendritic spine morphology. Tmod1 increases dendritic branching distal from the cell body and the number of filopodia/thin spines. Tmod2 increases dendritic branching proximal to the cell body and the number of mature dendritic spines. Tmods utilize two actin-binding sites and two tropomyosin (Tpm)-binding sites to cap F-actin. Overexpression of Tmods with disrupted Tpm-binding sites indicates that Tmod1 and Tmod2 differentially utilize their Tpm- and actin-binding sites to affect morphology. Disruption of Tmod1's Tpm-binding sites abolished the overexpression phenotype. In contrast, overexpression of the mutated Tmod2 caused the same phenotype as wild type overexpression. Proximity ligation assays indicate that the mutated Tmods are shuttled similarly to wild type Tmods. Our data begins to uncover the roles of Tmods in neural development and the mechanism by which Tmods alter neural morphology. These observations in combination with altered Tmod expression found in several neurological diseases also suggest that dysregulation of Tmod expression may be involved in the pathology of these diseases. © 2016 Wiley Periodicals, Inc. PMID:27126680

  8. B700, a murine melanoma-specific antigen, binds Vitamin D3; conservation of binding among albuminoid molecules

    International Nuclear Information System (INIS)

    B700, a murine melanoma-specific antigen, is a member of the serum albumin protein family. Other members of this family include serum albumin (SMA), a-fetoprotein (AFP), vitamin D binding protein (DBP), and C700. The primary structure and biochemical functions of B700, as well as its in vivo metabolic fate are largely unknown. The authors examined the functional characteristics of MSA, AFP, and DBP, and for their ability to specifically bind [3H]-1,25-dihydroxy-vitamin D3. Scatchard analysis revealed a single binding site for B700 with a Kd of 51,000 M and a Bmax of 4.51 x 10-7. There is no significant difference between the Kd and Bmax values among the albuminoid proteins. However, differences in the binding sites could be distinguished by competition of the 1,25-dihydroxy vitamin D3 with other steroids. 2nM of vitamin D3, vitamin D2, or estrogen competed for the specific binding of 1,25-dihydroxy vitamin D3 by B700 but not by DBP. The MSA binding site for 1,25 dihydroxy vitamin D3 more closely resembles that of DBP than B700. These data indicate that the binding function of the albuminoid proteins has been conserved in the B700 melanoma antigen

  9. Cellular internalization of a membrane binding two-photon probe by a complex of anionic diblock copolymer and cationic surfactant

    Science.gov (United States)

    Nag, Okhil Kumar; Woo, Han Young; Chen, Wei R.

    2012-03-01

    We report a two-photon (TP) absorbing molecular probe 1,4-bis(4'-(N,N-bis(6''-(N,N,N-trimethylammonium)hexyl)amino)-styryl)benzene tetrabromide (C1) and its interaction with cells upon encapsulation with polymeric vesicles. Two-photon microscopy (TPM) revealed that the free C1 specifically could bind to the plasma membrane and shows bright TP emission. However, C1 encapsulated with polymeric vesicles internalized into the cytosol. In addition, fluorescence quantum efficiency and TP cross section of encapsulated C1 enhanced by 2-fold. These results not only show useful guidelines for the development of efficient TP probes, but also underscore the possibility of using this type of nanostructure for intracellular delivery of the bioactive therapeutics.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  11. Characterization of the Escherichia coli prsA1-encoded mutant phosphoribosylpyrophosphate synthetase identifies a divalent cation-nucleotide binding site

    DEFF Research Database (Denmark)

    Bower, Stanley G.; Harlow, Kenneth W.; Switzer, Robert L.;

    1989-01-01

    The prsA1 allele, specifying a mutant Escherichia coli phosphoribosylpyrophosphate (PRPP) synthetase, has been cloned. The mutation was shown by nucleotide sequence analysis to result from substitution of Asp-128 (GAT) in the wild type by Ala (GCT) in prsA1. This alteration was confirmed......-fold lower affinity for Ca2+, as judged by the ability of Ca2+ to inhibit the reaction in the presence of 10 mM Mg2+. Wild type PRPP synthetase is subject to product inhibition by AMP, but AMP inhibition of the prsA1 mutant enzyme could not be detected. It has been previously proposed that a divalent...... cation binds to PRPP synthetase and serves as a bridge to the α-phosphate of ATP and AMP at the active site. The prsA1 mutation appears to alter this divalent cation site....

  12. Cationic Au Nanoparticle Binding with Plasma Membrane-like Lipid Bilayers: Potential Mechanism for Spontaneous Permeation to Cells Revealed by Atomistic Simulations

    DEFF Research Database (Denmark)

    Heikkila, E.; Martinez-Seara, H.; Gurtovenko, A. A.;

    2014-01-01

    Despite being chemically inert as a bulk material, nanoscale gold can pose harmful side effects to living organisms. In particular, cationic Au nanoparticles (AuNP+) of 2 nm diameter or less permeate readily through plasma membranes and induce cell death. We report atomistic simulations of cationic...... Au nanoparticles interacting with realistic membranes and explicit solvent using a model system that comprises two cellular compartments, extracellular and cytosolic, divided by two asymmetric lipid bilayers. The membrane-AuNP+ binding and membrane reorganization processes are discovered...... of the zwitterionic lipids and nanoparticle side groups in the contact area, giving rise to the initial stage of pore formation on the membrane surface. Such behavior is not seen on the cytosolic side, where AuNP+ is spontaneously captured by the negatively charged phosphatidylserine lipids that diffuse to enrich...

  13. Specific bindings of glycine peptides of distinctly different chain length on dynamic papain surfaces

    Science.gov (United States)

    Nishiyama, Katsuhiko

    2011-06-01

    We investigated the specific bindings of peptides of 1-10 glycine residues (1-10GLY) on dynamic papain surfaces via molecular dynamics and docking simulations. Although the binding specificities of 1-5GLY on papain fluctuated little with time, the binding specificities of 6-10GLY on papain considerably fluctuated with time. Some residues had a significant impact on bindings of 6-10GLY to sites near active center of papain, and some of their residues were specific for each 6GLY, 8GLY, and 10GLY. Modification of these specific residues should allow for control of binding specificity of 6GLY, 8GLY, and 10GLY to the active center.

  14. Statistical analysis of structural determinants for protein-DNA-binding specificity.

    Science.gov (United States)

    Corona, Rosario I; Guo, Jun-Tao

    2016-08-01

    DNA-binding proteins play critical roles in biological processes including gene expression, DNA packaging and DNA repair. They bind to DNA target sequences with different degrees of binding specificity, ranging from highly specific (HS) to nonspecific (NS). Alterations of DNA-binding specificity, due to either genetic variation or somatic mutations, can lead to various diseases. In this study, a comparative analysis of protein-DNA complex structures was carried out to investigate the structural features that contribute to binding specificity. Protein-DNA complexes were grouped into three general classes based on degrees of binding specificity: HS, multispecific (MS), and NS. Our results show a clear trend of structural features among the three classes, including amino acid binding propensities, simple and complex hydrogen bonds, major/minor groove and base contacts, and DNA shape. We found that aspartate is enriched in HS DNA binding proteins and predominately binds to a cytosine through a single hydrogen bond or two consecutive cytosines through bidentate hydrogen bonds. Aromatic residues, histidine and tyrosine, are highly enriched in the HS and MS groups and may contribute to specific binding through different mechanisms. To further investigate the role of protein flexibility in specific protein-DNA recognition, we analyzed the conformational changes between the bound and unbound states of DNA-binding proteins and structural variations. The results indicate that HS and MS DNA-binding domains have larger conformational changes upon DNA-binding and larger degree of flexibility in both bound and unbound states. Proteins 2016; 84:1147-1161. © 2016 Wiley Periodicals, Inc.

  15. Divalent cation tolerance protein binds to β-secretase and inhibits the processing of amyloid precursor protein

    Institute of Scientific and Technical Information of China (English)

    Runzhong Liu; Haibo Hou; Xuelian Yi; Shanwen Wu; Huan Zeng

    2013-01-01

    The deposition of amyloid-beta is a pathological hallmark of Alzheimer's disease. Amyloid-beta is derived from amyloid precursor protein through sequential proteolytic cleavages by β-secretase (beta-site amyloid precursor protein-cleaving enzyme 1) and γ-secretase. To further elucidate the roles of beta-site amyloid precursor protein-cleaving enzyme 1 in the development of Alzheimer's disease, a yeast two-hybrid system was used to screen a human embryonic brain cDNA library for proteins directly interacting with the intracellular domain of beta-site amyloid precursor protein-cleaving enzyme 1. A potential beta-site amyloid precursor protein-cleaving enzyme 1- interacting protein identified from the positive clones was divalent cation tolerance protein. Immunoprecipitation studies in the neuroblastoma cell line N2a showed that exogenous divalent cation tolerance protein interacts with endogenous beta-site amyloid precursor protein-cleaving enzyme 1. The overexpression of divalent cation tolerance protein did not affect beta-site amyloid precursor protein-cleaving enzyme 1 protein levels, but led to increased amyloid precursor protein levels in N2a/APP695 cells, with a concomitant reduction in the processing product amyloid precursor protein C-terminal fragment, indicating that divalent cation tolerance protein inhibits the processing of amyloid precursor protein. Our experimental findings suggest that divalent cation tolerance protein negatively regulates the function of beta-site amyloid precursor protein-cleaving enzyme 1. Thus, divalent cation tolerance protein could play a protective role in Alzheimer's disease.

  16. Rapid detection and purification of sequence specific DNA binding proteins using magnetic separation

    Directory of Open Access Journals (Sweden)

    TIJANA SAVIC

    2006-02-01

    Full Text Available In this paper, a method for the rapid identification and purification of sequence specific DNA binding proteins based on magnetic separation is presented. This method was applied to confirm the binding of the human recombinant USF1 protein to its putative binding site (E-box within the human SOX3 protomer. It has been shown that biotinylated DNA attached to streptavidin magnetic particles specifically binds the USF1 protein in the presence of competitor DNA. It has also been demonstrated that the protein could be successfully eluted from the beads, in high yield and with restored DNA binding activity. The advantage of these procedures is that they could be applied for the identification and purification of any high-affinity sequence-specific DNA binding protein with only minor modifications.

  17. An RNA aptamer possessing a novel monovalent cation-mediated fold inhibits lysozyme catalysis by inhibiting the binding of long natural substrates.

    Science.gov (United States)

    Padlan, Camille S; Malashkevich, Vladimir N; Almo, Steve C; Levy, Matthew; Brenowitz, Michael; Girvin, Mark E

    2014-04-01

    RNA aptamers are being developed as inhibitors of macromolecular and cellular function, diagnostic tools, and potential therapeutics. Our understanding of the physical nature of this emerging class of nucleic acid-protein complexes is limited; few atomic resolution structures have been reported for aptamers bound to their protein target. Guided by chemical mapping, we systematically minimized an RNA aptamer (Lys1) selected against hen egg white lysozyme. The resultant 59-nucleotide compact aptamer (Lys1.2minE) retains nanomolar binding affinity and the ability to inhibit lysozyme's catalytic activity. Our 2.0-Å crystal structure of the aptamer-protein complex reveals a helical stem stabilizing two loops to form a protein binding platform that binds lysozyme distal to the catalytic cleft. This structure along with complementary solution analyses illuminate a novel protein-nucleic acid interface; (1) only 410 Å(2) of solvent accessible surface are buried by aptamer binding; (2) an unusually small fraction (∼18%) of the RNA-protein interaction is electrostatic, consistent with the limited protein phosphate backbone contacts observed in the structure; (3) a single Na(+) stabilizes the loops that constitute the protein-binding platform, and consistent with this observation, Lys1.2minE-lysozyme complex formation takes up rather than displaces cations at low ionic strength; (4) Lys1.2minE inhibits catalysis of large cell wall substrates but not catalysis of small model substrates; and (5) the helical stem of Lys1.2minE can be shortened to four base pairs (Lys1.2minF) without compromising binding affinity, yielding a 45-nucleotide aptamer whose structure may be an adaptable protein binding platform.

  18. Synthesis and evaluation of [(18)F]-fluoromethyl triphenylphosphonium cation as a novel mitochondria-specific positron emission tomography tracer.

    Science.gov (United States)

    Zeng, Huahui; Wu, Xiangxiang; Song, Fahuan; Xu, Caiyun; Liu, Hao; Liu, Wendi

    2016-08-01

    We developed a radiosynthesis of the voltage sensitive tracer [(18)F]-fluoromethyltriphenylphosphonium cation ([(18)F]-FTPMP), giving high yield (30-34%, decay-corrected), radiochemical purity (>99%) and specific activity (about 760 GBq/μmol). [(18)F]-FTPMP had suitable lipophilicity (logP = 0.91 ± 0.03) and high in vivo/vitro stability. Biodistribution studies showed that [(18)F]-FTPMP had high heart uptake (>7%ID/g from 10 min to 120 min postinjection) and rapid clearance from the background. Clear cardiac images were obtained at different time periods, and the infarction areas could be detected sensitively with small-animal PET. The autoradiography and myocardial membrane potential studies confirmed the mitochondria specific of [(18)F]-FTPMP in rat myocardia. These excellent pharmacokinetic properties suggest [(18)F]-FTPMP is a promising mitochondria-specific tracer for clinical PET imaging of myocardial diseases associated with mitochondrial dysfunction. PMID:27123902

  19. Kinetic study of the binding of triplex-forming oligonucleotides containing partial cationic modifications to double-stranded DNA.

    Science.gov (United States)

    Hari, Yoshiyuki; Ijitsu, Shin; Akabane-Nakata, Masaaki; Yoshida, Takuya; Obika, Satoshi

    2014-07-15

    Several triplex-forming oligonucleotides (TFOs) partially modified with 2'-O-(2-aminoethyl)- or 2'-O-(2-guanidinoethyl)-nucleotides were synthesized and their association rate constants (kon) with double-stranded DNA were estimated by UV spectrophotometry. Introduction of cationic modifications in the 5'-region of the TFOs significantly increased the kon values compared to that of natural TFO, while no enhancement in the rate of triplex DNA formation was observed when the modifications were in the middle and at the 3'-region. The kon value of a TFO with three adjacent cationic modifications at the 5'-region was found to be 3.4 times larger than that of a natural one. These results provide useful information for overcoming the inherent sluggishness of triplex DNA formation. PMID:24865415

  20. Signal transduction of erythrocytes after specific binding of ecdysterone and cholesterol immobilized on nanodispersed magnetite

    Energy Technology Data Exchange (ETDEWEB)

    Mykhaylyk, O.M. E-mail: helek@iptelecom.net.ua; Kotzuruba, A.V.; Buchanevich, O.M.; Korduban, A.M.; Meged, E.F.; Gulaya, N.M

    2001-07-01

    Concurrent binding of cholesterol and ecdysterone immobilized on nanodispersed magnetite to intact rat erythrocytes was investigated. Several binding components on erythrocyte plasma membrane with different affinities were revealed in the range of 10{sup -15}-10{sup -8} M. The specific binding modulates signal transduction through adenylate cyclase and guanylate cyclase systems as manifested by the decrease in cAMP and increase in cGMP second messenger production.

  1. Target-specific binding of immunoliposomes in vivo

    International Nuclear Information System (INIS)

    Our group at the University of Tennessee has been concentrating on using monoclonal antibody for targeting of a liposomal drug carrier system. This paper discusses our initial effort to target these liposomes using an organ-specific monoclonal antibody. 9 refs., 9 figs

  2. Target-specific binding of immunoliposomes in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Holmberg, E.; Maruyama, K.; Kennel, S.; Klibanov, A.; Torchilin, V.; Ryan, U.; Huang, L.

    1989-01-01

    Our group at the University of Tennessee has been concentrating on using monoclonal antibody for targeting of a liposomal drug carrier system. This paper discusses our initial effort to target these liposomes using an organ-specific monoclonal antibody. 9 refs., 9 figs.

  3. 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.

  4. 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-...

  5. Characterization and DNA-binding specificities of Ralstonia TAL-like effectors

    KAUST Repository

    Li, Lixin

    2013-07-01

    Transcription activator-like effectors (TALEs) from Xanthomonas sp. have been used as customizable DNA-binding modules for genome-engineering applications. Ralstonia solanacearum TALE-like proteins (RTLs) exhibit similar structural features to TALEs, including a central DNA-binding domain composed of 35 amino acid-long repeats. Here, we characterize the RTLs and show that they localize in the plant cell nucleus, mediate DNA binding, and might function as transcriptional activators. RTLs have a unique DNA-binding architecture and are enriched in repeat variable di-residues (RVDs), which determine repeat DNA-binding specificities. We determined the DNA-binding specificities for the RVD sequences ND, HN, NP, and NT. The RVD ND mediates highly specific interactions with C nucleotide, HN interacts specifically with A and G nucleotides, and NP binds to C, A, and G nucleotides. Moreover, we developed a highly efficient repeat assembly approach for engineering RTL effectors. Taken together, our data demonstrate that RTLs are unique DNA-targeting modules that are excellent alternatives to be tailored to bind to user-selected DNA sequences for targeted genomic and epigenomic modifications. These findings will facilitate research concerning RTL molecular biology and RTL roles in the pathogenicity of Ralstonia spp. © 2013 The Author.

  6. CLONING AND CHARACTERIZATION OF A NUCLEAR, SITE-SPECIFIC SSDNA BINDING-PROTEIN

    NARCIS (Netherlands)

    SMIDT, MP; RUSSCHEN, B; SNIPPE, L; WIJNHOLDS, J; AB, G

    1995-01-01

    Estradiol inducible, liver-specific expression of the apoVLDL II gene is mediated through the estrogen receptor and a variety of other DNA-binding proteins. In the present study we report the cloning and characterisation of a single-strand DNA binding protein that interacts with the lower strand of

  7. Binding of ADAM12, a marker of skeletal muscle regeneration, to the muscle-specific actin-binding protein, alpha -actinin-2, is required for myoblast fusion

    DEFF Research Database (Denmark)

    Galliano, M F; Huet, C; Frygelius, J;

    2000-01-01

    differentiation. Using the yeast two-hybrid screen, we found that the muscle-specific alpha-actinin-2 strongly binds to the cytoplasmic tail of ADAM12. In vitro binding assays with GST fusion proteins confirmed the specific interaction. The major binding site for alpha-actinin-2 was mapped to a short sequence in...

  8. Multivalent glycobiomaterials for specific recognition and binding by lectins

    OpenAIRE

    Rosencrantz, Ruben R.

    2015-01-01

    Glycans are one of the most complex biomolecules and are used in nature for various tasks from cell-cell adhesions and communication to invasion or pathogenic processes. The most important term in protein-glycan interaction is the “multivalent effect”. This describes the boost in avidity as soon as the number of presented glycans in close proximity to each other is increased. In this work, we aimed for the design and evaluation of multivalent scaffolds based on polymers for the specific recog...

  9. Specificity and commonality of the phosphoinositide-binding proteome analyzed by quantitative mass spectrometry

    DEFF Research Database (Denmark)

    Jungmichel, Stephanie; Sylvestersen, Kathrine B; Choudhary, Chuna Ram;

    2014-01-01

    Phosphoinositides (PIPs) play key roles in signaling and disease. Using high-resolution quantitative mass spectrometry, we identified PIP-interacting proteins and profiled their binding specificities toward all seven PIP variants. This analysis revealed 405 PIP-binding proteins, which is greater...... than the total number of phospho- or ubiquitin-binding domains. Translocation and inhibitor assays of identified PIP-binding proteins confirmed that our methodology targets direct interactors. The PIP interactome encompasses proteins from diverse cellular compartments, prominently including the nucleus...

  10. Protective Action of Resveratrol in Human Skin: Possible Involvement of Specific Receptor Binding Sites

    OpenAIRE

    Stéphane Bastianetto; Yvan Dumont; Albert Duranton; Freya Vercauteren; Lionel Breton; Rémi Quirion

    2010-01-01

    BACKGROUND: Resveratrol is a plant-derived polyphenol with purported protecting action on various disorders associated with aging. It has been suggested that resveratrol could exert its protective action by acting on specific plasma membrane polyphenol binding sites (Han Y.S., et al. (2006) J Pharmacol Exp Ther 318:238-245). The purpose of this study was to investigate, in human skin, the possible existence of specific binding sites that mediate the protective action of resveratrol. METHODS A...

  11. Actinomycin D specifically inhibits the interaction between transcription factor Sp1 and its binding site.

    Science.gov (United States)

    Czyz, M; Gniazdowski, M

    1998-01-01

    The mode of action of many anticancer drugs involves DNA interactions. We here examine the ability of actinomycin D to alter the specific binding of transcription factors Spl and NFkappaB to their DNA sequences. Employing an electrophoretic mobility shift assay, it is shown that actinomycin D inhibits complex formation between nuclear proteins present in the extracts from stimulated human umbilical vein endothelial cells and the Sp1-binding site. Actinomycin D is also able to induce disruption of preformed DNA-protein complexes, pointing to the importance of an equilibrium of three components: actinomycin D, protein and DNA for drug action. The effect of actinomycin D is sequence-specific, since no inhibition is observed for interaction of nuclear proteins with the NFkappaB binding site. The results support the view that DNA-binding drugs displaying high sequence-selectivity can exhibit distinct effects on the interaction between DNA and different DNA-binding proteins. PMID:9701497

  12. Functional Equivalence of Retroviral MA Domains in Facilitating Psi RNA Binding Specificity by Gag

    Science.gov (United States)

    Rye-McCurdy, Tiffiny; Olson, Erik D.; Liu, Shuohui; Binkley, Christiana; Reyes, Joshua-Paolo; Thompson, Brian R.; Flanagan, John M.; Parent, Leslie J.; Musier-Forsyth, Karin

    2016-01-01

    Retroviruses specifically package full-length, dimeric genomic RNA (gRNA) even in the presence of a vast excess of cellular RNA. The “psi” (Ψ) element within the 5′-untranslated region (5′UTR) of gRNA is critical for packaging through interaction with the nucleocapsid (NC) domain of Gag. However, in vitro Gag binding affinity for Ψ versus non-Ψ RNAs is not significantly different. Previous salt-titration binding assays revealed that human immunodeficiency virus type 1 (HIV-1) Gag bound to Ψ RNA with high specificity and relatively few charge interactions, whereas binding to non-Ψ RNA was less specific and involved more electrostatic interactions. The NC domain was critical for specific Ψ binding, but surprisingly, a Gag mutant lacking the matrix (MA) domain was less effective at discriminating Ψ from non-Ψ RNA. We now find that Rous sarcoma virus (RSV) Gag also effectively discriminates RSV Ψ from non-Ψ RNA in a MA-dependent manner. Interestingly, Gag chimeras, wherein the HIV-1 and RSV MA domains were swapped, maintained high binding specificity to cognate Ψ RNAs. Using Ψ RNA mutant constructs, determinants responsible for promoting high Gag binding specificity were identified in both systems. Taken together, these studies reveal the functional equivalence of HIV-1 and RSV MA domains in facilitating Ψ RNA selectivity by Gag, as well as Ψ elements that promote this selectivity. PMID:27657107

  13. Monoclonal antibodies against rabbit mammary prolactin receptors. Specific antibodies to the hormone binding domain

    International Nuclear Information System (INIS)

    Three monoclonal antibodies (M110, A82, and A917) were obtained by fusing myeloma cells and spleen cells from mice immunized with partially purified rabbit mammary gland prolactin (PRL) receptors. All 3 antibodies were capable of complete inhibition of 125I-ovine prolactin (oPRL) binding to rabbit mammary PRL receptors in either particulate or soluble form. M110 showed slightly greater potency than oPRL in competing for 125I-oPRL binding. These antibodies also inhibited PRL binding to microsomal fractions from rabbit liver, kidney, adrenal, ovary, and pig mammary gland, although A82 showed poor inhibition in pig mammary gland. There was no cross-reaction of any of the 3 monoclonal antibodies (mAbs) for the other species tested: human (T-47D breast cancer cells) and rat (liver, ovary). In order to confirm that these antibodies are specific to the binding domain, antibodies were purified, iodinated, and binding characteristics were investigated. 125I-M110 and 125I-A82 binding was completely inhibited by lactogenic hormones, whereas nonlactogenic hormones did not cross-react. Competition of 125I-M110 by oPRL was comparable to that of 125I-oPRL by unlabeled oPRL, while 125I-A917 binding was only partially competed (30-60%) by lactogenic hormones. Tissue and species specificity of labeled antibody binding paralleled results of binding inhibition experiments using 125I-oPRL. In addition, A82 and A917 completely inhibited 125I-M110 binding. In contrast, 125I-A82 binding was stimulated by A917 and 125I-A917 binding was stimulated by A82

  14. 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

  15. Host-guest complexation. 18. Effects on cation binding of convergent ligand sites appended to macrocyclic polyethers

    International Nuclear Information System (INIS)

    Syntheses are reported for 16 new macrocyclic polyether ligand systems which contain potentially convergent side chains containing additional binding sites. The free energies of association of these systems in CDCl3 at 250C with Li+, Na+, K+, Rb+, Cs+, NH4+, CH3NH3+, and t-BuNH3+ picrates were determined

  16. New duel fluorescent 'on-off' and colorimetric sensor for Copper(II): Copper(II) binds through N coordination and pi cation interaction to sensor

    Science.gov (United States)

    Kumar, Jutika; Bhattacharyya, Pradip K.; Das, Diganta Kumar

    2015-03-01

    Schiff base derived from naphthylamine and benzil (L) binds to two Cu2+ ions, one by coordination through N of the Schiff base and another by pi cation interaction through benzil rings. This bonding pattern determined by DFT calculation has been proved by matching electronic spectrum obtained from TDDFT calculation to the experimental one. L acts as "on-off" fluorescent and bare eye detectable colorimetric (purple color) sensor for Cu2+ ion over the metal ions - Na+, K+, Ca2+ Mn2+, Co2+ Ni2+, Zn2+, Pb2+, Cd2+, Hg2+, Ag+, Hg2+ and Al3+ in 1:1 v/v CH3CN:H2O. These metal ions do not interfere the fluorescent/colorimetric sensing. As fluorescent sensor the linear range of detection is 5 × 10-5 to 3 × 10-4 M and detection limit 10-5 M.

  17. New duel fluorescent "on-off" and colorimetric sensor for Copper(II): Copper(II) binds through N coordination and pi cation interaction to sensor.

    Science.gov (United States)

    Kumar, Jutika; Bhattacharyya, Pradip K; Das, Diganta Kumar

    2015-03-01

    Schiff base derived from naphthylamine and benzil (L) binds to two Cu(2+) ions, one by coordination through N of the Schiff base and another by pi cation interaction through benzil rings. This bonding pattern determined by DFT calculation has been proved by matching electronic spectrum obtained from TDDFT calculation to the experimental one. L acts as "on-off" fluorescent and bare eye detectable colorimetric (purple color) sensor for Cu(2+) ion over the metal ions - Na(+), K(+), Ca(2+) Mn(2+), Co(2+) Ni(2+), Zn(2+), Pb(2+), Cd(2+), Hg(2+), Ag(+), Hg(2+) and Al(3+) in 1:1 v/v CH3CN:H2O. These metal ions do not interfere the fluorescent/colorimetric sensing. As fluorescent sensor the linear range of detection is 5×10(-5) to 3×10(-4)M and detection limit 10(-5)M. PMID:25479104

  18. Detection of site-specific binding and co-binding of ligands to macromolecules using sup 19 F NMR

    Energy Technology Data Exchange (ETDEWEB)

    Jenkins, B.G. (Massachusetts General Hospital and Harvard Medical School, Charlestown (USA))

    1991-01-01

    Study of ligand-macromolecular interactions by {sup 19}F nuclear magnetic resonance (NMR) spectroscopy affords many opportunities for obtaining molecular biochemical and pharmaceutical information. This is due to the absence of a background fluorine signal, as well as the relatively high sensitivity of {sup 19}F NMR. Use of fluorine-labeled ligands enables one to probe not only binding and co-binding phenomena to macromolecules, but also can provide data on binding constants, stoichiometries, kinetics, and conformational properties of these complexes. Under conditions of slow exchange and macromolecule-induced chemical shifts, multiple {sup 19}F NMR resonances can be observed for free and bound ligands. These shifted resonances are a direct correlate of the concentration of ligand bound in a specific state rather than the global concentrations of bound or free ligand which are usually determined using other techniques such as absorption spectroscopy or equilibrium dialysis. Examples of these interactions are demonstrated both from the literature and from interactions of 5-fluorotryptophan, 5-fluorosalicylic acid, flurbiprofen, and sulindac sulfide with human serum albumin. Other applications of {sup 19}F NMR to study of these interactions in vivo, as well for receptor binding and metabolic tracing of fluorinated drugs and proteins are discussed.

  19. The PickPocket method for predicting binding specificities for receptors based on receptor pocket similarities: application to MHC-peptide binding

    DEFF Research Database (Denmark)

    Zhang, H.; Lund, Ole; Nielsen, M.

    2009-01-01

    the polymorphic pocket residues in MHC molecules that are in close proximity to the peptide residue. For MHC molecules with known specificities, we established a library of pocket-residues and corresponding binding specificities. The binding specificity for a novel MHC molecule is calculated as the average...

  20. Conversion of MyoD to a Neurogenic Factor: Binding Site Specificity Determines Lineage

    Directory of Open Access Journals (Sweden)

    Abraham P. Fong

    2015-03-01

    Full Text Available MyoD and NeuroD2, master regulators of myogenesis and neurogenesis, bind to a “shared” E-box sequence (CAGCTG and a “private” sequence (CAGGTG or CAGATG, respectively. To determine whether private-site recognition is sufficient to confer lineage specification, we generated a MyoD mutant with the DNA-binding specificity of NeuroD2. This chimeric mutant gained binding to NeuroD2 private sites but maintained binding to a subset of MyoD-specific sites, activating part of both the muscle and neuronal programs. Sequence analysis revealed an enrichment for PBX/MEIS motifs at the subset of MyoD-specific sites bound by the chimera, and point mutations that prevent MyoD interaction with PBX/MEIS converted the chimera to a pure neurogenic factor. Therefore, redirecting MyoD binding from MyoD private sites to NeuroD2 private sites, despite preserved binding to the MyoD/NeuroD2 shared sites, is sufficient to change MyoD from a master regulator of myogenesis to a master regulator of neurogenesis.

  1. 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

  2. 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. PMID:8643634

  3. Kinetics and thermodynamics of binding reactions as exemplified by anthrax toxin channel blockage with a cationic cyclodextrin derivative.

    Science.gov (United States)

    Nestorovich, Ekaterina M; Karginov, Vladimir A; Berezhkovskii, Alexander M; Parsegian, V Adrian; Bezrukov, Sergey M

    2012-11-01

    The thermodynamics of binding reactions is usually studied in the framework of the linear van't Hoff analysis of the temperature dependence of the equilibrium constant. The logarithm of the equilibrium constant is plotted versus inverse temperature to discriminate between two terms: an enthalpic contribution that is linear in the inverse temperature, and a temperature-independent entropic contribution. When we apply this approach to a particular case-blockage of the anthrax PA(63) channel by a multicharged cyclodextrin derivative-we obtain a nearly linear behavior with a slope that is characterized by enthalpy of about 1 kcal/mol. In contrast, from blocker partitioning between the channel and the bulk, we estimate the depth of the potential well for the blocker in the channel to be at least 8 kcal/mol. To understand this apparent discrepancy, we use a simple model of particle interaction with the channel and show that this significant difference between the two estimates is due to the temperature dependence of the physical forces between the blocker and the channel. In particular, we demonstrate that if the major component of blocker-channel interaction is van der Waals interactions and/or Coulomb forces in water, the van't Hoff enthalpy of the binding reaction may be close to zero or even negative, including cases of relatively strong binding. The results are quite general and, therefore, of importance for studies of enzymatic reactions, rational drug design, small-molecule binding to proteins, protein-protein interactions, and protein folding, among others.

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

    Science.gov (United States)

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

    2016-06-01

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

  5. Transcriptional Regulation in Mammalian Cells by Sequence-Specific DNA Binding Proteins

    Science.gov (United States)

    Mitchell, Pamela J.; Tjian, Robert

    1989-07-01

    The cloning of genes encoding mammalian DNA binding transcription factors for RNA polymerase II has provided the opportunity to analyze the structure and function of these proteins. This review summarizes recent studies that define structural domains for DNA binding and transcriptional activation functions in sequence-specific transcription factors. The mechanisms by which these factors may activate transcriptional initiation and by which they may be regulated to achieve differential gene expression are also discussed.

  6. Signal transduction by erythrocytes on specific binding of doxorubicin immobilized on nanodispersed magnetite

    Energy Technology Data Exchange (ETDEWEB)

    Mykhaylyk, Olga [Institute Applied Problems Physics and Biophysics, NAS, Sluzhbova 3, UA-03142 Kyiv (Ukraine)]. E-mail: Olga.Mykhaylyk@gmx.net; Kotzuruba, Anatoliy [Institute of Biochemistry, NAS, Leontovicha 9, UA-01030 Kyiv (Ukraine); Dudchenko, Nataliya [Institute Applied Problems Physics and Biophysics, NAS, Sluzhbova 3, UA-03142 Kyiv (Ukraine); Toerok, Gyula [Research Institute for Solid State Physics and Optics, H-1525 Budapest, P.O. Box 49 (Hungary)

    2005-05-15

    Two specific binding sites for doxorubicin were revealed at the plasma membrane of human erythrocytes on investigation of the binding of doxorubicin magnetic nanoconjugates. Free and conjugated doxorubicins modulated signal transduction in erythrocytes in a similar way. Both up-regulated nitric oxide and cyclic GMP (cGMP) and down-regulated cyclic AMP (cAMP) production and stabilize the membranes of damaged erythrocytes.

  7. Specific binding and biological effects of tumor promoting phorbol esters on sponges.

    Science.gov (United States)

    Mazzorana, M; Garrone, R; Martel, N; Yamasaki, H

    1984-01-01

    Sponges grown in the presence of 12-O-tetradecanoyl phorbol-13-acetate (TPA) show deep alterations of their structure and development. Their aquiferous system (flagellated cells and canals) is largely altered and the tissues show an unusually high cell density. This focalized effect of TPA on the aquiferous system seems specific and is reversible at low concentrations (100 ng/ml). A toxic, non-specific effect is also noted, particularly at high concentrations (5000 ng/ml). Using 3H-phorbol-12, 13-dibutyrate (3H-PDBu), we demonstrate a class of specific binding sites for phorbol esters in the homogenates of sponges. These binding sites have high affinity (Kd = 26.0 nM) for PDBu and at saturation about 20 pmoles of 3H-PDBu is bound per mg protein of sponge homogenates. The binding of 3H-PDBu was inhibited by other phorbol esters and their congeners, and there was a good correlation between their potency in binding inhibition and their tumor promoting activity. It is concluded that sponges have a class of specific saturable and high affinity receptors for phorbol esters and that there is a very high conservation of these receptors during evolution. Such specific binding may be responsible for subsequent biological effect of TPA on sponges.

  8. Screening for PreS specific binding ligands with a phage displayed peptides library

    Institute of Scientific and Technical Information of China (English)

    Qiang Deng; Ming Zhuang; Yu-Ying Kong; You-Hua Xie; Yuan Wang

    2005-01-01

    AIM: To construct a random peptide phage display library and search for peptides that specifically bind to the PreS region of hepatitis B virus (HBV).METHODS: A phage display vector, pFuse8, based on the gene 8 product (pⅧ) of M13 phage was made and used to construct a random peptide library. E. coli derived thioredoxin-PreS was purified with Thio-bond beads, and exploited as the bait protein for library screening. Five rounds of bio-panning were performed. The PreS-binding specificities of enriched phages were characterized with phage ELISA assay.RESULTS: A phage display vector was successfully constructed as demonstrated to present a pⅧ fused HBV PreS1 epitope on the phage surface with a high efficiency.A cysteine confined random peptide library was constructed containing independent clones exceeding 5±108 clone forming unit (CFU). A pool of phages showing a PreS-binding specificity was obtained after the screening against thioPres with an enrichment of approximately 400 times. Five phages with high PreS-binding specificities were selected and characterized. Sequences of the peptides displayed on these phages were determined.CONCLUSION: A phage library has been constructed,with random peptides displaying as pⅧ-fusion proteins.Specific PreS-binding peptides have been obtained, which may be useful for developing antivirals against HBV infection.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1991-08-01

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

  10. Protein interactions and ligand binding: From protein subfamilies to functional specificity

    OpenAIRE

    Rausell, A.; de Juan, D.; Pazos, F; Valencia, A.

    2010-01-01

    The divergence accumulated during the evolution of protein families translates into their internal organization as subfamilies, and it is directly reflected in the characteristic patterns of differentially conserved residues. These specifically conserved positions in protein subfamilies are known as “specificity determining positions” (SDPs). Previous studies have limited their analysis to the study of the relationship between these positions and ligand-binding specificity, demonstrating sign...

  11. PEGylated cationic nanoemulsions can efficiently bind and transfect pIDUA in a mucopolysaccharidosis type I murine model.

    Science.gov (United States)

    Fraga, Michelle; Bruxel, Fernanda; Diel, Dirnete; de Carvalho, Talita Giacomet; Perez, Carlos Alberto; Magalhães-Paniago, Rogério; Malachias, Ângelo; Oliveira, Mônica Cristina; Matte, Ursula; Teixeira, Helder Ferreira

    2015-07-10

    Mucopolysaccharidosis type I (MPS I) is an autosomal disease caused by alpha-L-iduronidase deficiency. This study proposed the use of cationic nanoemulsions as non-viral vectors for a plasmid (pIDUA) containing the gene that codes for alpha-L-iduronidase. Nanoemulsions composed of medium chain triglycerides (MCT)/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)/1,2-dioleoyl-sn-glycero-3-trimethylammonium propane (DOTAP)/1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG) were prepared by high pressure homogenization. Formulations were prepared by the adsorption or encapsulation of preformed pIDUA-DOTAP complexes into the oil core of nanoemulsions at different charge ratios. pIDUA complexed was protected from enzymatic degradation by DNase I. The physicochemical characteristics of complexes in protein-containing medium were mainly influenced by the presence of DSPE-PEG. Bragg reflections corresponding to a lamellar organization were identified for blank formulations by energy dispersive X-ray diffraction, which could not be detected after pIDUA complexation. The intravenous injection of these formulations in MPS I knockout mice led to a significant increase in IDUA activity (fluorescence assay) and expression (RT-qPCR) in different organs, especially the lungs and liver. These findings were more significant for formulations prepared at higher charge ratios (+4/-), suggesting a correlation between charge ratio and transfection efficiency. The present preclinical results demonstrated that these nanocomplexes represent a potential therapeutic option for the treatment of MPS I. PMID:25886705

  12. Mechanism and specificity of lanthanide series cation transport by ionophores A23187, 4-BrA23187, and ionomycin.

    OpenAIRE

    Wang, E.; Taylor, R.W.; Pfeiffer, D R

    1998-01-01

    A23187, 4-BrA23187, and ionomycin transport several lanthanide series trivalent cations at efficiencies similar to Ca2+, when compared at cation concentrations of approximately 10(-5) M, ionophore concentrations of approximately 10(-6) M, and a pH of 7.00. Selectivity sequences and the range of relative rates are as follows: A23187, Nd3+ > La3+ > Eu3+ > Gd3+ > Er3+ > Yb3+ > Lu3+ (approximately 34-fold); 4-BrA23187, Nd3+ > Eu3+ > Gd3+ > La3+ > Er3+ > Yb3+ > Lu3+ (approximately 34-fold); ionomy...

  13. Cloning and characterisation of a nuclear, site specific ssDNA binding protein.

    Science.gov (United States)

    Smidt, M P; Russchen, B; Snippe, L; Wijnholds, J; Ab, G

    1995-07-11

    Estradiol inducible, liver-specific expression of the apoVLDL II gene is mediated through the estrogen receptor and a variety of other DNA-binding proteins. In the present study we report the cloning and characterisation of a single-strand DNA binding protein that interacts with the lower strand of a complex regulatory site, which includes the major estrogen responsive element and a site that resembles the rat albumin site D (apoVLDL II site D). Based on its binding specificity determined with electro-mobility shift assays, the protein is named single-strand D-box binding factor (ssDBF). Analysis of the deduced 302 amino acid sequence revealed that the protein belongs to the heteronuclear ribonucleoprotein A/B family (hnRNP A/B) and resembles other known eukaryotic single-strand DNA binding proteins. Transient transfection experiments in a chicken liver cell-line showed that the protein represses estrogen-induced transcription. A protein with similar binding characteristics is present in liver nuclear extract. The relevance of the occurrence of this protein to the expression of the apoVLDL II gene is discussed. PMID:7630716

  14. The Fanconi anemia associated protein FAAP24 uses two substrate specific binding surfaces for DNA recognition.

    Science.gov (United States)

    Wienk, Hans; Slootweg, Jack C; Speerstra, Sietske; Kaptein, Robert; Boelens, Rolf; Folkers, Gert E

    2013-07-01

    To maintain the integrity of the genome, multiple DNA repair systems exist to repair damaged DNA. Recognition of altered DNA, including bulky adducts, pyrimidine dimers and interstrand crosslinks (ICL), partially depends on proteins containing helix-hairpin-helix (HhH) domains. To understand how ICL is specifically recognized by the Fanconi anemia proteins FANCM and FAAP24, we determined the structure of the HhH domain of FAAP24. Although it resembles other HhH domains, the FAAP24 domain contains a canonical hairpin motif followed by distorted motif. The HhH domain can bind various DNA substrates; using nuclear magnetic resonance titration experiments, we demonstrate that the canonical HhH motif is required for double-stranded DNA (dsDNA) binding, whereas the unstructured N-terminus can interact with single-stranded DNA. Both DNA binding surfaces are used for binding to ICL-like single/double-strand junction-containing DNA substrates. A structural model for FAAP24 bound to dsDNA has been made based on homology with the translesion polymerase iota. Site-directed mutagenesis, sequence conservation and charge distribution support the dsDNA-binding model. Analogous to other HhH domain-containing proteins, we suggest that multiple FAAP24 regions together contribute to binding to single/double-strand junction, which could contribute to specificity in ICL DNA recognition.

  15. Selection of DMA aptamer that specific binding human carcinoembryonic antigen in vitro

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective:To select the specific aptamer of carcinoembryonic antigen (CEA), one of the most attractive molecule for cancer target therapy and imaging. Methods: Seven rounds in vitro selection were performed against the purified CEA protein. Ligand-mediated target purification and Co-immunoprecipitation were adopted to verify the specific binding of the aptamer to the purified and native protein separately. Results:The CEA-specific aptamer which can bind both the purified and native protein with the high specificity was obtained. Conclusion:This is the first time the CEA specific apatmer was produced. The results in this study provides the preliminary evidence for further investigation and application of CEA-aptamer in the future.

  16. Screening of a specific peptide binding to VPAC1 receptor from a phage display peptide library.

    Directory of Open Access Journals (Sweden)

    Bo Tang

    Full Text Available BACKGROUND/PURPOSE: The VPAC1 receptor, a member of the vasoactive intestinal peptide receptors (VIPRs, is overexpressed in the most frequently occurring malignant tumors and plays a major role in the progression and angiogenesis of a number of malignancies. Recently, phage display has become widely used for many applications, including ligand generation for targeted imaging, drug delivery and therapy. In this work, we developed a panning procedure using a phage display peptide library to select a peptide that specifically binds to the VPAC1 receptor to develop a novel targeted probe for molecular imaging and therapy. METHODS: CHO-K1 cells stably expressing VPAC1 receptors (CHO-K1/VPAC1 cells were used to select a VPAC1-binding peptide from a 12-mer phage peptide library. DNA sequencing and homologous analysis of the randomly selected phage clones were performed. A cellular ELISA was used to determine the most selectively binding peptide for further investigation. Binding specificity to the VPAC1 receptor was analyzed by competitive inhibition ELISA and flow cytometry. The binding ability of the selected peptide to CHO-K1/VPAC1 cells and colorectal cancer (CRC cell lines was confirmed using fluorescence microscopy and flow cytometry. RESULTS: A significant enrichment of phages that specifically bound to CHO-K1/VPAC1 cells was obtained after four rounds of panning. Of the selected phage clones, 16 out of 60 shared the same peptide sequence, GFRFGALHEYNS, which we termed the VP2 peptide. VP2 and vasoactive intestinal peptide (VIP competitively bound to the VPAC1 receptor. More importantly, we confirmed that VP2 specifically bound to CHO-K1/VPAC1 cells and several CRC cell lines. CONCLUSION: Our results demonstrate that the VP2 peptide could specifically bind to VPAC1 receptor and several CRC cell lines. And VP2 peptide may be a potential candidate to be developed as a useful diagnostic molecular imaging probe for early detection of CRC.

  17. Peptide binding landscapes: Specificity and homophilicity across sequence space in a lattice model

    Science.gov (United States)

    Jeon, Joohyun; Shell, M. Scott

    2016-10-01

    Peptide aggregation frequently involves sequences with strong homophilic binding character, i.e., sequences that self-assemble with like species in a crowded cellular environment, in the face of a multitude of other peptides or proteins as potential heterophilic binding partners. What kinds of sequences display a strong tendency towards homophilic binding and self-assembly, and what are the origins of this behavior? Here, we consider how sequence specificity in oligomerization processes plays out in a simple two-dimensional (2D) lattice statistical-thermodynamic peptide model that permits exhaustive examination of the entire sequence and configurational landscapes. We find that sequences with strong self-specificities have either alternating hydrophobic and hydrophilic residues or short patches of hydrophobic residues, both which minimize intramolecular hydrophobic interactions in part due to the constraints of the 2D lattice. We also find that these specificities are highly sensitive to entropic and free energetic features of the unbound conformational state, such that direct binding interaction energies alone do not capture the complete behavior. These results suggest that the ability of particular peptide sequences to self-assemble and aggregate in a many-protein environment reflects a precise balance of direct binding interactions and behavior in the unbound (monomeric) state.

  18. 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.

  19. Long-term reproducibility of in vivo measures of specific binding of radioligands in rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Kilbourn, Michael R. E-mail: mkilbour@umich.edu

    2004-07-01

    The long-term reproducibility of measures of in vivo specific binding of radiolabeled forms of (+)-{alpha}-dihydrotetrabenazine (DTBZ) and d-threo-methylphenidate (MPH) in rat brain was examined. All studies were done using a consistent bolus plus infusion protocol and calculation of equilibrium distribution volume ratios (DVR). Over a period of eight years striatal DVR values for DTBZ binding to the vesicular monoamine transporter 2 (VMAT2) in young adult (8-10 wks old) rats showed very good reproducibility (3.62{+-}0.33, N=35). Equivalent values were obtained using either tritiated or carbon-11 labeled DTBZ, and were irrespective of sex of animals. Older animals (78 wks old) showed losses (-45%) of specific binding. Striatal binding of MPH to the dopamine transporter (DAT) showed a similar reproducibility over a five year period (DVR=2.17{+-}0.39, N=52), again irrespective of radionuclide or sex. These studies demonstrate that use of a consistent in vivo technique can provide reliable measures of specific binding of radioligands to high affinity sites in the rat brain.

  20. Single-chain antibody-fragment M6P-1 possesses a mannose 6-phosphate monosaccharide-specific binding pocket that distinguishes N-glycan phosphorylation in a branch-specific manner†.

    Science.gov (United States)

    Blackler, Ryan J; Evans, Dylan W; Smith, David F; Cummings, Richard D; Brooks, Cory L; Braulke, Thomas; Liu, Xinyu; Evans, Stephen V; Müller-Loennies, Sven

    2016-02-01

    The acquisition of mannose 6-phosphate (Man6P) on N-linked glycans of lysosomal enzymes is a structural requirement for their transport from the Golgi apparatus to lysosomes mediated by the mannose 6-phosphate receptors, 300 kDa cation-independent mannose 6-phosphate receptor (MPR300) and 46 kDa cation-dependent mannose 6-phosphate receptor (MPR46). Here we report that the single-chain variable domain (scFv) M6P-1 is a unique antibody fragment with specificity for Man6P monosaccharide that, through an array-screening approach against a number of phosphorylated N-glycans, is shown to bind mono- and diphosphorylated Man6 and Man7 glycans that contain terminal αMan6P(1 → 2)αMan(1 → 3)αMan. In contrast to MPR300, scFv M6P-1 does not bind phosphodiesters, monophosphorylated Man8 or mono- or diphosphorylated Man9 structures. Single crystal X-ray diffraction analysis to 2.7 Å resolution of Fv M6P-1 in complex with Man6P reveals that specificity and affinity is achieved via multiple hydrogen bonds to the mannose ring and two salt bridges to the phosphate moiety. In common with both MPRs, loss of binding was observed for scFv M6P-1 at pH values below the second pKa of Man6P (pKa = 6.1). The structures of Fv M6P-1 and the MPRs suggest that the change of the ionization state of Man6P is the main driving force for the loss of binding at acidic lysosomal pH (e.g. lysosome pH ∼ 4.6), which provides justification for the evolution of a lysosomal enzyme transport pathway based on Man6P recognition. PMID:26503547

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

    Directory of Open Access Journals (Sweden)

    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.

  2. Thermodynamic and structural investigation of the specific SDS binding of humicola insolens cutinase

    DEFF Research Database (Denmark)

    Kold, David; Dauter, Zbigniew; Laustsen, Anne K;

    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...

  3. Porcine major histocompatibility complex (MHC) class I molecules and analysis of their peptide-binding specificities

    DEFF Research Database (Denmark)

    Pedersen, Lasse Eggers; Harndahl, Mikkel; Rasmussen, Michael;

    2011-01-01

    CTL staining and manipulation. This has enabled a complete mapping of all HLA-I specificities (“the Human MHC Project”). Here, we demonstrate that these approaches can be applied to other species. We systematically transferred domains of the frequently expressed swine MHC-I molecule, SLA-1*0401, onto...... a HLA-I molecule (HLA-A*11:01), thereby generating recombinant human/swine chimeric MHC-I molecules as well as the intact SLA-1*0401 molecule. Biochemical peptide-binding assays and positional scanning combinatorial peptide libraries were used to analyze the peptide-binding motifs of these molecules....... A pan-specific predictor of peptide–MHC-I binding, NetMHCpan, which was originally developed to cover the binding specificities of all known HLA-I molecules, was successfully used to predict the specificities of the SLA-1*0401 molecule as well as the porcine/human chimeric MHC-I molecules. These data...

  4. Reversible CO binding enables tunable CO/H₂ and CO/N₂ separations in metal-organic frameworks with exposed divalent metal cations.

    Science.gov (United States)

    Bloch, Eric D; Hudson, Matthew R; Mason, Jarad A; Chavan, Sachin; Crocellà, Valentina; Howe, Joshua D; Lee, Kyuho; Dzubak, Allison L; Queen, Wendy L; Zadrozny, Joseph M; Geier, Stephen J; Lin, Li-Chiang; Gagliardi, Laura; Smit, Berend; Neaton, Jeffrey B; Bordiga, Silvia; Brown, Craig M; Long, Jeffrey R

    2014-07-30

    Six metal-organic frameworks of the M2(dobdc) (M = Mg, Mn, Fe, Co, Ni, Zn; dobdc(4-) = 2,5-dioxido-1,4-benzenedicarboxylate) structure type are demonstrated to bind carbon monoxide reversibly and at high capacity. Infrared spectra indicate that, upon coordination of CO to the divalent metal cations lining the pores within these frameworks, the C-O stretching frequency is blue-shifted, consistent with nonclassical metal-CO interactions. Structure determinations reveal M-CO distances ranging from 2.09(2) Å for M = Ni to 2.49(1) Å for M = Zn and M-C-O angles ranging from 161.2(7)° for M = Mg to 176.9(6)° for M = Fe. Electronic structure calculations employing density functional theory (DFT) resulted in good agreement with the trends apparent in the infrared spectra and crystal structures. These results represent the first crystallographically characterized magnesium and zinc carbonyl compounds and the first high-spin manganese(II), iron(II), cobalt(II), and nickel(II) carbonyl species. Adsorption isotherms indicate reversible adsorption, with capacities for the Fe, Co, and Ni frameworks approaching one CO per metal cation site at 1 bar, corresponding to loadings as high as 6.0 mmol/g and 157 cm(3)/cm(3). The six frameworks display (negative) isosteric heats of CO adsorption ranging from 52.7 to 27.2 kJ/mol along the series Ni > Co > Fe > Mg > Mn > Zn, following the Irving-Williams stability order. The reversible CO binding suggests that these frameworks may be of utility for the separation of CO from various industrial gas mixtures, including CO/H2 and CO/N2. Selectivities determined from gas adsorption isotherm data using ideal adsorbed solution theory (IAST) over a range of gas compositions at 1 bar and 298 K indicate that all six M2(dobdc) frameworks could potentially be used as solid adsorbents to replace current cryogenic distillation technologies, with the choice of M dictating adsorbent regeneration energy and the level of purity of the resulting gases.

  5. Specific and Modular Binding Code for Cytosine Recognition in Pumilio/FBF (PUF) RNA-binding Domains

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Shuyun; Wang, Yang; Cassidy-Amstutz, Caleb; Lu, Gang; Bigler, Rebecca; Jezyk, Mark R.; Li, Chunhua; Tanaka Hall, Traci M.; Wang, Zefeng (NIH); (Beijing U); (UNC)

    2011-10-28

    Pumilio/fem-3 mRNA-binding factor (PUF) proteins possess a recognition code for bases A, U, and G, allowing designed RNA sequence specificity of their modular Pumilio (PUM) repeats. However, recognition side chains in a PUM repeat for cytosine are unknown. Here we report identification of a cytosine-recognition code by screening random amino acid combinations at conserved RNA recognition positions using a yeast three-hybrid system. This C-recognition code is specific and modular as specificity can be transferred to different positions in the RNA recognition sequence. A crystal structure of a modified PUF domain reveals specific contacts between an arginine side chain and the cytosine base. We applied the C-recognition code to design PUF domains that recognize targets with multiple cytosines and to generate engineered splicing factors that modulate alternative splicing. Finally, we identified a divergent yeast PUF protein, Nop9p, that may recognize natural target RNAs with cytosine. This work deepens our understanding of natural PUF protein target recognition and expands the ability to engineer PUF domains to recognize any RNA sequence.

  6. Binding of 125I-human growth hormone to specific receptors in human cultured lymphocytes

    International Nuclear Information System (INIS)

    The interaction of human growth hormone with human lymphocytes from an established culture (IM-9) was studied using 125I- human growth hormone. The binding of 125I-human growth hormone was rapid; with human growth hormone at 0.1 nM a steady state was observed in 90 min at 300. Bound labeled human growth hormone was dissociated rapidly by addition of excess unlabeled human growth hormone. Binding of 125I-human growth hormone to cultured lymphocytes was relatively insensitive to alterations in the pH and in the concentrations of Ca2+, Mg2+, EDTA. At 800 there was very little degradation of labeled human growth hormone or of the specific receptor sites. Tryptic digestion destroyed the capacity of cells to bind human growth hormone. The IM-9 cells bound all human growth hormone preparations but not unrelated hormones or nonprimate growth hormones. The binding of 125I-human growth hormone was inhibited 10 to 14 percent with 1 to 2 ng per ml of unlabeled human growth hormone and 50 percent with 30 to 40 ng per ml, well within the range of hormone concentrations in vivo. Analysis of steady state data revealed a single order of binding sites with an affinity constant of 1.3 x 109 M-1 and about 4000 binding sites per cell. Numerous human growth hormone preparations were assayed by use of this receptor system as well as by immunoassay and by bioassay in vivo. The po

  7. Specific induction of fibronectin binding activity by hemoglobin in Candida albicans grown in defined media.

    Science.gov (United States)

    Yan, S; Nègre, E; Cashel, J A; Guo, N; Lyman, C A; Walsh, T J; Roberts, D D

    1996-08-01

    Fibronectin (FN) is a major component of host extracellular matrix that may play an important role in the initiation and dissemination of Candida albicans infections. Expression of FN binding requires growth of C albicans blastoconidia in complex medium, and the regulation of FN receptor expression is poorly understood. We now demonstrate that hemoglobin is a potent and specific inducer of FN receptor expression and describe a defined medium supplemented with hemoglobin that greatly and stably enhances the binding activity of C. albicans for soluble FN. Enhancement of FN binding by hemoglobin in strain 44807 was concentration dependent and was maximal at 0.1% hemoglobin with 20- to 80-fold enhancement. The hemoglobin-induced FN binding to C. albicans was saturable, with a Kd of 2.7 X 10(-8) M. Enhancement required growth of C. albicans in hemoglobin-containing medium, since simply exposing blastoconidia to hemoglobin in a nongrowing status did not enhance binding. Induction was reversible following removal of hemoglobin from the growth medium and not associated with germination. Inorganic or protein-bound iron was not sufficient for the induction, since other iron-containing proteins or inorganic iron salts were inactive. Growth in the simple medium yeast nitrogen base supplemented with hemoglobin increased cell adhesion to immobilized FN and to cultured monolayers of bovine corneal endothelial cells. These data suggest that hemoglobin may be an important regulator of FN binding activity in C. albicans and thus may play a role in its pathogenesis. PMID:8757815

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

    Science.gov (United States)

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

    2016-01-01

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

  9. Determinants of BH3 binding specificity for Mcl-1 versus Bcl-xL.

    Science.gov (United States)

    Dutta, Sanjib; Gullá, Stefano; Chen, T Scott; Fire, Emiko; Grant, Robert A; Keating, Amy E

    2010-05-21

    Interactions among Bcl-2 family proteins are important for regulating apoptosis. Prosurvival members of the family interact with proapoptotic BH3 (Bcl-2-homology-3)-only members, inhibiting execution of cell death through the mitochondrial pathway. Structurally, this interaction is mediated by binding of the alpha-helical BH3 region of the proapoptotic proteins to a conserved hydrophobic groove on the prosurvival proteins. Native BH3-only proteins exhibit selectivity in binding prosurvival members, as do small molecules that block these interactions. Understanding the sequence and structural basis of interaction specificity in this family is important, as it may allow the prediction of new Bcl-2 family associations and/or the design of new classes of selective inhibitors to serve as reagents or therapeutics. In this work, we used two complementary techniques--yeast surface display screening from combinatorial peptide libraries and SPOT peptide array analysis--to elucidate specificity determinants for binding to Bcl-x(L)versus Mcl-1, two prominent prosurvival proteins. We screened a randomized library and identified BH3 peptides that bound to either Mcl-1 or Bcl-x(L) selectively or to both with high affinity. The peptides competed with native ligands for binding into the conserved hydrophobic groove, as illustrated in detail by a crystal structure of a specific peptide bound to Mcl-1. Mcl-1-selective peptides from the screen were highly specific for binding Mcl-1 in preference to Bcl-x(L), Bcl-2, Bcl-w, and Bfl-1, whereas Bcl-x(L)-selective peptides showed some cross-interaction with related proteins Bcl-2 and Bcl-w. Mutational analyses using SPOT arrays revealed the effects of 170 point mutations made in the background of a peptide derived from the BH3 region of Bim, and a simple predictive model constructed using these data explained much of the specificity observed in our Mcl-1 versus Bcl-x(L) binders. PMID:20363230

  10. Do Cation-π Interactions Exist in Bacteriorhodopsin

    Institute of Scientific and Technical Information of China (English)

    HU Kun-Sheng; WANG Guang-Yu; HE Jin-An

    2001-01-01

    Metal ions are essential to the structure and physiological functions of bacteriorhodopsin. Experimental evidence suggests the existence of specific cation binding to the negatively charged groups of Asp85 and Asp212 via an electrostatic interaction. However, only using electrostatic force is not enough to explain the role of the metal cations because the carboxylate of Asp85 is well known to be protonated in the M intermediate. Considering the presence of some aromatic amino acid residues in the vicinity of the retinal pocket, the existence of cation-π interactions between the metal cation and aromatic amino acid residues is suggested. Obviously, introduction of this kind of interaction is conducive to understanding the effects of the metal cations and aromatic amino acid residues inside the protein on the structural stability and proton pumping of bacteriorhodopsin.

  11. Autoradiographic localization of specific [3H]dexamethasone binding in fetal lung.

    Science.gov (United States)

    Beer, D G; Butley, M S; Cunha, G R; Malkinson, A M

    1984-10-01

    The cellular and subcellular localization of specific [3H]dexamethasone binding was examined in fetal mouse lung at various stages of development and in human fetal lung at 8 weeks of gestation using a rapid in vitro steroid incubation technique followed by thaw-mount autoradiography. Competition studies with unlabeled steroids demonstrate the specificity of [3H]dexamethasone labeling, and indicate that fetal lung mesenchyme is a primary glucocorticoid target during lung development. Quantitative binding studies, involving incubation of intact tissue with competing ligand and subsequent subcellular fractionation, show this to be specific, nuclear binding characteristic of glucocorticoid receptors. Autoradiographs of [3H]dexamethasone binding in lung tissue at early stages of development demonstrate that the mesenchyme directly adjacent to the more proximal portions of the bronchiolar network is heavily labeled. In contrast, the epithelium which will later differentiate into bronchi and bronchioles, is relatively unlabeled. Distal portions of the growing epithelium, destined to become alveolar ducts and alveoli, do show nuclear localization of [3H]dexamethasone. Because of the known importance of the mesenchyme in controlling lung development and the ability of glucocorticoids to stimulate lung development, these results suggest that many of the growth-promoting effects of glucocorticoids may be mediated through the mesenchyme. In addition, by utilizing a technique which allows the simultaneous examination of extracellular matrix components and [3H]dexamethasone binding, a relationship is observed between extensive mesenchymal [3H]dexamethasone binding and extensive extracellular matrix accumulation. Since glucocorticoids stimulate the synthesis of many extracellular matrix components, these results suggest a role for these hormones in affecting mesenchymal-epithelial interactions during lung morphogenesis.

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

    Science.gov (United States)

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

    2015-07-01

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

  13. Sequence specificity of formaldehyde-mediated covalent binding of anthracycline derivatives to DNA.

    Science.gov (United States)

    Szulawska, Agata; Gniazdowski, Marek; Czyz, Malgorzata

    2005-01-01

    Daunorubicin (DRB) and doxorubicin (DOX) in the presence of formaldehyde (CH2O) form covalent adducts with DNA. A G-specific adduct is formed by producing an aminal bridge between the C-3' of daunosamine and the C-2 of guanine. New derivatives of DRB, DOX and epidoxorubicin (EDOX) with an amidine group bonded to the C-3' of the daunosamine moiety, with either a morpholine or hexamethyleneimine ring attached to the amidine group, were studied in this paper. DNase I footprinting and analyses with restriction endonucleases were applied to compare the specificity of adduct formed by the amidine derivatives and their parent compounds. These approaches provide consistent results, proving that a GC pair is required for covalent binding of anthracycline derivatives to DNA and that different flanking sequences are able to modify the sequence preference of the drugs. The 5'-GC-3', 5'-CG-3' and 5'-TC-3' sequences were protected most efficiently by the parent compounds and their morpholine derivatives and some increased protection of 5'-TC-3' sequence was observed for morpholine analogues. Hexamethyleneimine derivatives bind to DNA with much lower efficiency. Finally, the sequence specificity of anthracycline derivatives was correlated with their ability to inhibit binding of transcription factors Sp1 and AP-1 to their DNA recognition sequences. The anthracycline derivatives were more potent in inhibiting Sp1 binding to its cognate GC box than in preventing AP-1 from binding to its mixed A.T and G.C site. Overall, the results indicate that the amidine derivatives of anthracyclines show similar, but not identical sequence specificity as parent compounds, though they exert their effect at a higher concentration. PMID:15588709

  14. Contribution of distinct homeodomain DNA binding specificities to Drosophila embryonic mesodermal cell-specific gene expression programs.

    Directory of Open Access Journals (Sweden)

    Brian W Busser

    Full Text Available Homeodomain (HD proteins are a large family of evolutionarily conserved transcription factors (TFs having diverse developmental functions, often acting within the same cell types, yet many members of this family paradoxically recognize similar DNA sequences. Thus, with multiple family members having the potential to recognize the same DNA sequences in cis-regulatory elements, it is difficult to ascertain the role of an individual HD or a subclass of HDs in mediating a particular developmental function. To investigate this problem, we focused our studies on the Drosophila embryonic mesoderm where HD TFs are required to establish not only segmental identities (such as the Hox TFs, but also tissue and cell fate specification and differentiation (such as the NK-2 HDs, Six HDs and identity HDs (I-HDs. Here we utilized the complete spectrum of DNA binding specificities determined by protein binding microarrays (PBMs for a diverse collection of HDs to modify the nucleotide sequences of numerous mesodermal enhancers to be recognized by either no or a single subclass of HDs, and subsequently assayed the consequences of these changes on enhancer function in transgenic reporter assays. These studies show that individual mesodermal enhancers receive separate transcriptional input from both I-HD and Hox subclasses of HDs. In addition, we demonstrate that enhancers regulating upstream components of the mesodermal regulatory network are targeted by the Six class of HDs. Finally, we establish the necessity of NK-2 HD binding sequences to activate gene expression in multiple mesodermal tissues, supporting a potential role for the NK-2 HD TF Tinman (Tin as a pioneer factor that cooperates with other factors to regulate cell-specific gene expression programs. Collectively, these results underscore the critical role played by HDs of multiple subclasses in inducing the unique genetic programs of individual mesodermal cells, and in coordinating the gene regulatory

  15. SPECIFIC BINDING OF HUMAN BONE MORPHOGENETIC PROTEIN (2A) WITH MOUSE OSTEOBLASTIC CELLS

    Institute of Scientific and Technical Information of China (English)

    刘新平; 陈苏民; 陈南春; 高磊; 赵忠良

    1996-01-01

    Human bone morphogenetic protein 2A (hBMP2A) cDNA terminal 567 nucleotides were cloned and expressed in a phage display vector pCSM2I. Hulnata BMP2A C-terminal peptide displayed on the surface of the phage can bind specifically to the sttrface of mouse osteoblastie cell (MC3T3) membrane. ELISA assay showed a positive signal of the binding by using antibody against M13 phage gene 8 protein. After labeling with 3HTdR,the counts of the binding groups were 3 to 10 times higher than the control groups. It suggests that the'surface of MC3T3 cells exist the recepzor for hBMP2A.

  16. Obesity risk gene TMEM18 encodes a sequence-specific DNA-binding protein.

    Directory of Open Access Journals (Sweden)

    Jaana M Jurvansuu

    Full Text Available Transmembrane protein 18 (TMEM18 has previously been connected to cell migration and obesity. However, the molecular function of the protein has not yet been described. Here we show that TMEM18 localises to the nuclear membrane and binds to DNA in a sequence-specific manner. The protein binds DNA with its positively charged C-terminus that contains also a nuclear localisation signal. Increase in the amount of TMEM18 in cells suppresses expression from a reporter vector with the TMEM18 target sequence. TMEM18 is a small protein of 140 residues and is predicted to be mostly alpha-helical with three transmembrane parts. As a consequence the DNA binding by TMEM18 would bring the chromatin very near to nuclear membrane. We speculate that this closed perinuclear localisation of TMEM18-bound DNA might repress transcription from it.

  17. Site-Specific Oligonucleotide Binding Represses Transcription of the Human c-myc Gene in vitro

    Science.gov (United States)

    Cooney, Michael; Czernuszewicz, Graznya; Postel, Edith H.; Flint, S. Jane; Hogan, Michael E.

    1988-07-01

    A 27-base-long DNA oligonucleotide was designed that binds to duplex DNA at a single site within the 5' end of the human c-myc gene, 115 base pairs upstream from the transcription origin P1. On the basis of the physical properties of its bound complex, it was concluded that the oligonucleotide forms a colinear triplex with the duplex binding site. By means of an in vitro assay system, it was possible to show a correlation between triplex formation at -115 base pairs and repression of c-myc transcription. The possibility is discussed that triplex formation (site-specific RNA binding to a DNA duplex) could serve as the basis for an alternative program of gene control in vivo.

  18. G-quadruplex RNA binding and recognition by the lysine-specific histone demethylase-1 enzyme.

    Science.gov (United States)

    Hirschi, Alexander; Martin, William J; Luka, Zigmund; Loukachevitch, Lioudmila V; Reiter, Nicholas J

    2016-08-01

    Lysine-specific histone demethylase 1 (LSD1) is an essential epigenetic regulator in metazoans and requires the co-repressor element-1 silencing transcription factor (CoREST) to efficiently catalyze the removal of mono- and dimethyl functional groups from histone 3 at lysine positions 4 and 9 (H3K4/9). LSD1 interacts with over 60 regulatory proteins and also associates with lncRNAs (TERRA, HOTAIR), suggesting a regulatory role for RNA in LSD1 function. We report that a stacked, intramolecular G-quadruplex (GQ) forming TERRA RNA (GG[UUAGGG]8UUA) binds tightly to the functional LSD1-CoREST complex (Kd ≈ 96 nM), in contrast to a single GQ RNA unit ([UUAGGG]4U), a GQ DNA ([TTAGGG]4T), or an unstructured single-stranded RNA. Stabilization of a parallel-stranded GQ RNA structure by monovalent potassium ions (K(+)) is required for high affinity binding to the LSD1-CoREST complex. These data indicate that LSD1 can distinguish between RNA and DNA as well as structured versus unstructured nucleotide motifs. Further, cross-linking mass spectrometry identified the primary location of GQ RNA binding within the SWIRM/amine oxidase domain (AOD) of LSD1. An ssRNA binding region adjacent to this GQ binding site was also identified via X-ray crystallography. This RNA binding interface is consistent with kinetic assays, demonstrating that a GQ-forming RNA can serve as a noncompetitive inhibitor of LSD1-catalyzed demethylation. The identification of a GQ RNA binding site coupled with kinetic data suggests that structured RNAs can function as regulatory molecules in LSD1-mediated mechanisms. PMID:27277658

  19. Structure-based redesign of the binding specificity of anti-apoptotic Bcl-x(L).

    Science.gov (United States)

    Chen, T Scott; Palacios, Hector; Keating, Amy E

    2013-01-01

    Many native proteins are multi-specific and interact with numerous partners, which can confound analysis of their functions. Protein design provides a potential route to generating synthetic variants of native proteins with more selective binding profiles. Redesigned proteins could be used as research tools, diagnostics or therapeutics. In this work, we used a library screening approach to reengineer the multi-specific anti-apoptotic protein Bcl-x(L) to remove its interactions with many of its binding partners, making it a high-affinity and selective binder of the BH3 region of pro-apoptotic protein Bad. To overcome the enormity of the potential Bcl-x(L) sequence space, we developed and applied a computational/experimental framework that used protein structure information to generate focused combinatorial libraries. Sequence features were identified using structure-based modeling, and an optimization algorithm based on integer programming was used to select degenerate codons that maximally covered these features. A constraint on library size was used to ensure thorough sampling. Using yeast surface display to screen a designed library of Bcl-x(L) variants, we successfully identified a protein with ~1000-fold improvement in binding specificity for the BH3 region of Bad over the BH3 region of Bim. Although negative design was targeted only against the BH3 region of Bim, the best redesigned protein was globally specific against binding to 10 other peptides corresponding to native BH3 motifs. Our design framework demonstrates an efficient route to highly specific protein binders and may readily be adapted for application to other design problems. PMID:23154169

  20. Specific RNA binding by amino-terminal peptides of alfalfa mosaic virus coat protein.

    Science.gov (United States)

    Baer, M L; Houser, F; Loesch-Fries, L S; Gehrke, L

    1994-01-01

    Specific RNA-protein interactions and ribonucleoprotein complexes are essential for many biological processes, but our understanding of how ribonucleoprotein particles form and accomplish their biological functions is rudimentary. This paper describes the interaction of alfalfa mosaic virus (A1MV) coat protein or peptides with viral RNA. A1MV coat protein is necessary both for virus particle formation and for the initiation of replication of the three genomic RNAs. We have examined protein determinants required for specific RNA binding and analyzed potential structural changes elicited by complex formation. The results indicate that the amino-terminus of the viral coat protein, which lacks primary sequence homology with recognized RNA binding motifs, is both necessary and sufficient for binding to RNA. Circular dichroism spectra and electrophoretic mobility shift experiments suggest that the RNA conformation is altered when amino-terminal coat protein peptides bind to the viral RNA. The peptide--RNA interaction is functionally significant because the peptides will substitute for A1MV coat protein in initiating RNA replication. The apparent conformational change that accompanies RNA--peptide complex formation may generate a structure which, unlike the viral RNA alone, can be recognized by the viral replicase. Images PMID:8313916

  1. Autoradiographic localization of specific [3H]dexamethasone binding in fetal lung

    International Nuclear Information System (INIS)

    The cellular and subcellular localization of specific [3H]dexamethasone binding was examined in fetal mouse lung at various stages of development and in human fetal lung at 8 weeks of gestation using a rapid in vitro steroid incubation technique followed by thaw-mount autoradiography. Competition studies with unlabeled steroids demonstrate the specificity of [3H]dexamethasone labeling, and indicate that fetal lung mesenchyme is a primary glucocorticoid target during lung development. Autoradiographs of [3H]dexamethasone binding in lung tissue at early stages of development demonstrate that the mesenchyme directly adjacent to the more proximal portions of the bronchiolar network is heavily labeled. In contrast, the epithelium which will later differentiate into bronchi and bronchioles, is relatively unlabeled. Distal portions of the growing epithelium, destined to become alveolar ducts and alveoli, do show nuclear localization of [3H]dexamethasone. In addition, by utilizing a technique which allows the simultaneous examination of extracellular matrix components and [3H]dexamethasone binding, a relationship is observed between extensive mesenchymal [3H]dexamethasone binding and extensive extracellular matrix accumulation. Since glucocorticoids stimulate the synthesis of many extracellular matrix components, these results suggest a role for these hormones in affecting mesenchymal-epithelial interactions during lung morphogenesis

  2. Study of the effect hydrogen binding in the solvation of alkaline earth cations with MeOH in nitromethane using 1 H NMR technique and determination of ionic solvation number

    CERN Document Server

    Alizadeh, N

    2001-01-01

    A proton NMR method for the study of the effect hydrogen binding and determination of solvation numbers of alkaline earth cations with methanol (MeOH) in in tromethane (NM) as diluent is described. The method is based on monitoring the resonance frequency of MeOH protons as a function of MeOH to metal ion mole ratio at constant metal ion concentration. the average solvation number of cation, n, at any MeOH/ metal ion mole ration was calculated from the NMR chemical shift-mole ration data and was plotted against the mole ration values. The solvation numbers of alkaline earth cations were obtained from the limiting values of the corresponding n, vs. mole ratio plots.

  3. Photoelectron Spectroscopy and Theoretical Studies of Anion-pi Interactions: Binding Strength and Anion Specificity

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Jian; Zhou, Bin; Sun, Zhenrong; Wang, Xue B.

    2015-01-01

    Proposed in theory and confirmed to exist, anion–π interactions have been recognized as new and important non-covalent binding forces. Despite extensive theoretical studies, numerous crystal structural identifications, and a plethora of solution phase investigations, intrinsic anion–π interaction strengths that are free from complications of condensed phases’ environments, have not been directly measured in the gas phase. Herein we present a joint photoelectron spectroscopic and theoretical study on this subject, in which tetraoxacalix[2]arene[2]triazine 1, an electron-deficient and cavity self-tunable macrocyclic was used as a charge-neutral molecular host to probe its interactions with a series of anions with distinctly different shapes and charge states (spherical halides Cl⁻, Br⁻, I⁻, linear thiocyanate SCN⁻, trigonal planar nitrate NO₃⁻, pyramidic iodate IO₃⁻, and tetrahedral sulfate SO₄²⁻). The binding energies of the resultant gaseous 1:1 complexes (1•Cl⁻,1•Br⁻, 1•I⁻, 1•SCN⁻, 1•NO₃⁻, 1•IO₃⁻ and 1•SO₄²⁻) were directly measured experimentally, exhibiting substantial non-covalent interactions with pronounced anion specific effects. The binding strengths of Cl⁻, NO₃⁻, IO₃⁻ with 1 are found to be strongest among all singly charged anions, amounting to ca. 30 kcal/mol, but only about 40% of that between 1 and SO₄²⁻. Quantum chemical calculations reveal that all anions reside in the center of the cavity of 1 with anion–π binding motif in the complexes’ optimized structures, where 1 is seen to be able to self-regulate its cavity structure to accommodate anions of different geometries and three-dimensional shapes. Electron density surface and natural bond orbital charge distribution analysis further support anion–π binding formation. The calculated binding energies of the anions and 1 nicely reproduce the experimentally estimated electron binding energy increase. This work

  4. Photoelectron spectroscopy and theoretical studies of anion-π interactions: binding strength and anion specificity.

    Science.gov (United States)

    Zhang, Jian; Zhou, Bin; Sun, Zhen-Rong; Wang, Xue-Bin

    2015-02-01

    Proposed in theory and then their existence confirmed, anion-π interactions have been recognized as new and important non-covalent binding forces. Despite extensive theoretical studies, numerous crystal structural identifications, and a plethora of solution phase investigations, anion-π interaction strengths that are free from complications of condensed-phase environments have not been directly measured in the gas phase. Herein we present a joint photoelectron spectroscopic and theoretical study on this subject, in which tetraoxacalix[2]arene[2]triazine 1, an electron-deficient and cavity self-tunable macrocyclic, was used as a charge-neutral molecular host to probe its interactions with a series of anions with distinctly different shapes and charge states (spherical halides Cl(-), Br(-), I(-), linear thiocyanate SCN(-), trigonal planar nitrate NO3(-), pyramidic iodate IO3(-), and tetrahedral sulfate SO4(2-)). The binding energies of the resultant gaseous 1 : 1 complexes (1·Cl(-), 1·Br(-), 1·I(-), 1·SCN(-), 1·NO3(-), 1·IO3(-) and 1·SO4(2-)) were directly measured experimentally, exhibiting substantial non-covalent interactions with pronounced anion-specific effects. The binding strengths of Cl(-), NO3(-), IO3(-) with 1 are found to be strongest among all singly charged anions, amounting to ca. 30 kcal mol(-1), but only about 40% of that between 1 and SO4(2-). Quantum chemical calculations reveal that all the anions reside in the center of the cavity of 1 with an anion-π binding motif in the complexes' optimized structures, where 1 is seen to be able to self-regulate its cavity structure to accommodate anions of different geometries and three-dimensional shapes. Electron density surface and charge distribution analyses further support anion-π binding formation. The calculated binding energies of the anions and 1 nicely reproduce the experimentally estimated electron binding energy increase. This work illustrates that size-selective photoelectron

  5. Identification of key residues in the A-Raf kinase important for phosphoinositide lipid binding specificity.

    Science.gov (United States)

    Johnson, Lindsey M; James, Kristy M; Chamberlain, M Dean; Anderson, Deborah H

    2005-03-01

    Raf kinases are involved in regulating cellular signal transduction pathways in response to a wide variety of external stimuli. Upstream signals generate activated Ras-GTP, important for the relocalization of Raf kinases to the membrane. Upon full activation, Raf kinases phosphorylate and activate downstream kinase in the mitogen-activated protein kinase (MAPK) signaling pathway. The Raf family of kinases has three members, Raf-1, B-Raf, and A-Raf. The ability of Raf-1 and B-Raf to bind phosphatidylserine (PS) and phosphatidic acid (PA) has been show to facilitate Raf membrane associations and regulate Raf kinase activity. We have characterized the lipid binding properties of A-Raf, as well as further characterized those of Raf-1. Both A-Raf and Raf-1 were found to bind to 3-, 4-, and 5-monophosphorylated phosphoinositides [PI(3)P, PI(4)P, and PI(5)P] as well as phosphatidylinositol 3,5-bisphosphate [PI(3,5)P(2)]. In addition, A-Raf also bound specifically to phosphatidylinositol 4,5- and 3,4-bisphosphates [PI(4,5)P(2) and PI(3,4)P(2)] and to PA. A mutational analysis of A-Raf localized the PI(4,5)P(2) binding site to two basic residues (K50 and R52) within the Ras binding domain. Additionally, an A-Raf mutant lacking the first 199 residues [i.e., the entire conserved region 1 (CR1) domain] bound the same phospholipids as full-length Raf-1. This suggests that a second region of A-Raf between amino acids 200 and 606 was responsible for interactions with the monophosphorylated PIs and PI(3,5)P(2). These results raise the possibility that Raf-1 and A-Raf bind to specific phosphoinositides as a mechanism to localize them to particular membrane microdomains rich in these phospholipids. Moreover, the differences in their lipid binding profiles could contribute to their proposed isoform-specific Raf functions.

  6. Quantitative Correlation of Conformational Binding Enthalpy with Substrate Specificity of Serine Proteases.

    Science.gov (United States)

    Waldner, Birgit J; Fuchs, Julian E; Huber, Roland G; von Grafenstein, Susanne; Schauperl, Michael; Kramer, Christian; Liedl, Klaus R

    2016-01-21

    Members of the same protease family show different substrate specificity, even if they share identical folds, depending on the physiological processes they are part of. Here, we investigate the key factors for subpocket and global specificity of factor Xa, elastase, and granzyme B which despite all being serine proteases and sharing the chymotrypsin-fold show distinct substrate specificity profiles. We determined subpocket interaction potentials with GRID for static X-ray structures and an in silico generated ensemble of conformations. Subpocket interaction potentials determined for static X-ray structures turned out to be insufficient to explain serine protease specificity for all subpockets. Therefore, we generated conformational ensembles using molecular dynamics simulations. We identified representative binding site conformations using distance-based hierarchical agglomerative clustering and determined subpocket interaction potentials for each representative conformation of the binding site. Considering the differences in subpocket interaction potentials for these representative conformations as well as their abundance allowed us to quantitatively explain subpocket specificity for the nonprime side for all three example proteases on a molecular level. The methods to identify key regions determining subpocket specificity introduced in this study are directly applicable to other serine proteases, and the results provide starting points for new strategies in rational drug design.

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

    OpenAIRE

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

    2011-01-01

    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 soluti...

  8. Protective action of resveratrol in human skin: possible involvement of specific receptor binding sites.

    Directory of Open Access Journals (Sweden)

    Stéphane Bastianetto

    Full Text Available BACKGROUND: Resveratrol is a plant-derived polyphenol with purported protecting action on various disorders associated with aging. It has been suggested that resveratrol could exert its protective action by acting on specific plasma membrane polyphenol binding sites (Han Y.S., et al. (2006 J Pharmacol Exp Ther 318:238-245. The purpose of this study was to investigate, in human skin, the possible existence of specific binding sites that mediate the protective action of resveratrol. METHODS AND FINDINGS: Using human skin tissue, we report here the presence of specific [(3H]-resveratrol binding sites (K(D  =  180 nM that are mainly located in the epidermis. Exposure of HaCaT cells to the nitric oxide free radical donor sodium nitroprusside (SNP; 0.3-3 mM resulted in cell death which was reduced by resveratrol (EC(50  =  14.7 µM, and to a much lesser extent by the resveratrol analogue piceatannol (EC(50  =  95 µM and epigallocatechin gallate (EC(50  =  200 µM, a green-tea derived polyphenol. The protective action of resveratrol likely relates to its anti-apoptotic effect since at the same range of concentration it was able to reduce both the number of apoptotic cells as well as mitochondrial apoptotic events triggered by SNP. CONCLUSION: Taken together, these findings suggest that resveratrol, by acting on specific polyphenol binding sites in epidermis, may be useful to prevent skin disorders associated with aging.

  9. Specific induction of fibronectin binding activity by hemoglobin in Candida albicans grown in defined media.

    OpenAIRE

    Yan, S.; Nègre, E; Cashel, J A; Guo, N; Lyman, C A; Walsh, T J; Roberts, D D

    1996-01-01

    Fibronectin (FN) is a major component of host extracellular matrix that may play an important role in the initiation and dissemination of Candida albicans infections. Expression of FN binding requires growth of C albicans blastoconidia in complex medium, and the regulation of FN receptor expression is poorly understood. We now demonstrate that hemoglobin is a potent and specific inducer of FN receptor expression and describe a defined medium supplemented with hemoglobin that greatly and stabl...

  10. A Novel DNA Binding Mechanism for maf Basic Region-Leucine Zipper Factors Inferred from a MafA-DNA Complex Structure and Binding Specificities

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Xun; Guanga, Gerald P; Wan, Cheng; Rose, Robert B [Z; (W Elec.); (NCSU)

    2012-11-13

    MafA is a proto-oncoprotein and is critical for insulin gene expression in pancreatic β-cells. Maf proteins belong to the AP1 superfamily of basic region-leucine zipper (bZIP) transcription factors. Residues in the basic helix and an ancillary N-terminal domain, the Extended Homology Region (EHR), endow maf proteins with unique DNA binding properties: binding a 13 bp consensus site consisting of a core AP1 site (TGACTCA) flanked by TGC sequences and binding DNA stably as monomers. To further characterize maf DNA binding, we determined the structure of a MafA–DNA complex. MafA forms base-specific hydrogen bonds with the flanking G–5C–4 and central C0/G0 bases, but not with the core-TGA bases. However, in vitro binding studies utilizing a pulse–chase electrophoretic mobility shift assay protocol revealed that mutating either the core-TGA or flanking-TGC bases dramatically increases the binding off rate. Comparing the known maf structures, we propose that DNA binding specificity results from positioning the basic helix through unique phosphate contacts. The EHR does not contact DNA directly but stabilizes DNA binding by contacting the basic helix. Collectively, these results suggest a novel multistep DNA binding process involving a conformational change from contacting the core-TGA to contacting the flanking-TGC bases.

  11. Natamycin blocks fungal growth by binding specifically to ergosterol without permeabilizing the membrane.

    Science.gov (United States)

    te Welscher, Yvonne M; ten Napel, Hendrik H; Balagué, Miriam Masià; Souza, Cleiton M; Riezman, Howard; de Kruijff, Ben; Breukink, Eefjan

    2008-03-01

    Natamycin is a polyene antibiotic that is commonly used as an antifungal agent because of its broad spectrum of activity and the lack of development of resistance. Other polyene antibiotics, like nystatin and filipin are known to interact with sterols, with some specificity for ergosterol thereby causing leakage of essential components and cell death. The mode of action of natamycin is unknown and is investigated in this study using different in vitro and in vivo approaches. Isothermal titration calorimetry and direct binding studies revealed that natamycin binds specifically to ergosterol present in model membranes. Yeast sterol biosynthetic mutants revealed the importance of the double bonds in the B-ring of ergosterol for the natamycin-ergosterol interaction and the consecutive block of fungal growth. Surprisingly, in strong contrast to nystatin and filipin, natamycin did not change the permeability of the yeast plasma membrane under conditions that growth was blocked. Also, in ergosterol containing model membranes, natamycin did not cause a change in bilayer permeability. This demonstrates that natamycin acts via a novel mode of action and blocks fungal growth by binding specifically to ergosterol. PMID:18165687

  12. The RFA regulatory sequence-binding protein in the promoter of prostate-specific antigen gene

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    To assure what sequence associated with the androgen regulation, a 15 bp region at the upstream of the ARE of prostate-specific antigen (PSA) promoter, termed RFA, was found indispensable for androgen receptor (AR)-mediated transactivation of PSA promoter. In transfection and CAT assays, some nucleotides substitution in RFA could significantly decrease the androgen inducibility for PSA promoter. The in vitro DNA binding assay demonstrated that RFA bound specifically with some non-receptor protein factors in prostate cell nucleus, but the mutant type of RFA lost this ability, so RFA might be a novel accessory cis-element. The RFA-binding proteins were isolated and purified by affinity chromatography using RFA probes. SDS-PAGE and preliminary protein identification showed these proteins possessed sequence high homology with multifunctional protein heterogeneous nuclear ribonucleoprotein A1, A2 (hnRNP A1, A2). RFA-binding proteins possibly cooperate with AR-mediated transactivation for PSA promoter as coactivator. The study results will facilitate further understanding the mechanism and tissue specificity of PSA promoter.

  13. Hydrophobic fluorescent probes introduce artifacts into single molecule tracking experiments due to non-specific binding.

    Directory of Open Access Journals (Sweden)

    Laura C Zanetti-Domingues

    Full Text Available Single-molecule techniques are powerful tools to investigate the structure and dynamics of macromolecular complexes; however, data quality can suffer because of weak specific signal, background noise and dye bleaching and blinking. It is less well-known, but equally important, that non-specific binding of probe to substrates results in a large number of immobile fluorescent molecules, introducing significant artifacts in live cell experiments. Following from our previous work in which we investigated glass coating substrates and demonstrated that the main contribution to this non-specific probe adhesion comes from the dye, we carried out a systematic investigation of how different dye chemistries influence the behaviour of spectrally similar fluorescent probes. Single-molecule brightness, bleaching and probe mobility on the surface of live breast cancer cells cultured on a non-adhesive substrate were assessed for anti-EGFR affibody conjugates with 14 different dyes from 5 different manufacturers, belonging to 3 spectrally homogeneous bands (491 nm, 561 nm and 638 nm laser lines excitation. Our results indicate that, as well as influencing their photophysical properties, dye chemistry has a strong influence on the propensity of dye-protein conjugates to adhere non-specifically to the substrate. In particular, hydrophobicity has a strong influence on interactions with the substrate, with hydrophobic dyes showing much greater levels of binding. Crucially, high levels of non-specific substrate binding result in calculated diffusion coefficients significantly lower than the true values. We conclude that the physic-chemical properties of the dyes should be considered carefully when planning single-molecule experiments. Favourable dye characteristics such as photostability and brightness can be offset by the propensity of a conjugate for non-specific adhesion.

  14. Mass-action equilibrium and non-specific interactions in protein binding networks

    Science.gov (United States)

    Maslov, Sergei

    2009-03-01

    Large-scale protein binding networks serve as a paradigm of complex properties of living cells. These networks are naturally weighted with edges characterized by binding strength and protein-nodes -- by their concentrations. However, the state-of-the-art high-throughput experimental techniques generate just a binary (yes or no) information about individual interactions. As a result, most of the previous research concentrated just on topology of these networks. In a series of recent publications [1-4] my collaborators and I went beyond purely topological studies and calculated the mass-action equilibrium of a genome-wide binding network using experimentally determined protein concentrations, localizations, and reliable binding interactions in baker's yeast. We then studied how this equilibrium responds to large perturbations [1-2] and noise [3] in concentrations of proteins. We demonstrated that the change in the equilibrium concentration of a protein exponentially decays (and sign-alternates) with its network distance away from the perturbed node. This explains why, despite a globally connected topology, individual functional modules in such networks are able to operate fairly independently. In a separate study [4] we quantified the interplay between specific and non-specific binding interactions under crowded conditions inside living cells. We show how the need to limit the waste of resources constrains the number of types and concentrations of proteins that are present at the same time and at the same place in yeast cells. [1] S Maslov, I. Ispolatov, PNAS 104:13655 (2007). [2] S. Maslov, K. Sneppen, I. Ispolatov, New J. of Phys. 9: 273 (2007). [3] K-K. Yan, D. Walker, S. Maslov, PRL accepted (2008). [4] J. Zhang, S. Maslov, and E. I. Shakhnovich, Mol Syst Biol 4, 210 (2008).

  15. Cations form sequence selective motifs within DNA grooves via a combination of cation-pi and ion-dipole/hydrogen bond interactions.

    Directory of Open Access Journals (Sweden)

    Mikaela Stewart

    Full Text Available The fine conformational subtleties of DNA structure modulate many fundamental cellular processes including gene activation/repression, cellular division, and DNA repair. Most of these cellular processes rely on the conformational heterogeneity of specific DNA sequences. Factors including those structural characteristics inherent in the particular base sequence as well as those induced through interaction with solvent components combine to produce fine DNA structural variation including helical flexibility and conformation. Cation-pi interactions between solvent cations or their first hydration shell waters and the faces of DNA bases form sequence selectively and contribute to DNA structural heterogeneity. In this paper, we detect and characterize the binding patterns found in cation-pi interactions between solvent cations and DNA bases in a set of high resolution x-ray crystal structures. Specifically, we found that monovalent cations (Tl⁺ and the polarized first hydration shell waters of divalent cations (Mg²⁺, Ca²⁺ form cation-pi interactions with DNA bases stabilizing unstacked conformations. When these cation-pi interactions are combined with electrostatic interactions a pattern of specific binding motifs is formed within the grooves.

  16. Cooperative and specific binding of Vif to the 5' region of HIV-1 genomic RNA.

    Science.gov (United States)

    Henriet, Simon; Richer, Delphine; Bernacchi, Serena; Decroly, Etienne; Vigne, Robert; Ehresmann, Bernard; Ehresmann, Chantal; Paillart, Jean-Christophe; Marquet, Roland

    2005-11-18

    The viral infectivity factor (Vif) protein of human immunodeficiency virus type 1 (HIV-1) is essential for viral replication in vivo. Packaging of Vif into viral particles is mediated by an interaction with viral genomic RNA and association with viral nucleoprotein complexes. Despite recent findings on the RNA-binding properties of Vif suggesting that Vif could be involved in retroviral assembly, no RNA sequence or structure specificity has been determined so far. To gain further insight into the mechanisms by which Vif might regulate viral replication, we studied the interactions of Vif with HIV-1 genomic RNA in vitro. Using extensive biochemical analysis, we have measured the affinity of recombinant Vif proteins for synthetic RNAs corresponding to various regions of the HIV-1 genome. We found that recombinant Vif proteins bind specifically to HIV-1 viral RNA fragments corresponding to the 5'-untranslated region (5'-UTR), gag and the 5' part of pol (K(d) between 45 nM and 65 nM). RNA encompassing nucleotides 1-497 or 499-996 of the HIV-1 genomic RNA bind 9+/-2 and 21+/-3 Vif molecules, respectively, and at least some of these proteins bind in a cooperative manner (Hill constant alpha(H) = 2.3). In contrast, RNAs corresponding to other parts of the HIV-1 genome or heterologous RNAs showed poor binding capacity and weak cooperativity (K(d) > 200 nM). Moreover, RNase T1 footprinting revealed a hierarchical binding of Vif, pointing to TAR and the poly(A) stem-loop structures as primary strong affinity targets, and downstream structures as secondary sites with moderate affinity. Taken together, our findings suggest that Vif may assist other proteins to maintain a correct folding of the genomic RNA in order to facilitate its packaging and further steps such as reverse transcription. Interestingly, our results suggest also that Vif could bind the viral RNA in order to protect it from the action of the antiviral factor APOBEC-3G/3F. PMID:16236319

  17. Identification of fluorescent compounds with non-specific binding property via high throughput live cell microscopy.

    Directory of Open Access Journals (Sweden)

    Sangeeta Nath

    Full Text Available INTRODUCTION: Compounds exhibiting low non-specific intracellular binding or non-stickiness are concomitant with rapid clearing and in high demand for live-cell imaging assays because they allow for intracellular receptor localization with a high signal/noise ratio. The non-stickiness property is particularly important for imaging intracellular receptors due to the equilibria involved. METHOD: Three mammalian cell lines with diverse genetic backgrounds were used to screen a combinatorial fluorescence library via high throughput live cell microscopy for potential ligands with high in- and out-flux properties. The binding properties of ligands identified from the first screen were subsequently validated on plant root hair. A correlative analysis was then performed between each ligand and its corresponding physiochemical and structural properties. RESULTS: The non-stickiness property of each ligand was quantified as a function of the temporal uptake and retention on a cell-by-cell basis. Our data shows that (i mammalian systems can serve as a pre-screening tool for complex plant species that are not amenable to high-throughput imaging; (ii retention and spatial localization of chemical compounds vary within and between each cell line; and (iii the structural similarities of compounds can infer their non-specific binding properties. CONCLUSION: We have validated a protocol for identifying chemical compounds with non-specific binding properties that is testable across diverse species. Further analysis reveals an overlap between the non-stickiness property and the structural similarity of compounds. The net result is a more robust screening assay for identifying desirable ligands that can be used to monitor intracellular localization. Several new applications of the screening protocol and results are also presented.

  18. 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.

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

    Directory of Open Access Journals (Sweden)

    Cicortas Gunnarsson Lavinia

    2009-10-01

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

  20. Pharmacological profile of the ATP-mediated increase in L-type calcium current amplitude and activation of a non-specific cationic current in rat ventricular cells.

    OpenAIRE

    Scamps, F.; Vassort, G

    1994-01-01

    1. The pharmacological profile of the ATP-induced increase in ICa amplitude and of ATP activation of a non-specific cationic current, IATP, was investigated in rat ventricular cells. 2. The EC50 values for ICa increase and IATP activation were 0.36 microM and 0.76 microM respectively. Suramin (10 microM) and cibacron blue (1 microM) competitively antagonized both effects of ATP. 3. The rank order of efficacy and potency of ATP analogues in increasing ICa amplitude was 2-methylthio-ATP approxi...

  1. Mechanism and specificity of lanthanide series cation transport by ionophores A23187, 4-BrA23187, and ionomycin.

    Science.gov (United States)

    Wang, E; Taylor, R W; Pfeiffer, D R

    1998-09-01

    A23187, 4-BrA23187, and ionomycin transport several lanthanide series trivalent cations at efficiencies similar to Ca2+, when compared at cation concentrations of approximately 10(-5) M, ionophore concentrations of approximately 10(-6) M, and a pH of 7.00. Selectivity sequences and the range of relative rates are as follows: A23187, Nd3+ > La3+ > Eu3+ > Gd3+ > Er3+ > Yb3+ > Lu3+ (approximately 34-fold); 4-BrA23187, Nd3+ > Eu3+ > Gd3+ > La3+ > Er3+ > Yb3+ > Lu3+ (approximately 34-fold); ionomycin, La3+ > Yb3+ > Nd3+ > Lu3+ > Er3+ > Eu3+ > Gd3+ (approximately 4-fold). At concentrations between 9 and 250 microM, La3+ is transported by an electroneutral mechanism, predominately through mixed complexes of the type (ionophore)2La-OH (A23187 and 4-BrA23187) or (ionophore)La-OH (ionomycin), when no membrane potential is present. For all three ionophores, an induced potential of approximately 160 mV accelerates transport by approximately 50-100%. However, measured values of H+/La3+ exchange indicate that only 4-BrA23187 displays a significant electrogenic activity under these conditions. At a La3+ concentration of 17 mM, transport by all three ionophores is electroneutral and apparently occurs through complexes of type (ionophore)3La (A23187 and 4-BrA23187) or (ionophore)La-OH (ionomycin). Analysis of these patterns in a context of comproportionation equilibria involving the transporting species and free La3+ indicates that the species containing three ionophore molecules are formed on the membrane when aqueous phase solution conditions would strongly favor a 1:1 complex, based upon previous studies in solution. The implications of this and other findings are discussed. PMID:9726927

  2. Related lectins from snowdrop and maize differ in their carbohydrate-binding specificity.

    Science.gov (United States)

    Fouquaert, Elke; Smith, David F; Peumans, Willy J; Proost, Paul; Balzarini, Jan; Savvides, Savvas N; Damme, Els J M Van

    2009-03-01

    Searches in an EST database from maize revealed the expression of a protein related to the Galanthus nivalis (GNA) agglutinin, referred to as GNA(maize). Heterologous expression of GNA(maize) in Pichia pastoris allowed characterization of the first nucleocytoplasmic GNA homolog from plants. GNA(maize) is a tetrameric protein which shares 64% sequence similarity with GNA. Glycan microarray analyses revealed important differences in the specificity. Unlike GNA, which binds strongly to high-mannose N-glycans, the lectin from maize reacts almost exclusively with more complex glycans. Interestingly, GNA(maize) prefers complex glycans containing beta1-2 GlcNAc residues. The obvious difference in carbohydrate-binding properties is accompanied by a 100-fold reduced anti-HIV activity. Although the sequences of GNA and GNA(maize) are clearly related they show only 28% sequence identity. Our results indicate that gene divergence within the family of GNA-related lectins leads to changes in carbohydrate-binding specificity, as shown on N-glycan arrays.

  3. A novel and highly specific phage endolysin cell wall binding domain for detection of Bacillus cereus.

    Science.gov (United States)

    Kong, Minsuk; Sim, Jieun; Kang, Taejoon; Nguyen, Hoang Hiep; Park, Hyun Kyu; Chung, Bong Hyun; Ryu, Sangryeol

    2015-09-01

    Rapid, specific and sensitive detection of pathogenic bacteria is crucial for public health and safety. Bacillus cereus is harmful as it causes foodborne illness and a number of systemic and local infections. We report a novel phage endolysin cell wall-binding domain (CBD) for B. cereus and the development of a highly specific and sensitive surface plasmon resonance (SPR)-based B. cereus detection method using the CBD. The newly discovered CBD from endolysin of PBC1, a B. cereus-specific bacteriophage, provides high specificity and binding capacity to B. cereus. By using the CBD-modified SPR chips, B. cereus can be detected at the range of 10(5)-10(8) CFU/ml. More importantly, the detection limit can be improved to 10(2) CFU/ml by using a subtractive inhibition assay based on the pre-incubation of B. cereus and CBDs, removal of CBD-bound B. cereus, and SPR detection of the unbound CBDs. The present study suggests that the small and genetically engineered CBDs can be promising biological probes for B. cereus. We anticipate that the CBD-based SPR-sensing methods will be useful for the sensitive, selective, and rapid detection of B. cereus.

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

    DEFF Research Database (Denmark)

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

    2008-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-03-23

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

  6. The specificity of the secondary DNA binding site of RecA protein defines its role in DNA strand exchange.

    OpenAIRE

    Mazin, A V; Kowalczykowski, S C

    1996-01-01

    The RecA protein-single-stranded DNA (ssDNA) filament can bind a second DNA molecule. Binding of ssDNA to this secondary site shows specificity, in that polypyrimidinic DNA binds to the RecA protein-ssDNA filament with higher affinity than polypurinic sequences. The affinity of ssDNA, which is identical in sequence to that bound in the primary site, is not always greater than that of nonhomologous DNA. Moreover, this specificity of DNA binding does not depend on the sequence of the DNA bound ...

  7. A cellular protein specifically binds to the 3'-terminal sequences of hepatitis C virus intermediate negative-strand RNA

    Institute of Scientific and Technical Information of China (English)

    王巍; 邓庆丽; 黄开红; 段朝晖; 邵静; 黄志清; 黄志明

    2003-01-01

    ObjectiveTo study the mechanism of the cellular proteins involved in the process of replication of hepatitis C virus (HCV) negative-strand RNA.MethodsUltraviolet (UV) cross-linking was used to identify the cellular proteins that would bind to the 3'-end of HCV negative-strand RNA. Competition experimentwas used to confirm the specificity of this binding, in which excess nonhomologous protein and RNA transcripts were used as competitors. The required binding sequence was determined by mapping, then the binding site was predicted through secondary structure analysis.ResultsA cellular protein of 45 kD (p45) was found to bind specifically to the 3'-endof HCV negative-strand RNA by UV cross-linking. nhomologous proteins and RNA transcripts could not compete out this binding, whereas the unlabeled 3'-endof HCV negative-strand RNA could. Mapping of the protein-binding site suggested that the 3'-end 131-278nt of HCV negative-strand RNA was the possible protein-binding region. Analysis of RNA secondary structure presumed that the potential binding site was located at 194-GAAAGAAC-201. ConclusionThe cellular protein p45 could specifically bind to the secondary structure of the 3'-end of HCV intermediate negative-strand RNA, and may play an important role in HCV RNA replication.

  8. Steric mechanism of auto-inhibitory regulation of specific and non-specific DNA binding by the ETS transcriptional repressor ETV6.

    Science.gov (United States)

    De, Soumya; Chan, Anson C K; Coyne, H Jerome; Bhachech, Niraja; Hermsdorf, Ulrike; Okon, Mark; Murphy, Michael E P; Graves, Barbara J; McIntosh, Lawrence P

    2014-04-01

    DNA binding by the ETS transcriptional repressor ETV6 (or TEL) is auto-inhibited ~50-fold due to an α-helix that sterically blocks its ETS domain binding interface. Using NMR spectroscopy, we demonstrate that this marginally stable helix is unfolded, and not displaced to a non-inhibitory position, when ETV6 is bound to DNA containing a consensus (5')GGAA(3') recognition site. Although significantly lower in affinity, binding to non-specific DNA is auto-inhibited ~5-fold and is also accompanied by helix unfolding. Based on NMR chemical shift perturbations, both specific and non-specific DNA are bound via the same canonical ETS domain interface. However, spectral perturbations are smaller for the non-specific complex, suggesting weaker and less well-defined interactions than in the specific complex. In parallel, the crystal structure of ETV6 bound to a specific DNA duplex was determined. The structure of this complex reveals that a non-conserved histidine residue in the ETS domain recognition helix helps establish the specificity of ETV6 for DNA-binding sites containing (5')GGAA(3')versus(5')GGAT(3'). These studies provide a unified steric mechanism for attenuating ETV6 binding to both specific and non-specific DNA and expand the repertoire of characterized auto-inhibitory strategies utilized to regulate ETS factors.

  9. Structural Basis for Metal Binding Specificity: the N-terminal Cadmium Binding Domain of the P1-type ATPase CadA

    OpenAIRE

    Banci, Lucia; Bertini, Ivano; Ciofi-Baffoni, Simone; Su, Xun-Cheng; Miras, Roger; Bal, Nathalie; Mintz, Elisabeth; Catty, Patrice; Shokes, Jacob E.; Scott, Robert A

    2005-01-01

    In bacteria, P1-type ATPases are responsible for resistance to di- and monovalent toxic heavy metals by taking them out of the cell. These ATPases have a cytoplasmic N terminus comprising metal binding domains defined by a βαββαβ fold and a CXXC metal binding motif. To check how the structural properties of the metal binding site in the N terminus can influence the metal specificity of the ATPase, the first structure of a Cd(II)-ATPase N terminus was determined by NMR and its coordination sph...

  10. Mycobacterium tuberculosis Rv2536 protein implicated in specific binding to human cell lines

    OpenAIRE

    García, Javier; Puentes, Alvaro; Rodríguez, Luis; Ocampo, Marisol; Curtidor, Hernando; Vera, Ricardo; Lopez, Ramses; Valbuena, John; Cortes, Jimena; Vanegas, Magnolia; Barrero, Carlos; Patarroyo, Manuel A; Urquiza, Mauricio; Patarroyo, Manuel E.

    2005-01-01

    The gene encoding the Mycobacterium tuberculosis Rv2536 protein is present in the Mycobacterium tuberculosis complex (as assayed by PCR) and transcribed (as determined by RT-PCR) in M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. bovis BCG, and M. africanum strains. Rabbits immunized with synthetic polymer peptides from this protein produced antibodies specifically recognizing a 25-kDa band in mycobacterial sonicate. U937 and A549 cells were used in binding assays involving 20-amino-acid-lon...

  11. Altered Specificity of DNA-Binding Proteins with Transition Metal Dimerization Domains

    Science.gov (United States)

    Cuenoud, Bernard; Schepartz, Alanna

    1993-01-01

    The bZIP motif is characterized by a leucine zipper domain that mediates dimerization and a basic domain that contacts DNA. A series of transition metal dimerization domains were used to alter systematically the relative orientation of basic domain peptides. Both the affinity and the specificity of the peptide-DNA interaction depend on domain orientation. These results indicate that the precise configuration linking the domains is important; dimerization is not always sufficient for DNA binding. This approach to studying the effect of orientation on protein function complements mutagenesis and could be used in many systems.

  12. Male tarsi specific odorant-binding proteins in the diving beetle Cybister japonicus sharp.

    Science.gov (United States)

    Song, Li-Mei; Jiang, Xiang; Wang, Xue-Min; Li, Jin-Dong; Zhu, Fang; Tu, Xiong-Bing; Zhang, Ze-Hua; Ban, Li-Ping

    2016-01-01

    Odorant binding proteins (OBPs) play critical roles in chemical communication of insects, as they recognize and transport environmental chemical signals to receptors. The diving beetle Cybister japonicus Sharp shows a remarkable sexual dimorphism. The foreleg tarsi of males are equipped with large suction cups, believed to help holding the female during underwater courtship and mating. Here, we identified two OBPs highly and specifically expressed in male tarsi, suggesting important functions of these structures in chemical communication. The first protein, CjapOBP1, exhibits the 6 conserved cysteines motif of classic OBPs, while the second, CjapOBP2, contains only four cysteines and can be assigned to the sub-class of C-minus OBPs. Both proteins were expressed in a bacterial system and the purified recombinant proteins were used to for antibodies preparation. Western Blot analysis showed that CjapOBP1 is predominantly expressed in male tarsi and could be also detected in antennae and palpi of both sexes, while CjapOBP2, besides male tarsi, is also present in testis. Ligand-binding experiments showed a good binding affinity between CjapOBP1, CjapOBP2 and citral and coniferyl aldehyde, respectively. These results support a possible function of these two OBPs in the male foreleg tarsi of diving beetles in chemical communication. PMID:27545810

  13. High specific activity enantiomerically enriched juvenile hormones: synthesis and binding assay

    Energy Technology Data Exchange (ETDEWEB)

    Prestwich, G.D.; Wawrzenczyk, C.

    1985-08-01

    A stereoselective total synthesis of chiral juvenile hormone I is described that allows stoichiometric introduction of two tritium atoms in the final step. Both optical antipodes of the pivotal epoxy alcohol intermediate were prepared in 95% enantiomeric excess by the Sharpless epoxidation of a (Z)-allylic alcohol. Elaboration of the hydroxy-methyl group to a vinyl group followed by selective homogeneous tritiation affords optically active juvenile hormone I analogs at 58 Ci/mmol. Competitive binding of the labeled 10R, 11S and 10S,11R enantiomers with unlabeled enantiomers to the hemolymph binding protein of Manduca sexta larvae was determined by using a dextran-coated charcoal assay. The natural 10R,11S enantiomer has twice the relative binding affinity of the 10S,11R enantiomer. The availability of such high specific activity optically pure hormones will contribute substantially to the search for high-affinity receptors for juvenile hormones in the nuclei of cells. Moreover, the chiral 12-hydroxy-(10R,11S)-epoxy intermediate allows modification of juvenile hormone for solid-phase biochemical and radioimmunochemical work without altering either the biologically important carbomethoxy or epoxy recognition sites.

  14. PTPRT regulates the interaction of Syntaxin-binding protein 1 with Syntaxin 1 through dephosphorylation of specific tyrosine residue

    Energy Technology Data Exchange (ETDEWEB)

    Lim, So-Hee; Moon, Jeonghee [Biomedical Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806 (Korea, Republic of); Lee, Myungkyu [Bionanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806 (Korea, Republic of); Lee, Jae-Ran, E-mail: leejr@kribb.re.kr [Biomedical Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806 (Korea, Republic of)

    2013-09-13

    Highlights: •PTPRT is a brain-specific, expressed, protein tyrosine phosphatase. •PTPRT regulated the interaction of Syntaxin-binding protein 1 with Syntaxin 1. •PTPRT dephosphorylated the specific tyrosine residue of Syntaxin-binding protein 1. •Dephosphorylation of Syntaxin-binding protein 1 enhanced the interaction with Syntaxin 1. •PTPRT appears to regulate the fusion of synaptic vesicle through dephosphorylation. -- Abstract: PTPRT (protein tyrosine phosphatase receptor T), a brain-specific tyrosine phosphatase, has been found to regulate synaptic formation and development of hippocampal neurons, but its regulation mechanism is not yet fully understood. Here, Syntaxin-binding protein 1, a key component of synaptic vesicle fusion machinery, was identified as a possible interaction partner and an endogenous substrate of PTPRT. PTPRT interacted with Syntaxin-binding protein 1 in rat synaptosome, and co-localized with Syntaxin-binding protein 1 in cultured hippocampal neurons. PTPRT dephosphorylated tyrosine 145 located around the linker between domain 1 and 2 of Syntaxin-binding protein 1. Syntaxin-binding protein 1 directly binds to Syntaxin 1, a t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein, and plays a role as catalysts of SNARE complex formation. Syntaxin-binding protein 1 mutant mimicking non-phosphorylation (Y145F) enhanced the interaction with Syntaxin 1 compared to wild type, and therefore, dephosphorylation of Syntaxin-binding protein 1 appeared to be important for SNARE-complex formation. In conclusion, PTPRT could regulate the interaction of Syntaxin-binding protein 1 with Syntaxin 1, and as a result, the synaptic vesicle fusion appeared to be controlled through dephosphorylation of Syntaxin-binding protein 1.

  15. Research progress in cation-π interactions

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Cation-π interaction is a potent intermolecular interaction between a cation and an aromatic system,which has been viewed as a new kind of binding force,as being compared with the classical interactions(e.g. hydrogen bonding,electrostatic and hydrophobic interactions). Cation-π interactions have been observed in a wide range of biological contexts. In this paper,we present an overview of the typical cation-π interactions in biological systems,the experimental and theoretical investigations on cation-π interactions,as well as the research results on cation-π interactions in our group.

  16. Research progress in cation-π interactions

    Institute of Scientific and Technical Information of China (English)

    CHENG JiaGao; LUO XiaoMin; YAN XiuHua; LI Zhong; TANG Yun; JIANG HuaLiang; ZHU WeiLiang

    2008-01-01

    Cation-π interaction is a potent intermolecular interaction between a cation and an aromatic system, which has been viewed as a new kind of binding force, as being compared with the classical interac-tions (e.g. hydrogen bonding, electrostatic and hydrophobic interactions). Cation-π interactions have been observed in a wide range of biological contexts. In this paper, we present an overview of the typi-cal cation-π interactions in biological systems, the experimental and theoretical investigations on cation-π interactions, as well as the research results on cation-π interactions in our group.

  17. Molecular Determinants Underlying Binding Specificities of the ABL Kinase Inhibitors: Combining Alanine Scanning of Binding Hot Spots with Network Analysis of Residue Interactions and Coevolution.

    Directory of Open Access Journals (Sweden)

    Amanda Tse

    Full Text Available Quantifying binding specificity and drug resistance of protein kinase inhibitors is of fundamental importance and remains highly challenging due to complex interplay of structural and thermodynamic factors. In this work, molecular simulations and computational alanine scanning are combined with the network-based approaches to characterize molecular determinants underlying binding specificities of the ABL kinase inhibitors. The proposed theoretical framework unveiled a relationship between ligand binding and inhibitor-mediated changes in the residue interaction networks. By using topological parameters, we have described the organization of the residue interaction networks and networks of coevolving residues in the ABL kinase structures. This analysis has shown that functionally critical regulatory residues can simultaneously embody strong coevolutionary signal and high network centrality with a propensity to be energetic hot spots for drug binding. We have found that selective (Nilotinib and promiscuous (Bosutinib, Dasatinib kinase inhibitors can use their energetic hot spots to differentially modulate stability of the residue interaction networks, thus inhibiting or promoting conformational equilibrium between inactive and active states. According to our results, Nilotinib binding may induce a significant network-bridging effect and enhance centrality of the hot spot residues that stabilize structural environment favored by the specific kinase form. In contrast, Bosutinib and Dasatinib can incur modest changes in the residue interaction network in which ligand binding is primarily coupled only with the identity of the gate-keeper residue. These factors may promote structural adaptability of the active kinase states in binding with these promiscuous inhibitors. Our results have related ligand-induced changes in the residue interaction networks with drug resistance effects, showing that network robustness may be compromised by targeted mutations

  18. Mineralocorticoid specificity of renal type I receptors: in vivo binding studies

    Energy Technology Data Exchange (ETDEWEB)

    Sheppard, K.; Funder, J.W.

    1987-02-01

    The authors have injected rats with (TH)aldosterone or (TH) corticosterone, plus 100-fold excess of the highly specific glucocorticoid RU 28362, with or without excess unlabeled aldosterone or corticosterone and compared type I receptor occupancy in kidney and hippocampus. Thirty minutes after subcutaneous injection (TH)aldosterone was well retained in renal papilla-inner medulla, renal cortex-outer medulla, and hippocampus; in contrast, (TH)corticosterone was well retained only in hippocampus. Competition studies for (TH)aldosterone binding sites showed corticosterone to be a poor competitor in the kidney compared with hippocampus. Time-course studies, with rats killed 10-180 min after tracer administration, showed very low uptake/retention of (TH)corticosterone by kidney; in hippocampus (TH)corticosterone retention was similar to that of (TH)aldosterone in kidney, and retention of (TH)aldosterone by hippocampus was much more prolonged than of either tracer in any other tissue. Studies in 10-day-old rats, with very low levels of corticosteroid binding globulin (CBG), showed a high degree of aldosterone selectivity in both zones of the kidney, whereas 9TH)aldosterone and (TH)corticosterone were equivalently bound in hippocampus. They interpret these data as evidenced for a mechanism unrelated to extravascular CBG conferring mineralocorticoid specificity on renal type I receptors and propose two models derived from their findings consistent with such differential selectivity.

  19. Molecular and Functional Characterization of ssDNA Aptamers that Specifically Bind Leishmania infantum PABP.

    Directory of Open Access Journals (Sweden)

    Natalia Guerra-Pérez

    Full Text Available A poly (A-binding protein from Leishmania infantum (LiPABP has been recently cloned and characterized in our laboratory. Although this protein shows a very high homology with PABPs from other eukaryotic organisms including mammals and other parasites, exist divergences along the sequence that convert them in potential diagnostic markers and/or therapeutics targets. Aptamers are oligonucleotide ligands that are selected in vitro by their affinity and specificity for the target as a consequence of the particular tertiary structure that they are able to acquire depending on their sequence. Development of high-affinity molecules with the ability to recognize specifically Leishmania proteins is essential for the progress of this kind of study.We have selected a ssDNA aptamer population against a recombinant 6xHIS-LiPABP protein (rLiPABP that is able to recognize the target with a low Kd. Cloning, sequencing and in silico analysis of the aptamers obtained from the population yielded three aptamers (ApPABP#3, ApPABP#7 and ApPABP#11 that significantly bound to PABP with higher affinity than the naïve population. These aptamers were analyzed by ELONA and slot blot to establish affinity and specificity for rLiPABP. Results demonstrated that the three aptamers have high affinity and specificity for the target and that they are able to detect an endogenous LiPABP (eLiPABP protein amount corresponding to 2500 L. infantum promastigotes in a significant manner. The functional analysis of the aptamers also revealed that ApPABP#11 disrupts the binding of both Myc-LiPABP and eLiPABP to poly (A in vitro. On the other hand, these aptamers are able to bind and purify LiPABP from complex mixes.Results presented here demonstrate that aptamers represent new reagents for characterization of LiPABP and that they can affect LiPABP activity. At this respect, the use of these aptamers as therapeutic tool affecting the physiological role of PABP has to be analyzed.

  20. Improved pan-specific MHC class I peptide-binding predictions using a novel representation of the MHC-binding cleft environment

    DEFF Research Database (Denmark)

    Carrasco Pro, S.; Zimic, M.; Nielsen, Morten

    2014-01-01

    Major histocompatibility complex (MHC) molecules play a key role in cell-mediated immune responses presenting bounded peptides for recognition by the immune system cells. Several in silico methods have been developed to predict the binding affinity of a given peptide to a specific MHC molecule. One...... of the current state-of-the-art methods for MHC class I is NetMHCpan, which has a core ingredient for the representation of the MHC class I molecule using a pseudo-sequence representation of the binding cleft amino acid environment. New and large MHC-peptide-binding data sets are constantly being made available......, and also new structures of MHC class I molecules with a bound peptide have been published. In order to test if the NetMHCpan method can be improved by integrating this novel information, we created new pseudo-sequence definitions for the MHC-binding cleft environment from sequence and structural analyses...

  1. Binding of the Cationic Peptide (KL)4K to Lipid Monolayers at the Air-Water Interface: Effect of Lipid Headgroup Charge, Acyl Chain Length, and Acyl Chain Saturation.

    Science.gov (United States)

    Hädicke, André; Blume, Alfred

    2016-04-28

    The binding of the cationic peptide (KL)4K to monolayers of different anionic lipids was determined by adsorption experiments. The chemical structure of the anionic phospholipids was changed in different ways. First, the hydrophobic region of phosphatidylglycerols was altered by elongation of the acyl chain length. Second, an unsaturated chain was introduced. Third, lipids with negatively charged headgroups of different chemical structure were compared. (KL)4K itself shows no surface activity and does not bind to monolayers of zwitterionic lipids. Analysis of (KL)4K binding to anionic lipid monolayers reveals a competition between two binding processes: (i) incorporation of the peptide into the acyl chain region (surface pressure increase) and (ii) electrostatic interaction screening the negative charges with reduction of charge repulsion (surface pressure decrease due to monolayer condensation). The lipid acyl chain length and the chemical structure of the headgroup have minor effects on the binding properties. However, a strong dependence on the phase state of the monolayer was observed. In the liquid-expanded (LE) phase, the fluid monolayer provides enough space, so that peptide insertion due to hydrophobic interactions dominates. For monolayers in the liquid-condensed (LC) phase, peptide binding followed by monolayer condensation is the main effect. PMID:27049846

  2. Directed evolution of estrogen receptor proteins with altered ligand-binding specificities.

    Science.gov (United States)

    Islam, Kazi Mohammed Didarul; Dilcher, Meik; Thurow, Corinna; Vock, Carsten; Krimmelbein, Ilga Kristine; Tietze, Lutz Friedjan; Gonzalez, Victor; Zhao, Huimin; Gatz, Christiane

    2009-01-01

    Transcriptional activators that respond to ligands with no cellular targets are powerful tools that can confer regulated expression of a transgene in almost all biological systems. In this study, we altered the ligand-binding specificity of the human estrogen receptor alpha (hER alpha) so that it would recognize a non-steroidal synthetic compound with structural similarities to the phytoestrogen resveratrol. For this purpose, we performed iterative rounds of site-specific saturation mutagenesis of a fixed set of ligand-contacting residues and subsequent random mutagenesis of the entire ligand-binding domain. Selection of the receptor mutants and quantification of the interaction were carried out by exploiting a yeast two-hybrid system that reports the ligand-dependent interaction between hER alpha and steroid receptor coactivator-1 (SRC-1). The screen was performed with a synthetic ligand (CV3320) that promoted growth of the reporter yeast strain to half maximal levels at a concentration of 3.7 microM. The optimized receptor mutant (L384F/L387M/Y537S) showed a 67-fold increased activity to the synthetic ligand CV3320 (half maximal yeast growth at 0.055 microM) and a 10-fold decreased activity to 17beta-estradiol (E2; half maximal yeast growth at 4 nM). The novel receptor-ligand pair partially fulfills the requirements for a specific 'gene switch' as it responds to concentrations of the synthetic ligand which do not activate the wildtype receptor. Due to its residual responsiveness to E2 at concentrations (4 nM) that might occur in vivo, further improvements have to be performed to render the system applicable in organisms with endogenous E2 synthesis.

  3. Accurate pan-specific prediction of peptide-MHC class II binding affinity with improved binding core identification

    DEFF Research Database (Denmark)

    Andreatta, Massimo; Karosiene, Edita; Rasmussen, Michael;

    2015-01-01

    A key event in the generation of a cellular response against malicious organisms through the endocytic pathway is binding of peptidic antigens by major histocompatibility complex class II (MHC class II) molecules. The bound peptide is then presented on the cell surface where it can be recognized ...

  4. Characterization of a Single-Stranded DNA-Binding-Like Protein from Nanoarchaeum equitans--A Nucleic Acid Binding Protein with Broad Substrate Specificity.

    Directory of Open Access Journals (Sweden)

    Marcin Olszewski

    Full Text Available SSB (single-stranded DNA-binding proteins play an essential role in all living cells and viruses, as they are involved in processes connected with ssDNA metabolism. There has recently been an increasing interest in SSBs, since they can be applied in molecular biology techniques and analytical methods. Nanoarchaeum equitans, the only known representative of Archaea phylum Nanoarchaeota, is a hyperthermophilic, nanosized, obligatory parasite/symbiont of Ignicoccus hospitalis.This paper reports on the ssb-like gene cloning, gene expression and characterization of a novel nucleic acid binding protein from Nanoarchaeum equitans archaeon (NeqSSB-like protein. This protein consists of 243 amino acid residues and one OB fold per monomer. It is biologically active as a monomer like as SSBs from some viruses. The NeqSSB-like protein displays a low sequence similarity to the Escherichia coli SSB, namely 10% identity and 29% similarity, and is the most similar to the Sulfolobus solfataricus SSB (14% identity and 32% similarity. The NeqSSB-like protein binds to ssDNA, although it can also bind mRNA and, surprisingly, various dsDNA forms, with no structure-dependent preferences as evidenced by gel mobility shift assays. The size of the ssDNA binding site, which was estimated using fluorescence spectroscopy, is 7 ± 1 nt. No salt-dependent binding mode transition was observed. NeqSSB-like protein probably utilizes a different model for ssDNA binding than the SSB proteins studied so far. This protein is highly thermostable; the half-life of the ssDNA binding activity is 5 min at 100 °C and melting temperature (T(m is 100.2 °C as shown by differential scanning calorimetry (DSC analysis.NeqSSB-like protein is a novel highly thermostable protein which possesses a unique broad substrate specificity and is able to bind all types of nucleic acids.

  5. Characterization of a Single-Stranded DNA-Binding-Like Protein from Nanoarchaeum equitans—A Nucleic Acid Binding Protein with Broad Substrate Specificity

    Science.gov (United States)

    Olszewski, Marcin; Balsewicz, Jan; Nowak, Marta; Maciejewska, Natalia; Cyranka-Czaja, Anna; Zalewska-Piątek, Beata; Piątek, Rafał; Kur, Józef

    2015-01-01

    Background SSB (single-stranded DNA-binding) proteins play an essential role in all living cells and viruses, as they are involved in processes connected with ssDNA metabolism. There has recently been an increasing interest in SSBs, since they can be applied in molecular biology techniques and analytical methods. Nanoarchaeum equitans, the only known representative of Archaea phylum Nanoarchaeota, is a hyperthermophilic, nanosized, obligatory parasite/symbiont of Ignicoccus hospitalis. Results This paper reports on the ssb-like gene cloning, gene expression and characterization of a novel nucleic acid binding protein from Nanoarchaeum equitans archaeon (NeqSSB-like protein). This protein consists of 243 amino acid residues and one OB fold per monomer. It is biologically active as a monomer like as SSBs from some viruses. The NeqSSB-like protein displays a low sequence similarity to the Escherichia coli SSB, namely 10% identity and 29% similarity, and is the most similar to the Sulfolobus solfataricus SSB (14% identity and 32% similarity). The NeqSSB-like protein binds to ssDNA, although it can also bind mRNA and, surprisingly, various dsDNA forms, with no structure-dependent preferences as evidenced by gel mobility shift assays. The size of the ssDNA binding site, which was estimated using fluorescence spectroscopy, is 7±1 nt. No salt-dependent binding mode transition was observed. NeqSSB-like protein probably utilizes a different model for ssDNA binding than the SSB proteins studied so far. This protein is highly thermostable; the half-life of the ssDNA binding activity is 5 min at 100°C and melting temperature (Tm) is 100.2°C as shown by differential scanning calorimetry (DSC) analysis. Conclusion NeqSSB-like protein is a novel highly thermostable protein which possesses a unique broad substrate specificity and is able to bind all types of nucleic acids. PMID:25973760

  6. Stage-specific adhesion of Leishmania promastigotes to sand fly midguts assessed using an improved comparative binding assay.

    Directory of Open Access Journals (Sweden)

    Raymond Wilson

    forms, but is absent in the early blood meal and final stages (procyclic and metacyclic forms. Further they show that although gut binding may be necessary for parasite establishment, in several vector-parasite pairs the specificity of such in vitro binding alone is insufficient to explain overall vector specificity. Other significant barriers to development must exist in certain refractory Leishmania parasite-sand fly vector combinations. A re-appraisal of the specificity of the Leishmania-sand fly relationship is required.

  7. Effect of water coordination on competition between π and non-π cation binding sites in aromatic amino acids: L-phenylalanine, L-tyrosine, and L-tryptophan Li+, Na +, and K+ complexes.

    Science.gov (United States)

    Remko, Milan; Šoralová, Stanislava

    2012-04-01

    Quantum chemistry methods have been applied to charged complexes of the alkali metals Li(+), Na(+), and K(+) with the aromatic amino acids (AAAs) phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp). The geometries of 72 different complexes (Phe·M, Tyr·M, Trp·M, M is Li(+), Na(+), or K(+)) were completely optimized at the B3LYP/6-311+G(d,p) level of density functional theory. The solvent effect on the geometry and stability of individual complexes was studied by making use of a microsolvation model. The interaction enthalpies, entropies, and Gibbs energies of nine different complexes of the systems Phe·M, Tyr·M, and Trp·M (M is Li(+), Na(+), or K(+)) were also determined at the B3LYP density functional level of theory. The calculated Gibbs binding energies of the M(+)-AAA complexes follow the order Phe < Tyr < Trp for all three metal cations studied. Among the three AAAs studied, the indole ring of Trp is the best π donor for alkali metal cations. Our calculations demonstrated the existence of strong cation-π interactions between the alkali metals and the aromatic side chains of the three AAAs. These AAAs comprise about 8% of all known protein sequences. Thus, besides the potential for hydrogen-bond interaction, aromatic residues of Phe, Tyr, and Trp show great potential for π-donor interactions. The existence of cation-π interaction in proteins has also been demonstrated experimentally. However, more complex experimental studies of metal cation-π interaction in diverse biological systems will no doubt lead to more exact validation of these investigations.

  8. Structures of 5-Methylthioribose Kinase Reveal Substrate Specificity and Unusual Mode of Nucleotide Binding

    Energy Technology Data Exchange (ETDEWEB)

    Ku,S.; Yip, P.; Cornell, K.; Riscoe, M.; Behr, J.; Guillerm, G.; Howell, P.

    2007-01-01

    The methionine salvage pathway is ubiquitous in all organisms, but metabolic variations exist between bacteria and mammals. 5-Methylthioribose (MTR) kinase is a key enzyme in methionine salvage in bacteria and the absence of a mammalian homolog suggests that it is a good target for the design of novel antibiotics. The structures of the apo-form of Bacillus subtilis MTR kinase, as well as its ADP, ADP-PO4, AMPPCP, and AMPPCP-MTR complexes have been determined. MTR kinase has a bilobal eukaryotic protein kinase fold but exhibits a number of unique features. The protein lacks the DFG motif typically found at the beginning of the activation loop and instead coordinates magnesium via a DXE motif (Asp{sup 250}-Glu{sup 252}). In addition, the glycine-rich loop of the protein, analogous to the 'Gly triad' in protein kinases, does not interact extensively with the nucleotide. The MTR substrate-binding site consists of Asp{sup 233} of the catalytic HGD motif, a novel twin arginine motif (Arg{sup 340}/Arg{sup 341}), and a semi-conserved W-loop, which appears to regulate MTR binding specificity. No lobe closure is observed for MTR kinase upon substrate binding. This is probably because the enzyme lacks the lobe closure/inducing interactions between the C-lobe of the protein and the ribosyl moiety of the nucleotide that are typically responsible for lobe closure in protein kinases. The current structures suggest that MTR kinase has a dissociative mechanism.

  9. On the specificity of heparin/heparan sulfate binding to proteins. Anion-binding sites on antithrombin and thrombin are fundamentally different.

    Directory of Open Access Journals (Sweden)

    Philip D Mosier

    Full Text Available BACKGROUND: The antithrombin-heparin/heparan sulfate (H/HS and thrombin-H/HS interactions are recognized as prototypic specific and non-specific glycosaminoglycan (GAG-protein interactions, respectively. The fundamental structural basis for the origin of specificity, or lack thereof, in these interactions remains unclear. The availability of multiple co-crystal structures facilitates a structural analysis that challenges the long-held belief that the GAG binding sites in antithrombin and thrombin are essentially similar with high solvent exposure and shallow surface characteristics. METHODOLOGY: Analyses of solvent accessibility and exposed surface areas, gyrational mobility, symmetry, cavity shape/size, conserved water molecules and crystallographic parameters were performed for 12 X-ray structures, which include 12 thrombin and 16 antithrombin chains. Novel calculations are described for gyrational mobility and prediction of water loci and conservation. RESULTS: The solvent accessibilities and gyrational mobilities of arginines and lysines in the binding sites of the two proteins reveal sharp contrasts. The distribution of positive charges shows considerable asymmetry in antithrombin, but substantial symmetry for thrombin. Cavity analyses suggest the presence of a reasonably sized bifurcated cavity in antithrombin that facilitates a firm 'hand-shake' with H/HS, but with thrombin, a weaker 'high-five'. Tightly bound water molecules were predicted to be localized in the pentasaccharide binding pocket of antithrombin, but absent in thrombin. Together, these differences in the binding sites explain the major H/HS recognition characteristics of the two prototypic proteins, thus affording an explanation of the specificity of binding. This provides a foundation for understanding specificity of interaction at an atomic level, which will greatly aid the design of natural or synthetic H/HS sequences that target proteins in a specific manner.

  10. Nucleotide binding database NBDB – a collection of sequence motifs with specific protein-ligand interactions

    Science.gov (United States)

    Zheng, Zejun; Goncearenco, Alexander; Berezovsky, Igor N.

    2016-01-01

    NBDB database describes protein motifs, elementary functional loops (EFLs) that are involved in binding of nucleotide-containing ligands and other biologically relevant cofactors/coenzymes, including ATP, AMP, ATP, GMP, GDP, GTP, CTP, PAP, PPS, FMN, FAD(H), NAD(H), NADP, cAMP, cGMP, c-di-AMP and c-di-GMP, ThPP, THD, F-420, ACO, CoA, PLP and SAM. The database is freely available online at http://nbdb.bii.a-star.edu.sg. In total, NBDB contains data on 249 motifs that work in interactions with 24 ligands. Sequence profiles of EFL motifs were derived de novo from nonredundant Uniprot proteome sequences. Conserved amino acid residues in the profiles interact specifically with distinct chemical parts of nucleotide-containing ligands, such as nitrogenous bases, phosphate groups, ribose, nicotinamide, and flavin moieties. Each EFL profile in the database is characterized by a pattern of corresponding ligand–protein interactions found in crystallized ligand–protein complexes. NBDB database helps to explore the determinants of nucleotide and cofactor binding in different protein folds and families. NBDB can also detect fragments that match to profiles of particular EFLs in the protein sequence provided by user. Comprehensive information on sequence, structures, and interactions of EFLs with ligands provides a foundation for experimental and computational efforts on design of required protein functions. PMID:26507856

  11. Nucleotide binding database NBDB--a collection of sequence motifs with specific protein-ligand interactions.

    Science.gov (United States)

    Zheng, Zejun; Goncearenco, Alexander; Berezovsky, Igor N

    2016-01-01

    NBDB database describes protein motifs, elementary functional loops (EFLs) that are involved in binding of nucleotide-containing ligands and other biologically relevant cofactors/coenzymes, including ATP, AMP, ATP, GMP, GDP, GTP, CTP, PAP, PPS, FMN, FAD(H), NAD(H), NADP, cAMP, cGMP, c-di-AMP and c-di-GMP, ThPP, THD, F-420, ACO, CoA, PLP and SAM. The database is freely available online at http://nbdb.bii.a-star.edu.sg. In total, NBDB contains data on 249 motifs that work in interactions with 24 ligands. Sequence profiles of EFL motifs were derived de novo from nonredundant Uniprot proteome sequences. Conserved amino acid residues in the profiles interact specifically with distinct chemical parts of nucleotide-containing ligands, such as nitrogenous bases, phosphate groups, ribose, nicotinamide, and flavin moieties. Each EFL profile in the database is characterized by a pattern of corresponding ligand-protein interactions found in crystallized ligand-protein complexes. NBDB database helps to explore the determinants of nucleotide and cofactor binding in different protein folds and families. NBDB can also detect fragments that match to profiles of particular EFLs in the protein sequence provided by user. Comprehensive information on sequence, structures, and interactions of EFLs with ligands provides a foundation for experimental and computational efforts on design of required protein functions. PMID:26507856

  12. Rational steering of insulin binding specificity by intra-chain chemical crosslinking

    Science.gov (United States)

    Viková, Jitka; Collinsová, Michaela; Kletvíková, Emília; Buděšínský, Miloš; Kaplan, Vojtěch; Žáková, Lenka; Veverka, Václav; Hexnerová, Rozálie; Aviñó, Roberto J. Tarazona; Straková, Jana; Selicharová, Irena; Vaněk, Václav; Wright, Daniel W.; Watson, Christopher J.; Turkenburg, Johan P.; Brzozowski, Andrzej M.; Jiráček, Jiří

    2016-01-01

    Insulin is a key hormone of human metabolism with major therapeutic importance for both types of diabetes. New insulin analogues with more physiological profiles and better glycemic control are needed, especially analogues that preferentially bind to the metabolic B-isoform of insulin receptor (IR-B). Here, we aimed to stabilize and modulate the receptor-compatible conformation of insulin by covalent intra-chain crosslinking within its B22-B30 segment, using the CuI-catalyzed Huisgen 1,3-dipolar cycloaddition reaction of azides and alkynes. This approach resulted in 14 new, systematically crosslinked insulin analogues whose structures and functions were extensively characterized and correlated. One of the analogues, containing a B26-B29 triazole bridge, was highly active in binding to both IR isoforms, with a significant preference for IR-B. Our results demonstrate the potential of chemistry-driven modulation of insulin function, also shedding new light on the functional importance of hormone’s B-chain C-terminus for its IR-B specificity.

  13. Analysis of Specific Binding and Subcellular Localization of Wheat ERF Transcription Factor W17

    Institute of Scientific and Technical Information of China (English)

    ZHAO Yun-xiang; LIU Pei; XU Zhao-shi; CHEN Ming; LI Lian-cheng; CHEN Yao-feng; XIONG Xiang-jin; MA You-zhi

    2008-01-01

    The study aims to detect the subcellular localization of ERF (ethylene-responsive element binding factor) transcription factor W17 protein, the interaction between W17 and cis-acting regulatory elements GCC-box and DRE in vitro, the binding and transactivating ability in vivo, and the role of W17 in higher plant stress-signal pathway. Recombinant plasmid W17/163hGFP was introduced into onion epidermal cells by the particle bombardment method with a PDS1000/He. Transformed cells were incubated for 24h at 22℃ in the dark and green fluorescence was monitored under a confocal microscope. The gene W17 was fused N-terminus of GST (glutathione-S-transferase) in prokaryotic expression vector pGEX-4T-1 and then transformed into E. coli strain BL21 (DE3). IPTG (0.5mmol L-1) was added to induce the expression of recombinant GST/W17 for 3h. The fused proteins were purified by GST purification columns, and then subjected to gel retardation assay with a 32P-labeled GCC or DRE sequence. The different reporter and effector plasmids were introduced into tobacco leaves through agroinfiltration, then transformed leaves stained by X-Gluc, faded with 75% alcohol and monitored under a Stereozooming microscope. The GFP fused with W17 protein was localized in the nuclei; SDS-PAGE assay demonstrated that the fused protein GST/W17 could be induced and purified with molecular weight at around 42.2kD under the induction of IPTG. Purified fused protein was able to specifically bind to both the wild-type GCC-box and DRE element, but had no interaction with either the mutant DRE or GCC-box; W17 protein can bind to GCC-box and transactive downstream GUS gene in vivo. W17 can localize into the nuclei, and it may be involved not only in biotic stresses controlled by GCC-box, but also in abiotic stresses (e. g., salt-) induced signaling pathway.

  14. 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.

  15. Detection of the specific binding on protein microarrays by oblique-incidence reflectivity difference method

    Science.gov (United States)

    Lu, Heng; Wen, Juan; Wang, Xu; Yuan, Kun; Li, Wei; Lu, Huibin; Zhou, Yueliang; Jin, Kuijuan; Ruan, Kangcheng; Yang, Guozhen

    2010-09-01

    The specific binding between Cy5-labeled goat anti-mouse Immunoglobulin G (IgG) and mouse IgG with a concentration range from 625 to 104 µg ml - 1 has been detected successfully by the oblique-incidence reflectivity difference (OI-RD) method in each procedure of microarray fabrication. The experimental data prove that the OI-RD method can be employed not only to distinguish the different concentrations in label-free fashion but also to detect the antibody-antigen capture. In addition, the differential treatment of the OI-RD signals can decrease the negative influences of glass slide as the microarray upholder. Therefore the OI-RD technique has promising applications for the label-free and high-throughput detection of protein microarrays.

  16. Detection of the specific binding on protein microarrays by oblique-incidence reflectivity difference method

    International Nuclear Information System (INIS)

    The specific binding between Cy5-labeled goat anti-mouse Immunoglobulin G (IgG) and mouse IgG with a concentration range from 625 to 104 µg ml−1 has been detected successfully by the oblique-incidence reflectivity difference (OI-RD) method in each procedure of microarray fabrication. The experimental data prove that the OI-RD method can be employed not only to distinguish the different concentrations in label-free fashion but also to detect the antibody–antigen capture. In addition, the differential treatment of the OI-RD signals can decrease the negative influences of glass slide as the microarray upholder. Therefore the OI-RD technique has promising applications for the label-free and high-throughput detection of protein microarrays

  17. The hydrophobic core of twin-arginine signal sequences orchestrates specific binding to Tat-pathway related chaperones.

    Directory of Open Access Journals (Sweden)

    Anitha Shanmugham

    Full Text Available Redox enzyme maturation proteins (REMPs bind pre-proteins destined for translocation across the bacterial cytoplasmic membrane via the twin-arginine translocation system and enable the enzymatic incorporation of complex cofactors. Most REMPs recognize one specific pre-protein. The recognition site usually resides in the N-terminal signal sequence. REMP binding protects signal peptides against degradation by proteases. REMPs are also believed to prevent binding of immature pre-proteins to the translocon. The main aim of this work was to better understand the interaction between REMPs and substrate signal sequences. Two REMPs were investigated: DmsD (specific for dimethylsulfoxide reductase, DmsA and TorD (specific for trimethylamine N-oxide reductase, TorA. Green fluorescent protein (GFP was genetically fused behind the signal sequences of TorA and DmsA. This ensures native behavior of the respective signal sequence and excludes any effects mediated by the mature domain of the pre-protein. Surface plasmon resonance analysis revealed that these chimeric pre-proteins specifically bind to the cognate REMP. Furthermore, the region of the signal sequence that is responsible for specific binding to the corresponding REMP was identified by creating region-swapped chimeric signal sequences, containing parts of both the TorA and DmsA signal sequences. Surprisingly, specificity is not encoded in the highly variable positively charged N-terminal region of the signal sequence, but in the more similar hydrophobic C-terminal parts. Interestingly, binding of DmsD to its model substrate reduced membrane binding of the pre-protein. This property could link REMP-signal peptide binding to its reported proofreading function.

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

    Science.gov (United States)

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

    2015-07-01

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

  19. Binding of Sperm to the Zona Pellucida Mediated by Sperm Carbohydrate-Binding Proteins is not Species-Specific in Vitro between Pigs and Cattle

    Directory of Open Access Journals (Sweden)

    Minoru Nakano

    2013-01-01

    Full Text Available Carbohydrates are candidates for the basis of species-selective interaction of gametes during mammalian fertilization. In this study, we sought to clarify the roles of sugar residues in the species-selective, sperm–oocyte interaction in pigs and cattle. Acrosome-intact porcine and bovine sperm exhibited their strongest binding affinities for β-Gal and α-Man residues, respectively. Porcine-sperm specificity changed from β-Gal to α-Man after the acrosome reaction, while bovine-sperm specificity did not. Binding of acrosome-intact and acrosome-reacted sperm decreased after trypsinization, indicating that the carbohydrate-binding components are proteins. While immature oocytes bound homologous sperm preferentially to heterologous sperm, oocytes matured in vitro bound similar numbers of homologous and heterologous sperm. Lectin staining revealed the aggregation of α-Man residues on the outer surface of the porcine zona during maturation. In both species, zona-free, mature oocytes bound homologous sperm preferentially to heterologous sperm. The lectin-staining patterns of the zona pellucida and zona-free oocytes coincided with the carbohydrate-binding specificities of acrosome-intact and acrosome-reacted sperm, respectively, supporting the involvement of carbohydrates in gamete recognition in pigs and cattle. These results also indicate that sperm-zona pellucida and sperm–oolemma bindings are not strictly species-specific in pigs and cattle, and further suggest that sperm penetration into the zona and/or fusion with oolemma may be species-specific between pigs and cattle.

  20. Binding of Sperm to the Zona Pellucida Mediated by Sperm Carbohydrate-Binding Proteins is not Species-Specific in Vitro between Pigs and Cattle.

    Science.gov (United States)

    Takahashi, Kazuya; Kikuchi, Kazuhiro; Uchida, Yasuomi; Kanai-Kitayama, Saeko; Suzuki, Reiichiro; Sato, Reiko; Toma, Kazunori; Geshi, Masaya; Akagi, Satoshi; Nakano, Minoru; Yonezawa, Naoto

    2013-01-01

    Carbohydrates are candidates for the basis of species-selective interaction of gametes during mammalian fertilization. In this study, we sought to clarify the roles of sugar residues in the species-selective, sperm-oocyte interaction in pigs and cattle. Acrosome-intact porcine and bovine sperm exhibited their strongest binding affinities for β-Gal and α-Man residues, respectively. Porcine-sperm specificity changed from β-Gal to α-Man after the acrosome reaction, while bovine-sperm specificity did not. Binding of acrosome-intact and acrosome-reacted sperm decreased after trypsinization, indicating that the carbohydrate-binding components are proteins. While immature oocytes bound homologous sperm preferentially to heterologous sperm, oocytes matured in vitro bound similar numbers of homologous and heterologous sperm. Lectin staining revealed the aggregation of α-Man residues on the outer surface of the porcine zona during maturation. In both species, zona-free, mature oocytes bound homologous sperm preferentially to heterologous sperm. The lectin-staining patterns of the zona pellucida and zona-free oocytes coincided with the carbohydrate-binding specificities of acrosome-intact and acrosome-reacted sperm, respectively, supporting the involvement of carbohydrates in gamete recognition in pigs and cattle. These results also indicate that sperm-zona pellucida and sperm-oolemma bindings are not strictly species-specific in pigs and cattle, and further suggest that sperm penetration into the zona and/or fusion with oolemma may be species-specific between pigs and cattle.

  1. Fragile X mental retardation protein (FMRP) binds specifically to the brain cytoplasmic RNAs BC1/BC200 via a novel RNA-binding motif

    OpenAIRE

    Zalfa, F.; S. Adinolfi; Napoli, I; Kuhn-Holsken, E; Urlaub, H.; Achsel, Tilmann; Pastore, A; Bagni, Claudia

    2005-01-01

    Fragile X mental retardation protein (FMRP), the protein responsible for the fragile X syndrome, is an RNA-binding protein involved in localization and translation of neuronal mRNAs. One of the RNAs known to interact with FMRP is the dendritic non-translatable brain cytoplasmic RNA 1 BC1 RNA that works as an adaptor molecule linking FMRP and some of its regulated mRNAs. Here, we showed that the N terminus of FMRP binds strongly and specifically to BC1 and to its potential human analog BC200. ...

  2. Effect of cation doping on low-temperature specific heat of LaCoO{sub 3} cobaltate

    Energy Technology Data Exchange (ETDEWEB)

    Thakur, Rasna, E-mail: rasnathakur@yahoo.com [Department of Physics, Barkatullah University, Bhopal 462026 (India); Gaur, N.K.; Thakur, Rajesh K. [Department of Physics, Barkatullah University, Bhopal 462026 (India)

    2013-01-15

    We have computed the effect of strontium doping on the thermophysical properties of perovskite structure rare earth cobaltate, La{sub 1-x}Sr{sub x}CoO{sub 3} (0.0{<=}x{<=}1.0) by means of a rigid ion model (RIM) in the temperature 0 K{sup 2}{<=}T{sup 2}{<=}100 K{sup 2}. The trends of variation of our computed specific heat with temperature are in agreement with corresponding experimental data. A meticulous attention was given to the investigation of the assumption that these cobaltate perovskites are Debye-like solids and obeys T{sup 3} law at low temperature (T<{theta}{sub D}/50). Present investigations reaffirm the presence of strong electron-phonon interactions in these compounds. An interesting phenomenon was observed in C{sub p}/T vs. T{sup 2} plot for x{<=}0.2 below 15 K where linearity deviates, which suggests the gradual freezing of the entropy on cooling. In addition, the results on bulk modulus (B), cohesive energy ({phi}), molecular force constant (f), restsrahlen frequency ({upsilon}), and Debye temperature({theta}{sub D}) are also presented. The scope of further extension of the present model has also been discussed. - Highlights: Black-Right-Pointing-Pointer The negative values of cohesive energy show the stability of La{sub 1-x}Sr{sub x}CoO{sub 3} compounds. Black-Right-Pointing-Pointer Our results on specific heat are in good agreement with the experimental data. Black-Right-Pointing-Pointer The stability of La{sub 1-x}Sr{sub x}CoO{sub 3} (0.0{<=}x{<=}1.0) perovskite increases with the Sr substitution.

  3. LBD1 of Vitellogenin Receptor Specifically Binds to the Female-Specific Storage Protein SP1 via LBR1 and LBR3

    Science.gov (United States)

    Liu, Lina; Wang, Yejing; Li, Yu; Lin, Ying; Hou, Yong; Zhang, Yan; Wei, Shuguang; Zhao, Peng; Zhao, Ping; He, Huawei

    2016-01-01

    Storage proteins are the major protein synthesized in the fat body, released into hemolymph and re-sequestered into the fat body before pupation in most insect species. Storage proteins are important amino acid and nutrition resources during the non-feeding pupal period and play essential roles for the metamorphosis and oogenesis of insects. The sequestration of storage protein is a selective, specific receptor-mediated process. However, to date, the potential receptor mediating the sequestration of storage protein has not been determined in Bombyx mori. In this study, we expressed and purified the first ligand binding domain of Bombyx mori vitellogenin receptor (BmVgR), LBD1, and found LBD1 could bind with an unknown protein from the hemolymph of the ultimate silkworm larval instar via pull-down assay. This unknown protein was subsequently identified to be the female-specific storage protein SP1 by mass spectrometry. Furthermore, far western blotting assay, immunoprecipitation and isothermal titration calorimetry analysis demonstrated LBD1 specifically bound with the female-specific SP1, rather than another unisex storage protein SP2. The specific binding of LBD1 with SP1 was dependent on the presence of Ca2+ as it was essential for the proper conformation of LBD1. Deletion mutagenesis and ITC analysis revealed the first and third ligand binding repeats LBR1 and LBR3 were indispensable for the binding of LBD1 with SP1, and LBR2 and LBR4 also had a certain contribution to the specific binding. Our results implied BmVgR may mediate the sequestration of SP1 from hemolymph into the fat body during the larval-pupal transformation of Bombyx mori. PMID:27637099

  4. Generation of a haptoglobin-hemoglobin complex-specific Fab antibody blocking the binding of the complex to CD163

    DEFF Research Database (Denmark)

    Horn, Ivo R; Nielsen, Marianne Jensby; Madsen, Mette;

    2003-01-01

    During intravascular hemolysis hemoglobin (Hb) binds to haptoglobin (Hp) leading to endocytosis of the complex by the macrophage receptor, CD163. In the present study, we used a phage-display Fab antibody strategy to explore if the complex formation between Hp and Hb leads to exposure of antigenic...... epitopes specific for the complex. By Hp-Hb-affinity screening of a phage-Fab library, we isolated a phage clone against the ligand complex. Surface plasmon resonance analyses of the Fab part expressed as a recombinant protein revealed a high affinity binding (KD = 3.9 nm) to Hp-Hb, whereas no binding...... was measured for non-complexed Hp or Hb. The Fab antibody completely inhibited the binding of 125I-labeled Hp-Hb complexes to CD163 and blocked their uptake in CD163-transfected cells. In conclusion, we have raised a receptor-blocking antibody specifically recognizing the Hp-Hb complex. In addition to provide...

  5. Squalamine, a novel cationic steroid, specifically inhibits the brush-border Na+/H+ exchanger isoform NHE3.

    Science.gov (United States)

    Akhter, S; Nath, S K; Tse, C M; Williams, J; Zasloff, M; Donowitz, M

    1999-01-01

    Squalamine, an endogenous molecule found in the liver and other tissues of Squalus acanthias, has antibiotic properties and causes changes in endothelial cell shape. The latter suggested that its potential targets might include transport proteins that control cell volume or cell shape. The effect of purified squalamine was examined on cloned Na+/H+ exchanger isoforms NHE1, NHE2, and NHE3 stably transfected in PS120 fibroblasts. Squalamine (1-h pretreatment) decreased the maximal velocity of rabbit NHE3 in a concentration-dependent manner (13, 47, and 57% inhibition with 3, 5, and 7 micrograms/ml, respectively) and also increased K'[H+]i. Squalamine did not affect rabbit NHE1 or NHE2 function. The inhibitory effect of squalamine was 1) time dependent, with no effect of immediate addition and maximum effect with 1 h of exposure, and 2) fully reversible. Squalamine pretreatment of the ileum for 60 min inhibited brush-border membrane vesicle Na+/H+ activity by 51%. Further investigation into the mechanism of squalamine's effects showed that squalamine required the COOH-terminal 76 amino acids of NHE3. Squalamine had no cytotoxic effect at the concentrations studied, as indicated by monitoring lactate dehydrogenase release. These results indicate that squalamine 1) is a specific inhibitor of the brush-border NHE isoform NHE3 and not NHE1 or NHE2, 2) acts in a nontoxic and fully reversible manner, and 3) has a delayed effect, indicating that it may influence brush-border Na+/H+ exchanger function indirectly, through an intracellular signaling pathway or by acting as an intracellular modulator. PMID:9886929

  6. NITRIC OXIDE BINDS TO AND MODULATES THE ACTIVITY OF A POLLEN SPECIFIC ARABIDOPSIS DIACYLGLYCEROL KINASE

    KAUST Repository

    Wong, Aloysius Tze

    2014-06-01

    Nitric oxide (NO) is an important signaling molecule in plants. In the pollen of Arabidopsis thaliana, NO causes re-orientation of the growing tube and this response is mediated by 3′,5′-cyclic guanosine monophosphate (cGMP). However, in plants, NO-sensors have remained somewhat elusive. Here, the findings of an NO-binding candidate, Arabidopsis thaliana DIACYLGLYCEROL KINASE 4 (ATDGK4; AT5G57690) is presented. In addition to the annotated diacylglycerol kinase domain, this molecule also harbors a predicted heme-NO/oxygen (H-NOX) binding site and a guanylyl cyclase (GC) catalytic domain which have been identified based on the alignment of functionally conserved amino acid residues across species. A 3D model of the molecule was constructed, and from which the locations of the kinase catalytic center, the ATP-binding site, the GC and H-NOX domains were estimated. Docking of ATP to the kinase catalytic center was also modeled. The recombinant ATDGK4 demonstrated kinase activity in vitro, catalyzing the ATP-dependent conversion of sn-1,2-diacylglycerol (DAG) to phosphatidic acid (PA). This activity was inhibited by the mammalian DAG kinase inhibitor R59949 and importantly also by the NO donors diethylamine NONOate (DEA NONOate) and sodium nitroprusside (SNP). Recombinant ATDGK4 also has GC activity in vitro, catalyzing the conversion of guanosine-5\\'-triphosphate (GTP) to cGMP. The catalytic domains of ATDGK4 kinase and GC may be independently regulated since the kinase but not the GC, was inhibited by NO while Ca2+ only stimulates the GC. It is likely that the DAG kinase product, PA, causes the release of Ca2+ from the intracellular stores and Ca2+ in turn activates the GC domain of ATDGK4 through a feedback mechanism. Analysis of publicly available microarray data has revealed that ATDGK4 is highly expressed in the pollen. Here, the pollen tubes of mis-expressing atdgk4 recorded slower growth rates than the wild-type (Col-0) and importantly, they showed altered

  7. Selective binding of specific mouse genomic DNA fragments by mouse vimentin filaments in vitro.

    Science.gov (United States)

    Wang, X; Tolstonog, G; Shoeman, R L; Traub, P

    1996-03-01

    Mouse vimentin intermediate filaments (IFs) reconstituted in vitro were analyzed for their capacity to select certain DNA sequences from a mixture of about 500-bp-long fragments of total mouse genomic DNA. The fragments preferentially bound by the IFs and enriched by several cycles of affinity binding and polymerase chain reaction (PCR) amplification were cloned and sequenced. In general, they were G-rich and highly repetitive in that they often contained Gn, (GT)n, and (GA)n repeat elements. Other, more complex repeat sequences were identified as well. Apart from the capacity to adopt a Z-DNA and triple helix configuration under superhelical tension, many fragments were potentially able to form cruciform structures and contained consensus binding sites for various transcription factors. All of these sequence elements are known to occur in introns and 5'/3'-flanking regions of genes and to play roles in DNA transcription, recombination and replication. A FASTA search of the EMBL data bank indeed revealed that sequences homologous to the mouse repetitive DNA fragments are commonly associated with gene-regulatory elements. Unexpectedly, vimentin IFs also bound a large number of apparently overlapping, AT-rich DNA fragments that could be aligned into a composite sequence highly homologous to the 234-bp consensus centromere repeat sequence of gamma-satellite DNA. Previous experiments have shown a high affinity of vimentin for G-rich, repetitive telomere DNA sequences, superhelical DNA, and core histones. Taken together, these data support the hypothesis that, after penetration of the double nuclear membrane via an as yet unidentified mechanism, vimentin IFs cooperatively fix repetitive DNA sequence elements in a differentiation-specific manner in the nuclear periphery subjacent to the nuclear lamina and thus participate in the organization of chromatin and in the control of transcription, replication, and recombination processes. This includes aspects of global

  8. Nuclear Factor 90 uses an ADAR2-like binding mode to recognize specific bases in dsRNA

    OpenAIRE

    Jayachandran, Uma; Grey, Heather; Cook, Atlanta

    2016-01-01

    Nuclear factors 90 and 45 (NF90 and NF45) form a protein complex involved in the posttranscriptional control of many genes in vertebrates. NF90 is a member of the dsRNA binding domain (dsRBD) family of proteins. RNA binding partners identified so far include elements in 3' untranslated regions of specific mRNAs and several noncoding RNAs. In NF90, a tandem pair of dsRBDs separated by a natively unstructured segment confers dsRNA binding activity. We determined a crystal structure of the tande...

  9. Nuclear factor 90 uses an ADAR2-like binding mode to recognize specific bases in dsRNA

    OpenAIRE

    Jayachandran, Uma; Grey, Heather; Cook, Atlanta

    2015-01-01

    Nuclear factors 90 and 45 (NF90 and NF45) form a protein complex involved in the post-transcriptional control of many genes in vertebrates. NF90 is a member of the dsRNA binding domain (dsRBD) family of proteins. RNA binding partners identified so far include elements in 3′ untranslated regions of specific mRNAs and several non-coding RNAs. In NF90, a tandem pair of dsRBDs separated by a natively unstructured segment confers dsRNA binding activity. We determined a crystal structure of the tan...

  10. Electromotive force and impedance studies of cellulose acetate membranes: Evidence for two binding sites for divalent cations and for an alveolar structure of the skin layer

    DEFF Research Database (Denmark)

    Smith Sørensen, T.; Jensen, J.B.; Malmgren-Hansen, B.

    1991-01-01

    . Alveoles in dense membranes or in the skin layer of asymmetric membranes seem to have a mean radius corresponding to ca. 70 angstrom. The dielectric constant in the alveoles is ca. 30 in contrast to a dielectric constant of ca. 16 in the >>lamellar phaseconstant...... of ca. 30 in the alveolar phase is also supported by a simple dielectric calculation of the Nernst distribution of mono- and divalent ions between external water and the alveolar solution. Corrections for activity coefficients only seems important above 0.5 M. The Onsager-Samaras dielectric repulsion...... may temporarily transform the membranes from weak cationic exchange membranes to weak anionic exchange membranes. The divalent cations may be washed out, but the rate of dissociation is very low. There seems to be two relaxations, the slower being of the order of weeks, the faster being of the order...

  11. Advances in Research on Plasma-Membrane Associated Cation-Bind-ing Proteins%细胞膜相联钙结合蛋白的结构和功能研究进展

    Institute of Scientific and Technical Information of China (English)

    何伟; 王斐; 闫海芳

    2014-01-01

    细胞膜相联钙结合蛋白(PCaP)是定位于细胞膜上的一类新的Ca2+结合蛋白。简要综述了PCaP的蛋白结构、调控Ca2+/磷脂酰肌醇3-磷酸信号通路的分子机制、调节微管微丝稳定的能力,以及其在植物顶端生长极性中的功能研究进展。%The plasma-membrane associated cation-binding protein(PCaP) of Arabidopsis thaliana is a kind of cation-binding protein which is located in plasma membrane. In this review, we mainly describled the advances re-search on the protein structure, the molecular mechanism of regulating Ca2+/PtdInsP(phosphatidylinositol 3-phos-phate) signaling pathways, the ability of adjusting microtube, and its biological functions in plant tip growth polari-ty of PCaP.

  12. DNA-binding protein prediction using plant specific support vector machines: validation and application of a new genome annotation tool.

    Science.gov (United States)

    Motion, Graham B; Howden, Andrew J M; Huitema, Edgar; Jones, Susan

    2015-12-15

    There are currently 151 plants with draft genomes available but levels of functional annotation for putative protein products are low. Therefore, accurate computational predictions are essential to annotate genomes in the first instance, and to provide focus for the more costly and time consuming functional assays that follow. DNA-binding proteins are an important class of proteins that require annotation, but current computational methods are not applicable for genome wide predictions in plant species. Here, we explore the use of species and lineage specific models for the prediction of DNA-binding proteins in plants. We show that a species specific support vector machine model based on Arabidopsis sequence data is more accurate (accuracy 81%) than a generic model (74%), and based on this we develop a plant specific model for predicting DNA-binding proteins. We apply this model to the tomato proteome and demonstrate its ability to perform accurate high-throughput prediction of DNA-binding proteins. In doing so, we have annotated 36 currently uncharacterised proteins by assigning a putative DNA-binding function. Our model is publically available and we propose it be used in combination with existing tools to help increase annotation levels of DNA-binding proteins encoded in plant genomes. PMID:26304539

  13. The N-terminal part of Als1 protein from Candida albicans specifically binds fucose-containing glycans.

    Science.gov (United States)

    Donohue, Dagmara S; Ielasi, Francesco S; Goossens, Katty V Y; Willaert, Ronnie G

    2011-06-01

    The opportunistic pathogen Candida albicans expresses on its surface Als (Agglutinin like sequence) proteins, which play an important role in the adhesion to host cells and in the development of candidiasis. The binding specificity of these proteins is broad, as they can bind to various mammalian proteins, such as extracellular matrix proteins, and N- and E-cadherins. The N-terminal part of Als proteins constitutes the substrate-specific binding domain and is responsible for attachment to epithelial and endothelial cells. We have used glycan array screening to identify possible glycan receptors for the binding domain of Als1p-N. Under those conditions, Als1p-N binds specifically to fucose-containing glycans, which adds a lectin function to the functional diversity of the Als1 protein. The binding between Als1p-N and BSA-fucose glycoconjugate was quantitatively characterized using surface plasmon resonance, which demonstrated a weak millimolar affinity between Als1p-N and fucose. Furthermore, we have also quantified the affinity of Als1p-N to the extracellular matrix proteins proteins fibronectin and laminin, which is situated in the micromolar range. Surface plasmon resonance characterization of Als1p-N-Als1p-N interaction was in the micromolar affinity range.

  14. Specific binding of a dihydropyrimidinone derivative with DNA: Spectroscopic, calorimetric and modeling investigations

    Energy Technology Data Exchange (ETDEWEB)

    Wang Gongke, E-mail: wanggongke@126.com [School of Chemistry and Environmental Science, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, Henan Normal University, Xinxiang, Henan 453007 (China); Yan Changling; Wang Dongchao; Li Dan [School of Chemistry and Environmental Science, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, Henan Normal University, Xinxiang, Henan 453007 (China); Lu Yan, E-mail: yanlu2001@sohu.com [School of Chemistry and Environmental Science, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, Henan Normal University, Xinxiang, Henan 453007 (China)

    2012-07-15

    One of the dihydropyrimidinone derivative 5-(ethoxycarbonyl)-6-methyl-4-(4-methoxyphenyl) -3,4-dihydropyrimidin-2(1H)-one (EMMD) was synthesized, and its binding properties with calf-thymus DNA (ctDNA) were investigated using spectroscopic, viscometric, isothermal titration calorimetric (ITC) and molecular modeling techniques. Fluorescence spectra suggested that the fluorescence enhancement of the binding interaction of EMMD to ctDNA was a static process with ground state complex formation. The binding constant determined with spectroscopic titration and ITC was found to be in the same order of 10{sup 4} M{sup -1}. According to the results of the viscosity analysis, fluorescence competitive binding experiment, fluorescence quenching studies, absorption spectral and ITC investigations, it can be concluded that EMMD is intercalative binding to ctDNA. Furthermore, the results of molecular modeling confirmed those obtained from spectroscopic, viscosimetric and ITC investigations. Additionally, ITC studies also indicated that the binding interaction is predominantly enthalpy driven. - Highlights: Black-Right-Pointing-Pointer Medically important dihydropyrimidinones derivative EMMD is synthesized. Black-Right-Pointing-Pointer EMMD is intercalative binding into ctDNA helix. Black-Right-Pointing-Pointer Hydrogen bonding may play an essential role in the binding of EMCD with ctDNA. Black-Right-Pointing-Pointer This binding interaction is predominantly enthalpy driven.

  15. Bassoon specifically controls presynaptic P/Q-type Ca(2+) channels via RIM-binding protein.

    Science.gov (United States)

    Davydova, Daria; Marini, Claudia; King, Claire; Klueva, Julia; Bischof, Ferdinand; Romorini, Stefano; Montenegro-Venegas, Carolina; Heine, Martin; Schneider, Romy; Schröder, Markus S; Altrock, Wilko D; Henneberger, Christian; Rusakov, Dmitri A; Gundelfinger, Eckart D; Fejtova, Anna

    2014-04-01

    Voltage-dependent Ca(2+) channels (CaVs) represent the principal source of Ca(2+) ions that trigger evoked neurotransmitter release from presynaptic boutons. Ca(2+) influx is mediated mainly via CaV2.1 (P/Q-type) and CaV2.2 (N-type) channels, which differ in their properties. Their relative contribution to synaptic transmission changes during development and tunes neurotransmission during synaptic plasticity. The mechanism of differential recruitment of CaV2.1 and CaV2.2 to release sites is largely unknown. Here, we show that the presynaptic scaffolding protein Bassoon localizes specifically CaV2.1 to active zones via molecular interaction with the RIM-binding proteins (RBPs). A genetic deletion of Bassoon or an acute interference with Bassoon-RBP interaction reduces synaptic abundance of CaV2.1, weakens P/Q-type Ca(2+) current-driven synaptic transmission, and results in higher relative contribution of neurotransmission dependent on CaV2.2. These data establish Bassoon as a major regulator of the molecular composition of the presynaptic neurotransmitter release sites. PMID:24698275

  16. Structure-guided development of specific pyruvate dehydrogenase kinase inhibitors targeting the ATP-binding pocket.

    Science.gov (United States)

    Tso, Shih-Chia; Qi, Xiangbing; Gui, Wen-Jun; Wu, Cheng-Yang; Chuang, Jacinta L; Wernstedt-Asterholm, Ingrid; Morlock, Lorraine K; Owens, Kyle R; Scherer, Philipp E; Williams, Noelle S; Tambar, Uttam K; Wynn, R Max; Chuang, David T

    2014-02-14

    Pyruvate dehydrogenase kinase isoforms (PDKs 1-4) negatively regulate activity of the mitochondrial pyruvate dehydrogenase complex by reversible phosphorylation. PDK isoforms are up-regulated in obesity, diabetes, heart failure, and cancer and are potential therapeutic targets for these important human diseases. Here, we employed a structure-guided design to convert a known Hsp90 inhibitor to a series of highly specific PDK inhibitors, based on structural conservation in the ATP-binding pocket. The key step involved the substitution of a carbonyl group in the parent compound with a sulfonyl in the PDK inhibitors. The final compound of this series, 2-[(2,4-dihydroxyphenyl)sulfonyl]isoindoline-4,6-diol, designated PS10, inhibits all four PDK isoforms with IC50 = 0.8 μM for PDK2. The administration of PS10 (70 mg/kg) to diet-induced obese mice significantly augments pyruvate dehydrogenase complex activity with reduced phosphorylation in different tissues. Prolonged PS10 treatments result in improved glucose tolerance and notably lessened hepatic steatosis in the mouse model. The results support the pharmacological approach of targeting PDK to control both glucose and fat levels in obesity and type 2 diabetes. PMID:24356970

  17. Lectin domains of polypeptide GalNAc transferases exhibit glycopeptide binding specificity.

    Science.gov (United States)

    Pedersen, Johannes W; Bennett, Eric P; Schjoldager, Katrine T-B G; Meldal, Morten; Holmér, Andreas P; Blixt, Ola; Cló, Emiliano; Levery, Steven B; Clausen, Henrik; Wandall, Hans H

    2011-09-16

    UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferases (GalNAc-Ts) constitute a family of up to 20 transferases that initiate mucin-type O-glycosylation. The transferases are structurally composed of catalytic and lectin domains. Two modes have been identified for the selection of glycosylation sites by GalNAc-Ts: confined sequence recognition by the catalytic domain alone, and concerted recognition of acceptor sites and adjacent GalNAc-glycosylated sites by the catalytic and lectin domains, respectively. Thus far, only the catalytic domain has been shown to have peptide sequence specificity, whereas the primary function of the lectin domain is to increase affinity to previously glycosylated substrates. Whether the lectin domain also has peptide sequence selectivity has remained unclear. Using a glycopeptide array with a library of synthetic and recombinant glycopeptides based on sequences of mucins MUC1, MUC2, MUC4, MUC5AC, MUC6, and MUC7 as well as a random glycopeptide bead library, we examined the binding properties of four different lectin domains. The lectin domains of GalNAc-T1, -T2, -T3, and -T4 bound different subsets of small glycopeptides. These results indicate an additional level of complexity in the initiation step of O-glycosylation by GalNAc-Ts.

  18. Myocyte-specific enhancer binding factor 2A expression is downregulated during temporal lobe epilepsy.

    Science.gov (United States)

    Huang, Yunyi; Wu, Xuling; Guo, Jing; Yuan, Jinxian

    2016-09-01

    Myocyte-specific enhancer binding factor 2A (MEF2A) is a multifunctional nuclear protein that regulates synaptogenesis, dendritic morphogenesis, and neuronal survival. This study aimed to investigate the expression pattern of MEF2A in epileptogenic processes. MEF2A expression was detected in 20 temporal neocortex tissue samples from patients with temporal lobe epilepsy (TLE) and 20 samples from trauma patients without epilepsy by real-time quantitative polymerase chain reaction, immunohistochemistry, double-label immunofluorescent staining, and western blot analysis. In addition, the expression patterns of MEF2A in the hippocampus and adjacent cortex of a lithium-pilocarpine-induced TLE rat model and control rats were examined. MEF2A was found to be expressed in the nuclei of neurons but not in the dendrites of neurons and astrocytes. MEF2A expression was significantly downregulated in temporal neocortex of humans and rats with TLE compared to the control groups. In addition, in the lithium-pilocarpine-induced TLE model, MEF2A expression dynamically decreased within 2 months. Taken together, these data suggest that MEF2A is involved in the pathogenesis of TLE. PMID:26439092

  19. Specific binding of DNA to aggregated forms of Alzheimer's disease amyloid peptides.

    Science.gov (United States)

    Camero, Sergio; Ayuso, Jose M; Barrantes, Alejandro; Benítez, María J; Jiménez, Juan S

    2013-04-01

    Anomalous protein aggregation is closely associated to age-related mental illness. Extraneuronal plaques, mainly composed of aggregated amyloid peptides, are considered as hallmarks of Alzheimer's disease. According to the amyloid cascade hypothesis, this disease starts as a consequence of an abnormal processing of the amyloid precursor protein resulting in an excess of amyloid peptides. Nuclear localization of amyloid peptide aggregates together with amyloid-DNA interaction, have been repeatedly reported. In this paper we have used surface plasmon resonance and electron microscopy to study the structure and behavior of different peptides and proteins, including β-lactoglobulin, bovine serum albumin, myoglobin, histone, casein and the amyloid-β peptides related to Alzheimer's disease Aβ25-35 and Aβ1-40. The main purpose of this study is to investigate whether proneness to DNA interaction is a general property displayed by aggregated forms of proteins, or it is an interaction specifically related to the aggregated forms of those particular proteins and peptides related to neurodegenerative diseases. Our results reveal that those aggregates formed by amyloid peptides show a particular proneness to interact with DNA. They are the only aggregated structures capable of binding DNA, and show more affinity for DNA than for other polyanions like heparin and polyglutamic acid, therefore strengthening the hypothesis that amyloid peptides may, by means of interaction with nuclear DNA, contribute to the onset of Alzheimer's disease.

  20. The Non-Specific Binding of Fluorescent-Labeled MiRNAs on Cell Surface by Hydrophobic Interaction.

    Directory of Open Access Journals (Sweden)

    Ting Lu

    Full Text Available MicroRNAs are small noncoding RNAs about 22 nt long that play key roles in almost all biological processes and diseases. The fluorescent labeling and lipofection are two common methods for changing the levels and locating the position of cellular miRNAs. Despite many studies about the mechanism of DNA/RNA lipofection, little is known about the characteristics, mechanisms and specificity of lipofection of fluorescent-labeled miRNAs.Therefore, miRNAs labeled with different fluorescent dyes were transfected into adherent and suspension cells using lipofection reagent. Then, the non-specific binding and its mechanism were investigated by flow cytometer and laser confocal microscopy. The results showed that miRNAs labeled with Cy5 (cyanine fluorescent dye could firmly bind to the surface of adherent cells (Hela and suspended cells (K562 even without lipofection reagent. The binding of miRNAs labeled with FAM (carboxyl fluorescein to K562 cells was obvious, but it was not significant in Hela cells. After lipofectamine reagent was added, most of the fluorescently labeled miRNAs binding to the surface of Hela cells were transfected into intra-cell because of the high transfection efficiency, however, most of them were still binding to the surface of K562 cells. Moreover, the high-salt buffer which could destroy the electrostatic interactions did not affect the above-mentioned non-specific binding, but the organic solvent which could destroy the hydrophobic interactions eliminated it.These results implied that the fluorescent-labeled miRNAs could non-specifically bind to the cell surface by hydrophobic interaction. It would lead to significant errors in the estimation of transfection efficiency only according to the cellular fluorescence intensity. Therefore, other methods to evaluate the transfection efficiency and more appropriate fluorescent dyes should be used according to the cell types for the accuracy of results.

  1. Carbohydrate/glycan-binding specificity of legume lectins in respect to their proposed biological functions

    Directory of Open Access Journals (Sweden)

    Márcio Viana Ramos

    2000-01-01

    Full Text Available The lectins, proteins which specifically recognize carbohydrate moieties, have been extensively studied in many biochemical and structural aspects in order to establish the molecular basis of this non-catalytic event. On the other hand, their clinical and agricultural potentials have been growing fast. Although lectins, mainly those from legume plants, had been investigated for biological properties, studies about the physiological functions of lectins are scarce in literature. Therefore, despite the accumulated data on lectins (as proteins, the role played by these signalizing molecules is poorly discussed. In the light of our accumulated results on legume lectins, specially those obtained from plants belonging to the Diocleinae sub-tribe and available data in literature, we discuss here the main hypothesis of their functions according to their carbohydrate/glycan-binding specificity.As lectinas, proteinas que especificamente reconhecem estruturas que contém carboidratos, têm sido extensivamente estudadas em muitos aspectos bioquímicos e estruturais, objetivando estabelecer as bases moleculares deste evento não-catalítico. Por outro lado, os potenciais clínicos e agriculturais destas proteínas têm crescido rapidamente. Embora as lectinas, principalmente aquelas de legumes tenham sido bastante investigadas em suas propriedades biológicas, estudos sobre as funcões fisiológicas de lectinas são escassos na literatura. Além disto, a despeito da quantidade de dados acumulados sobre lectinas (como proteínas, o papel desempenhado por estas moléculas de sinalização é pobremente discutido. Valendo-se de nossos estudos sobre lectinas de leguminosas, principalmente da sub-tribo Diocleinae, e outros dados presentes na literatura, discutimos aqui, as principais hipóteses de suas funções com base na especificidade por carboidratos e glicanos complexos.

  2. Motility assays using myosin attached to surfaces through specific binding to monoclonal antibodies.

    Science.gov (United States)

    Winkelmann, D A; Bourdieu, L; Kinose, F; Libchaber, A

    1995-04-01

    We have analyzed the dependence of actin filament movement on the mode of myosin attachment to surfaces. Monoclonal antibodies that bind to three distinct sites were used to tether myosin to nitrocellulose-coated glass. One antibody reacts with an epitope on the regulatory light chain located at the head-rod junction. The other two react with sites in the rod domain, one in the S2 region near the S2-LMM hinge, and the other at the C terminus of the myosin rod. These monoclonal antibodies were used to provide increasing flexibility in the mode of attachment. Fast skeletal muscle myosin monomers were bound to the surfaces through the specific interaction with these monoclonal antibodies and the sliding movement of fluorescently labeled actin filaments analyzed by video microscopy. Each of these antibodies produced stable, myosin-coated surfaces that supported uniform movement of actin over the course of several hours. Attachment of myosin through the anti-S2 and anti-LMM monoclonal antibodies yielded a maximum velocity of 10 microns/s at 30 degrees C, whereas attachment through anti-LC2 produced a lower velocity of 4-5 microns/s. Each antibody showed a characteristic minimum myosin density below which sliding movement was no longer supported and an exponential dependence of actin filament velocity on myosin surface density below Vmax. Maximum sliding velocity was achieved over a range of myosin surface densities. Thus, the specific mode of attachment can influence the characteristic velocity of actin filament movement and the surface density needed to support movement. These data are being used to analyze the dynamics of sliding filament assays and evaluate estimates of the average number of motor molecules per unit length of actin required to support movement.

  3. Quantitative characterization of conformational-specific protein-DNA binding using a dual-spectral interferometric imaging biosensor

    Science.gov (United States)

    Zhang, Xirui; Daaboul, George G.; Spuhler, Philipp S.; Dröge, Peter; Ünlü, M. Selim

    2016-03-01

    DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are not fully understood. Recently, it was discovered that DNA-binding proteins recognize specific binding sites to carry out their functions through an indirect readout mechanism by recognizing and capturing DNA conformational flexibility and deformation. High-throughput DNA microarray-based methods that provide large-scale protein-DNA binding information have shown effective and comprehensive analysis of protein-DNA binding affinities, but do not provide information of DNA conformational changes in specific protein-DNA complexes. Building on the high-throughput capability of DNA microarrays, we demonstrate a quantitative approach that simultaneously measures the amount of protein binding to DNA and nanometer-scale DNA conformational change induced by protein binding in a microarray format. Both measurements rely on spectral interferometry on a layered substrate using a single optical instrument in two distinct modalities. In the first modality, we quantitate the amount of binding of protein to surface-immobilized DNA in each DNA spot using a label-free spectral reflectivity technique that accurately measures the surface densities of protein and DNA accumulated on the substrate. In the second modality, for each DNA spot, we simultaneously measure DNA conformational change using a fluorescence vertical sectioning technique that determines average axial height of fluorophores tagged to specific nucleotides of the surface-immobilized DNA. The approach presented in this paper, when combined with current high-throughput DNA microarray-based technologies, has the potential to serve as a rapid and simple method for quantitative and large-scale characterization of conformational specific protein-DNA interactions.DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are

  4. Non-specific recognition is achieved in Pot1pC through the use of multiple binding modes

    Science.gov (United States)

    Dickey, Thayne H.; McKercher, Marissa A.; Wuttke, Deborah S.

    2012-01-01

    Summary Pot1 is the protein responsible for binding to and protecting the 3’ single-stranded DNA (ssDNA) overhang at most eukaryotic telomeres. Here we present the crystal structure of one of the two OB-folds (Pot1pC) that make up the ssDNA-binding domain in S. pombe Pot1. Comparison with the homologous human domain reveals unexpected structural divergence in the mode of ligand binding that explains the differing ligand requirements between species. Despite the presence of apparently base-specific hydrogen bonds, Pot1pC is able to bind a wide range of ssDNA sequences with thermodynamic equivalence. To address how Pot1pC binds ssDNA with little to no specificity, multiple structures of Pot1pC bound to non-cognate ssDNA ligands were solved. These structures reveal that this promiscuity is implemented through new binding modes that thermodynamically compensate for base-substitutions through alternate stacking interactions and new H-bonding networks. PMID:23201273

  5. Effects of a Variety of Food Extracts and Juices on the Specific Binding Ability of Norovirus GII.4 P Particles

    Science.gov (United States)

    LI, DAN; BAERT, LEEN; XIA, MING; ZHONG, WEIMING; JIANG, XI; UYTTENDAELE, MIEKE

    2014-01-01

    The effects of 13 food extracts and juices, including shellfish, fruits, and vegetables, on the binding ability of human norovirus (NoV) were examined, using P particles of human NoV GII.4 as a research surrogate. The enhancements (positive values) or reductions (negative values) of NoV P particle detection (changes in optical density at 450 nm) in the presence of different food extracts and juices as compared with P particles diluted in phosphate-buffered saline were tested by saliva-binding, enzyme-linked immunosorbent assay in triplicate. In the presence of different food extracts and juices at different concentrations, an increase or decrease of the receptor-binding ability of the NoV P particles was observed. Due to a higher specific binding and thus a higher accumulation of the viral particles, oysters may be contaminated with human NoV more often than other shellfish species (mussel, hard clams, and razor clams). Cranberry and pomegranate juices were shown to reduce the specific binding ability of human NoV P particles. No such binding inhibition effects were observed for the other tested extracts of fresh produce (strawberry, blackberry, blueberry, cherry tomato, spinach, romaine lettuce) or, notably, for raspberry, which has been associated with human NoV outbreaks. PMID:22980024

  6. Binding specificity of Bacillus thuringiensis Cry1Aa for purified, native Bombyx mori aminopeptidase N and cadherin-like receptors

    Directory of Open Access Journals (Sweden)

    Jenkins Jeremy L

    2001-10-01

    Full Text Available Abstract Background To better understand the molecular interactions of Bt toxins with non-target insects, we have examined the real-time binding specificity and affinity of Cry1 toxins to native silkworm (Bombyx mori midgut receptors. Previous studies on B. mori receptors utilized brush border membrane vesicles or purifed receptors in blot-type assays. Results The Bombyx mori (silkworm aminopeptidase N (APN and cadherin-like receptors for Bacillus thuringiensis insecticidal Cry1Aa toxin were purified and their real-time binding affinities for Cry toxins were examined by surface plasmon resonance. Cry1Ab and Cry1Ac toxins did not bind to the immobilized native receptors, correlating with their low toxicities. Cry1Aa displayed moderate affinity for B. mori APN (75 nM, and unusually tight binding to the cadherin-like receptor (2.6 nM, which results from slow dissociation rates. The binding of a hybrid toxin (Aa/Aa/Ac was identical to Cry1Aa. Conclusions These results indicate domain II of Cry1Aa is essential for binding to native B. mori receptors and for toxicity. Moreover, the high-affinity binding of Cry1Aa to native cadherin-like receptor emphasizes the importance of this receptor class for Bt toxin research.

  7. Screening of specific binding peptide targeting blood vessel of human esophageal cancer in vivo in mice

    Institute of Scientific and Technical Information of China (English)

    ZHI Min; WU Kai-chun; HAO Zhi-ming; GUO Chang-cun; YAO Jia-yin

    2011-01-01

    Background Cancer of the esophagus and gastroesophageal junction remains a virulent malignancy with poor prognosis. Rapid progresses were made in chemotherapeutic agents and the development of molecular markers allowed better identification of candidates for targeted therapy. This study aimed to identify the candidate peptides used for anti-angiogenic therapy of esophageal cancer by in vivo screening C7C peptide library for peptides binding specifically to blood vessels of human esophageal cancer.Methods The phage displayed C7C peptide library was injected intravenously into mice bearing human esophageal tumor xenografts under renal capsule. After 5 rounds of screening, 13 clones were picked up individually and sequenced.During each round of screening, titers of phage recovery were calculated from tumor xenograft and control tissues.Homing of these 9 peptides to tumor vessel was detected by calculating phage titers in the tumor xenograft and control tissues (lung and spleen) after each phage was injected into mice model, and compared with the distribution of phage M13 and Ⅷ-related antigen in tumor xenograft by immunohistochemical staining. Comparisons among groups of data were made using one-way analysis of variance (ANOVA), followed by the Bonferroni multiple comparisons test.Results The number of phage recovered from tumor tissue of each round increased gradually in tumor group while decreased in control groups (P <0.01 in tumor and spleen, P <0.05 in lung). Immunohistochemical staining showed similar staining pattern with M13 antibody or Ⅷ-related antigen antibody, suggesting that phages displaying the selected peptides could home to blood vessel of human esophageal cancer. According to their DNA, 9 corresponding peptide sequences were deduced. And the homing ability to blood vessel of phages displaying the selected peptides was confirmed by comparing with their recovery in tumor and control tissues. Two motifs, YSXNXW and PXNXXN, were also obtained by

  8. Alteration of the carbohydrate-binding specificity of a C-type lectin CEL-I mutant with an EPN carbohydrate-binding motif.

    Science.gov (United States)

    Hatakeyama, Tomomitsu; Ishimine, Tomohiro; Baba, Tomohiro; Kimura, Masanari; Unno, Hideaki; Goda, Shuichiro

    2013-07-01

    CEL-I is a Gal/GalNAc-specific C-type lectin isolated from the sea cucumber Cucumaria echinata. This lectin is composed of two carbohydrate-recognition domains (CRDs) with the carbohydrate-recognition motif QPD (Gln-Pro- Asp), which is generally known to exist in galactose-specific C-type CRDs. In the present study, a mutant CEL-I with EPN (Glu-Pro-Asn) motif, which is thought to be responsible for the carbohydrate-recognition of mannose-specific Ctype CRDs, was produced in Escherichia coli, and its effects on the carbohydrate-binding specificity were examined using polyamidoamine dendrimer (PD) conjugated with carbohydrates. Although wild-type CEL-I effectively formed complexes with N-acetylgalactosamine (GalNAc)-PD but not with mannose-PD, the mutant CEL-I showed relatively weak but definite affinity for mannose-PD. These results indicated that the QPD and EPN motifs play a significant role in the carbohydrate-recognition mechanism of CEL-I, especially in the discrimination of galactose and mannose. Additional mutations in the recombinant CEL-I binding site may further increase its specificity for mannose, and should provide insights into designing novel carbohydrate-recognition proteins. PMID:23157284

  9. Distorted octahedral coordination of tungstate in a subfamily of specific binding proteins

    NARCIS (Netherlands)

    Hollenstein, K.; Comellas-Bigler, M.; Bevers, L.E.; Feiters, M.C.; Meyer-Klaucke, W.; Hagedoorn, P.-L.; Locher, K.P.

    2009-01-01

    Bacteria and archaea import molybdenum and tungsten from the environment in the form of the oxyanions molybdate (MoO4 2−) and tungstate (WO4 2−). These substrates are captured by an external, high-affinity binding protein, and delivered to ATP binding cassette transporters, which move them across th

  10. DNA-binding specificity and molecular functions of NAC transcription factors

    DEFF Research Database (Denmark)

    Olsen, Addie Nina; Ernst, Heidi Asschenfeldt; Lo Leggio, Leila;

    2005-01-01

    The family of NAC (NAM/ATAF1,2/CUC2) transcription factors has been implicated in a wide range of plant processes, but knowledge on the DNA-binding properties of the family is limited. Using a reiterative selection procedure on random oligonucleotides, we have identified consensus binding sites...

  11. Multiple individual and cross-specific indiotypes on 13 levan-binding myeloma proteins of BALB/c mice

    Science.gov (United States)

    1975-01-01

    13 leven-binding myeloma proteins (LBMP) of BALB/c origin were classified into two groups with different binding specificities; one group of 11 proteins bound beta2 leads to 1 fructosans, a second group of two proteins bound fructosans probably of beta2 leads to 6 linkage. Anti-idiotypic sera prepared to 10 of the proteins in the appropriate strains of mice identified numerous idiotypic determinants. Each protein used for immunization had its own unique individual idiotypic specificities (IdI) and in addition most of the proteins carried two- nine cross-specific or shared idiotypes (IdX) that were found only among LBMP, and not found in 106 non-LBMP. Most of the IdX determinants and only four of the IdI determinants of the beta2 leads to 1 fructosan binding group were located in the antigen-binding site. The multiplicity of antigenic differences in this functionally related group of immunoglobulins reveals an unexpected degree of heterogeneity in V-regions that appears to be unrelated to binding. PMID:1151286

  12. Identification and binding mechanism of phage displayed peptides with specific affinity to acid-alkali treated titanium.

    Science.gov (United States)

    Sun, Yuhua; Tan, Jing; Wu, Baohua; Wang, Jianxin; Qu, Shuxin; Weng, Jie; Feng, Bo

    2016-10-01

    Acid-alkali treatment is one of means widely used for preparing bioactive titanium surfaces. Peptides with specific affinity to titanium surface modified by acid-alkali two-steps treatment were obtained via phage display technology. Out of the eight new unique peptides, titanium-binding peptide 54 displayed by monoclonal M13 phage at its pIII coat protein (TBP54-M13 phage) was proved to have higher binding affinity to the substrate. The binding interaction occurred at the domain from phenylalanine at position 1 to arginine at position 6 in the sequences of TBP54 (FAETHRGFHFSF) mainly via the reaction of these residues with the Ti surface. Together the coordination and electrostatic interactions controlled the specific binding of the phage to the substrate. The binding affinity was dependent on the surface basic hydroxyl group content. In addition, the phage showed a different interaction way with the Ti surface without acid-alkali treatment along with an impaired affinity. This study could provide more understanding of the interaction mechanism between the selected peptide and its specific substrate, and develop a promising method for the biofunctionalization of titanium. PMID:27371890

  13. Sensitive and direct detection of receptor binding specificity of highly pathogenic avian influenza A virus in clinical samples.

    Directory of Open Access Journals (Sweden)

    Tadanobu Takahashi

    Full Text Available Influenza A virus (IAV recognizes two types of N-acetylneuraminic acid (Neu5Ac by galactose (Gal linkages, Neu5Acα2,3Gal and Neu5Acα2,6Gal. Avian IAV preferentially binds to Neu5Acα2,3Gal linkage, while human IAV preferentially binds to Neu5Acα2,6Gal linkage, as a virus receptor. Shift in receptor binding specificity of avian IAV from Neu5Acα2,3Gal linkage to Neu5Acα2,6Gal linkage is generally believed to be a critical factor for its transmission ability among humans. Surveillance of this shift of highly pathogenic H5N1 avian IAV (HPAI is thought to be a very important for prediction and prevention of a catastrophic pandemic of HPAI among humans. In this study, we demonstrated that receptor binding specificity of IAV bound to sialo-glycoconjugates was sensitively detected by quantifying the HA gene with real-time reverse-transcription-PCR. The new assay enabled direct detection of receptor binding specificity of HPAIs in chicken clinical samples including trachea and cloaca swabs in only less than 4 h.

  14. Receptor binding proteins of Listeria monocytogenes bacteriophages A118 and P35 recognize serovar-specific teichoic acids

    International Nuclear Information System (INIS)

    Adsorption of a bacteriophage to the host requires recognition of a cell wall-associated receptor by a receptor binding protein (RBP). This recognition is specific, and high affinity binding is essential for efficient virus attachment. The molecular details of phage adsorption to the Gram-positive cell are poorly understood. We present the first description of receptor binding proteins and a tail tip structure for the siphovirus group infecting Listeria monocytogenes. The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. Two proteins were identified as RBPs in phage A118. Rhamnose residues in wall teichoic acids represent the binding ligands for both proteins. In phage P35, protein gp16 could be identified as RBP and the role of both rhamnose and N-acetylglucosamine in phage adsorption was confirmed. Immunogold-labeling and transmission electron microscopy allowed the creation of a topological model of the A118 phage tail. - Highlights: • We present the first description of receptor binding proteins and a tail tip structure for the Siphovirus group infecting Listeria monocytogenes. • The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. • Rhamnose residues in wall teichoic acids represent the binding ligands for both receptor binding proteins in phage A118. • Rhamnose and N-acetylglucosamine are required for adsorption of phage P35. • We preset a topological model of the A118 phage tail

  15. Receptor binding proteins of Listeria monocytogenes bacteriophages A118 and P35 recognize serovar-specific teichoic acids

    Energy Technology Data Exchange (ETDEWEB)

    Bielmann, Regula; Habann, Matthias; Eugster, Marcel R. [Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 7, 8092 Zurich (Switzerland); Lurz, Rudi [Max-Planck Institute for Molecular Genetics, 14195 Berlin (Germany); Calendar, Richard [Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202 (United States); Klumpp, Jochen, E-mail: jochen.klumpp@hest.ethz.ch [Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 7, 8092 Zurich (Switzerland); Loessner, Martin J. [Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 7, 8092 Zurich (Switzerland)

    2015-03-15

    Adsorption of a bacteriophage to the host requires recognition of a cell wall-associated receptor by a receptor binding protein (RBP). This recognition is specific, and high affinity binding is essential for efficient virus attachment. The molecular details of phage adsorption to the Gram-positive cell are poorly understood. We present the first description of receptor binding proteins and a tail tip structure for the siphovirus group infecting Listeria monocytogenes. The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. Two proteins were identified as RBPs in phage A118. Rhamnose residues in wall teichoic acids represent the binding ligands for both proteins. In phage P35, protein gp16 could be identified as RBP and the role of both rhamnose and N-acetylglucosamine in phage adsorption was confirmed. Immunogold-labeling and transmission electron microscopy allowed the creation of a topological model of the A118 phage tail. - Highlights: • We present the first description of receptor binding proteins and a tail tip structure for the Siphovirus group infecting Listeria monocytogenes. • The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. • Rhamnose residues in wall teichoic acids represent the binding ligands for both receptor binding proteins in phage A118. • Rhamnose and N-acetylglucosamine are required for adsorption of phage P35. • We preset a topological model of the A118 phage tail.

  16. /sup 125/I-human epidermal growth factor specific binding to placentas and fetal membranes from varoius pregnancy states

    Energy Technology Data Exchange (ETDEWEB)

    Hofmann, G.E.; Siddiqi, T.A.; Rao, Ch. V.; Carman, F.R.

    1988-01-01

    Specific binding of /sup 125/I-human epidermal growth factor (hEGF) to homogenates of term human placentas and fetal membranes from normal and appropriate for gestational age (N = 20), intrauterine growth retarded (N = 9), twin (N = 11), White class AB diabetic (N = 12), and large for gestational age (N = 13) pregnancies was measured. In all pregnancy states, placentas bound approximately four times more /sup 125/I-hEGF than did fetal membranes (P<0.0001). There was no significant differnce in /sup 125/I-hEGF binding to fetal membranes from the various pregnancy states (P<0.05). /sup 125/I-hEGF specific binding to placentas from intrauterine growth retarded or twin pregnancies was significantly greater compared with placentas from normal and appropriate for gestational age pregnancies (P<0.05). The binding to placentas from pregnancies complicated by White class AB diabetes or large for gestational age infants, on the other hand, was not significantly different from that to placentas from normal and appropriate for gestational age pregnancies. /sup 125/I-hEGF specific binding did not differ between placentas from intrauterine growth retarded or twin pregnancies (P<0.05). Placental and fetal membrane /sup 125/I-hEGF binding did not vary with fetal sex, maternal race, placental weight, or gestational age between 37 to 42 weeks (P<0.05). Placental but not fetal membrane /sup 125/I-hEGF binding increased with increasing infant weight when appropriate for gestational age and large for gestational age infants were included (P<0.05, r = 0.38, N = 32) but not for intrauterine growth retarded, appropriate for gestational age, or large for gestational age infants alone.

  17. Specific binding and laterality of human extrastriatal dopamine D2/D3 receptors in late onset type 1 alcoholic patients.

    Science.gov (United States)

    Kuikka, J T; Repo, E; Bergström, K A; Tupala, E; Tiihonen, J

    2000-09-29

    Late onset type 1 alcoholism has been suggested to be associated with decreased dopaminergic transmission. Our hypothesis was that late onset type 1 alcoholics have also abnormal extrastriatal dopamine D(2)/D(3) receptor distribution. We performed binding, heterogeneity and laterality analysis of extrastriatal and striatal dopamine D(2)/D(3) receptors in nine late onset male alcoholics and in 12 age-matched healthy males. A radioligand, [(123)I]epidepride was used in high resolution single-photon emission tomography (SPET). Specific binding of epidepride in the left temporal pole was significantly (Pepidepride distribution observed in control males (0.89+/-0.19 vs. 1.10+/-0.19; P<0.05). The results suggest that the specific binding of dopamine D(2)/D(3) receptors in late type 1 alcoholics is decreased and its laterality in the temporal brain is altered from normal. PMID:10996449

  18. Allele-specific locus binding and genome editing by CRISPR at the p16INK4a locus

    Science.gov (United States)

    Fujita, Toshitsugu; Yuno, Miyuki; Fujii, Hodaka

    2016-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR) system has been adopted for a wide range of biological applications including genome editing. In some cases, dissection of genome functions requires allele-specific genome editing, but the use of CRISPR for this purpose has not been studied in detail. In this study, using the p16INK4a gene in HCT116 as a model locus, we investigated whether chromatin states, such as CpG methylation, or a single-nucleotide gap form in a target site can be exploited for allele-specific locus binding and genome editing by CRISPR in vivo. First, we showed that allele-specific locus binding and genome editing could be achieved by targeting allele-specific CpG-methylated regions, which was successful for one, but not all guide RNAs. In this regard, molecular basis underlying the success remains elusive at this stage. Next, we demonstrated that an allele-specific single-nucleotide gap form could be employed for allele-specific locus binding and genome editing by CRISPR, although it was important to avoid CRISPR tolerance of a single nucleotide mismatch brought about by mismatched base skipping. Our results provide information that might be useful for applications of CRISPR in studies of allele-specific functions in the genomes. PMID:27465215

  19. Assessment of altered binding specificity of bacteriophage for ciprofloxacin-induced antibiotic-resistant Salmonella Typhimurium.

    Science.gov (United States)

    Kim, Jeongjin; Jo, Ara; Ding, Tian; Lee, Hyeon-Yong; Ahn, Juhee

    2016-08-01

    This study describes a new effort toward understanding the interaction mechanisms between antibiotic-resistant Salmonella Typhimurium and phages. The antibiotic susceptibility, β-lactamase activity, bacterial motility, gene expression, and lytic activity were evaluated in ciprofloxacin-induced antibiotic-sensitive Salmonella Typhimurium (ASST(CIP)) and ciprofloxacin-induced antibiotic-resistant S. Typhimurium (ARST(CIP)), which were compared to the wild-type strains (ASST(WT) and ARST(WT)). The MIC values of ampicillin, norfloxacin, chloramphenicol, and tetracycline were significantly increased to > 512, 16, 16, and 256 μg/ml, respectively, in the ARST(CIP). The lowest and highest extracellular lactamase activities were observed in ASST(WT) (6.85 μmol/min/ml) and ARST(CIP) (48.83 μmol/min/ml), respectively. The acrA, lpfE, and hilA genes were significantly upregulated by more than tenfold in both ASST(CIP) and ARST(CIP). The induction of multiple antibiotic resistance resulted from the increased efflux pump activity (AcrAB-TolC). The highest phage adsorption rates were more than 95 % for ASST(WT), ASST(CIP), and ARST(WT), while the lowest adsorption rate was 52 % for ARST(CIP) at 15 min of infection. The least lytic activity of phage was 20 % against the ARST(CIP), followed by ASST(CIP) (30 %). The adsorption rate of phage against ARST(CIP) was 52 % at 15 min of infection, which resulted in the decrease in lytic activity (12 %). Understanding the interaction of phage and bacteria is essential for the practical application of phage to control and detect antibiotic-resistant bacteria. The results provide useful information for understanding the binding specificity of phages for multiple antibiotic-resistant pathogens. PMID:27000396

  20. FE65 binds Teashirt, inhibiting expression of the primate-specific caspase-4.

    Directory of Open Access Journals (Sweden)

    Yuji Kajiwara

    Full Text Available The Alzheimer disease (AD amyloid protein precursor (APP can bind the FE65 adaptor protein and this complex can regulate gene expression. We carried out yeast two-hybrid studies with a PTB domain of FE65, focusing on those genes that might be involved in nuclear signaling, and identified and validated Teashirt proteins as FE65 interacting proteins in neurons. Using reporter systems, we observed that FE65 could simultaneously recruit SET, a component of the inhibitor of acetyl transferase, and Teashirt, which in turn recruited histone deacetylases, to produce a powerful gene-silencing complex. We screened stable cell lines with a macroarray focusing on AD-related genes and identified CASP4, encoding caspase-4, as a target of this silencing complex. Chromatin immunoprecipitation showed a direct interaction of FE65 and Teashirt3 with the promoter region of CASP4. Expression studies in postmortem samples demonstrated decreasing expression of Teashirt and increasing expression of caspase-4 with progressive cognitive decline. Importantly, there were significant increases in caspase-4 expression associated with even the earliest neuritic plaque changes in AD. We evaluated a case-control cohort and observed evidence for a genetic association between the Teashirt genes TSHZ1 and TSHZ3 and AD, with the TSHZ3 SNP genotype correlating with expression of Teashirt3. The results were consistent with a model in which reduced expression of Teashirt3, mediated by genetic or other causes, increases caspase-4 expression, leading to progression of AD. Thus the cell biological, gene expression and genetic data support a role for Teashirt/caspase-4 in AD biology. As caspase-4 shows evidence of being a primate-specific gene, current models of AD and other neurodegenerative conditions may be incomplete because of the absence of this gene in the murine genome.

  1. Identification of amino acid residues in PEPHC1 important for binding to the tumor-specific receptor EGFRvIII

    DEFF Research Database (Denmark)

    Hansen, Charlotte Lund; Hansen, Paul Robert; Pedersen, Nina;

    2008-01-01

    EGFRvIII is a cancer-specific epidermal growth factor tyrosine kinase receptor mutation, expressed in different kinds of cancer, in particular ovarian, glioblastomas, and breast cancer. A peptide, PEPHC1, has previously been shown to bind selectively to EGFRvIII. An alanine scan was performed...

  2. The conserved PA14 domain of cell wall-associated fungal adhesins governs their glycan-binding specificity

    NARCIS (Netherlands)

    P.W.J. de Groot; F.M. Klis

    2008-01-01

    Yeast cell wall-associated, lectin-like adhesins form large families that mediate flocculation and host cell recognition. The glycan specificity of individual adhesins is largely unknown. Zupancic et al. (this issue of Molecular Microbiology) used glycan microarrays to compare the glycan-binding cha

  3. Structural Analysis of Semi-specific Oligosaccharide Recognition by a Cellulose-binding Protein of Thermotoga maritima Reveals Adaptations for Functional Diversification of the Oligopeptide Periplasmic Binding Protein Fold

    Energy Technology Data Exchange (ETDEWEB)

    Cuneo, Matthew J.; Beese, Lorena S.; Hellinga, Homme W.; (Duke)

    2010-05-25

    Periplasmic binding proteins (PBPs) constitute a protein superfamily that binds a wide variety of ligands. In prokaryotes, PBPs function as receptors for ATP-binding cassette or tripartite ATP-independent transporters and chemotaxis systems. In many instances, PBPs bind their cognate ligands with exquisite specificity, distinguishing, for example, between sugar epimers or structurally similar anions. By contrast, oligopeptide-binding proteins bind their ligands through interactions with the peptide backbone but do not distinguish between different side chains. The extremophile Thermotoga maritima possesses a remarkable array of carbohydrate-processing metabolic systems, including the hydrolysis of cellulosic polymers. Here, we present the crystal structure of a T. maritima cellobiose-binding protein (tm0031) that is homologous to oligopeptide-binding proteins. T. maritima cellobiose-binding protein binds a variety of lengths of {beta}(1 {yields} 4)-linked glucose oligomers, ranging from two rings (cellobiose) to five (cellopentaose). The structure reveals that binding is semi-specific. The disaccharide at the nonreducing end binds specifically; the other rings are located in a large solvent-filled groove, where the reducing end makes several contacts with the protein, thereby imposing an upper limit of the oligosaccharides that are recognized. Semi-specific recognition, in which a molecular class rather than individual species is selected, provides an efficient solution for the uptake of complex mixtures.

  4. Specific binding of a fungal glucan phytoalexin elicitor to membrane fractions from soybean Glycine max

    International Nuclear Information System (INIS)

    Treatment of soybean tissues with elicitors results in the production of phytoalexins, one of a number of inducible plant defense reactions against microbial infections. The present study uses a β-1,3-[3H] glucan elicitor fraction from Phytophthora megasperma f.sp. glycinea, a fungal pathogen of soybean, to identify putative elicitor targets in soybean tissues. Use of the radiolabeled elicitor disclosed saturable high-affinity elicitor binding site(s) in membrane fractions of soybean roots. Highest binding activity is associated with a plasma membrane-enriched fraction. The apparent K/sub d/ value for β-glucan elicitor binding is ≅ 0.2 x 10-6 M and the maximum number of binding sites is 0.5 pmol per mg of protein. Competition studies the [3H]glucan elicitor and a number of polysaccharides demonstrate that only polysaccharides of a branched β-glucan type effectively displace the radiolabeled ligand from membrane binding. Differential displacing activity of the glucans on P. megasperma elicitor binding corresponds closely to their respective ability to elicit phytoalexin production in a cotyledon bioassay

  5. Anomer-Specific Recognition and Dynamics in a Fucose-Binding Lectin.

    Science.gov (United States)

    Antonik, Paweł M; Volkov, Alexander N; Broder, Ursula N; Re, Daniele Lo; van Nuland, Nico A J; Crowley, Peter B

    2016-03-01

    Sugar binding by a cell surface ∼29 kDa lectin (RSL) from the bacterium Ralstonia solanacearum was characterized by NMR spectroscopy. The complexes formed with four monosaccharides and four fucosides were studied. Complete resonance assignments and backbone dynamics were determined for RSL in the sugar-free form and when bound to l-fucose or d-mannose. RSL was found to interact with both the α- and the β-anomer of l-fucose and the "fucose like" sugars d-arabinose and l-galactose. Peak splitting was observed for some resonances of the binding site residues. The assignment of the split signals to the α- or β-anomer was confirmed by comparison with the spectra of RSL bound to methyl-α-l-fucoside or methyl-β-l-fucoside. The backbone dynamics of RSL were sensitive to the presence of ligand, with the protein adopting a more compact structure upon binding to l-fucose. Taking advantage of tryptophan residues in the binding sites, we show that the indole resonance is an excellent reporter on ligand binding. Each sugar resulted in a distinct signature of chemical shift perturbations, suggesting that tryptophan signals are a sufficient probe of sugar binding.

  6. Identification of Plasmodium falciparum RhopH3 protein peptides that specifically bind to erythrocytes and inhibit merozoite invasion

    Science.gov (United States)

    Pinzón, Carlos Giovanni; Curtidor, Hernando; Reyes, Claudia; Méndez, David; Patarroyo, Manuel Elkin

    2008-01-01

    The identification of sequences involved in binding to erythrocytes is an important step for understanding the molecular basis of merozoite–erythrocyte interactions that take place during invasion of the Plasmodium falciparum malaria parasite into host cells. Several molecules located in the apical organelles (micronemes, rhoptry, dense granules) of the invasive-stage parasite are essential for erythrocyte recognition, invasion, and establishment of the nascent parasitophorous vacuole. Particularly, it has been demonstrated that rhoptry proteins play an important role in binding to erythrocyte surface receptors, among which is the PfRhopH3 protein, which triggers important immune responses in patients from endemic regions. It has also been reported that anti-RhopH3 antibodies inhibit in vitro invasion of erythrocytes, further supporting its direct involvement in erythrocyte invasion processes. In this study, PfRhopH3 consecutive peptides were synthesized and tested in erythrocyte binding assays for identifying those regions mediating binding to erythrocytes. Fourteen PfRhopH3 peptides presenting high specific binding activity were found, whose bindings were saturable and presented nanomolar dissociation constants. These high-activity binding peptides (HABPs) were characterized by having α-helical structural elements, as determined by circular dichroism, and having receptors of a possible sialic acid-dependent and/or glycoprotein-dependent nature, as evidenced in enzyme-treated erythrocyte binding assays and further corroborated by cross-linking assay results. Furthermore, these HABPs inhibited merozoite in vitro invasion of normal erythrocytes at 200 μM by up to 60% and 90%, suggesting that some RhopH3 protein regions are involved in the P. falciparum erythrocyte invasion. PMID:18593818

  7. Cations Stiffen Actin Filaments by Adhering a Key Structural Element to Adjacent Subunits.

    Science.gov (United States)

    Hocky, Glen M; Baker, Joseph L; Bradley, Michael J; Sinitskiy, Anton V; De La Cruz, Enrique M; Voth, Gregory A

    2016-05-26

    Ions regulate the assembly and mechanical properties of actin filaments. Recent work using structural bioinformatics and site-specific mutagenesis favors the existence of two discrete and specific divalent cation binding sites on actin filaments, positioned in the long axis between actin subunits. Cation binding at one site drives polymerization, while the other modulates filament stiffness and plays a role in filament severing by the regulatory protein, cofilin. Existing structural methods have not been able to resolve filament-associated cations, and so in this work we turn to molecular dynamics simulations to suggest a candidate binding pocket geometry for each site and to elucidate the mechanism by which occupancy of the "stiffness site" affects filament mechanical properties. Incorporating a magnesium ion in the "polymerization site" does not seem to require any large-scale change to an actin subunit's conformation. Binding of a magnesium ion in the "stiffness site" adheres the actin DNase-binding loop (D-loop) to its long-axis neighbor, which increases the filament torsional stiffness and bending persistence length. Our analysis shows that bound D-loops occupy a smaller region of accessible conformational space. Cation occupancy buries key conserved residues of the D-loop, restricting accessibility to regulatory proteins and enzymes that target these amino acids. PMID:27146246

  8. The cationic peptide LL-37 binds Mac-1 (CD11b/CD18) with a low dissociation rate and promotes phagocytosis.

    Science.gov (United States)

    Zhang, Xianwei; Bajic, Goran; Andersen, Gregers R; Christiansen, Stig Hill; Vorup-Jensen, Thomas

    2016-05-01

    As a broad-spectrum anti-microbial peptide, LL-37 plays an important role in the innate immune system. A series of previous reports implicates LL-37 as an activator of various cell surface receptor-mediated functions, including chemotaxis in integrin CD11b/CD18 (Mac-1)-expressing cells. However, evidence is scarce concerning the direct binding of LL-37 to these receptors and investigations on the associated binding kinetics is lacking. Mac-1, a member of the β2 integrin family, is mainly expressed in myeloid leukocytes. Its critical functions include phagocytosis of complement-opsonized pathogens. Here, we report on interactions of LL-37 and its fragment FK-13 with the ligand-binding domain of Mac-1, the α-chain I domain. LL-37 bound the I-domain with an affinity comparable to the complement fragment C3d, one of the strongest known ligands for Mac-1. In cell adhesion assays both LL-37 and FK-13 supported binding by Mac-1 expressing cells, however, with LL-37-coupled surfaces supporting stronger cell adhesion than FK-13. Likewise, in phagocytosis assays with primary human monocytes both LL-37 and FK-13 enhanced uptake of particles coupled with these ligands but with a tendency towards a stronger uptake by LL-37.

  9. Novel interactions of ankyrins-G at the costameres: The muscle-specific Obscurin/Titin-Binding-related Domain (OTBD) binds plectin and filamin C

    Energy Technology Data Exchange (ETDEWEB)

    Maiweilidan, Yimingjiang; Klauza, Izabela; Kordeli, Ekaterini, E-mail: ekaterini.kordeli@inserm.fr

    2011-04-01

    Ankyrins, the adapters of the spectrin skeleton, are involved in local accumulation and stabilization of integral proteins to the appropriate membrane domains. In striated muscle, tissue-dependent alternative splicing generates unique Ank3 gene products (ankyrins-G); they share the Obscurin/Titin-Binding-related Domain (OTBD), a muscle-specific insert of the C-terminal domain which is highly conserved among ankyrin genes, and binds obscurin and titin to Ank1 gene products. We previously proposed that OTBD sequences constitute a novel domain of protein-protein interactions which confers ankyrins with specific cellular functions in muscle. Here we searched for muscle proteins binding to ankyrin-G OTBD by yeast two hybrid assay, and we found plectin and filamin C, two organizing elements of the cytoskeleton with essential roles in myogenesis, muscle cell cytoarchitecture, and muscle disease. The three proteins coimmunoprecipitate from skeletal muscle extracts and colocalize at costameres in adult muscle fibers. During in vitro myogenesis, muscle ankyrins-G are first expressed in postmitotic myocytes undergoing fusion to myotubes. In western blots of subcellular fractions from C2C12 cells, the majority of muscle ankyrins-G appear associated with membrane compartments. Occasional but not extensive co-localization at nascent costameres suggested that ankyrin-G interactions with plectin and filamin C are not involved in costamere assembly; they would rather reinforce stability and/or modulate molecular interactions in sarcolemma microdomains by establishing novel links between muscle-specific ankyrins-G and the two costameric dystrophin-associated glycoprotein and integrin-based protein complexes. These results report the first protein-protein interactions involving the ankyrin-G OTBD domain and support the hypothesis that OTBD sequences confer ankyrins with a gain of function in vertebrates, bringing further consolidation and resilience of the linkage between sarcomeres

  10. [Application of aspartic acid as a non-specific binding inhibitor in the enrichment of phosphopeptides with titanium dioxide].

    Science.gov (United States)

    Chi, Ming; Bi, Wei; Lu, Zhuang; Song, Lina; Jia, Wei; Zhang, Yangjun; Qian, Xiaohong; Cai, Yun

    2010-02-01

    Titanium dioxide (TiO2) is one of metal oxides widely used for phosphopeptide enrichment in phosphoproteomic research nowadays. However it can bind to some non-phosphorylated peptides containing one or more aspartic acid residues and/or glutamic acid residues. These non-phosphorylated peptides can be eluted along with phosphorylated peptides and cause the reduction of the selectivity. Conventional inhibitors for the non-specific binding of non-phosphorylated peptides can often contaminate the ion source of mass spectrometry and therefore their applications are limited in liquid chromatography-mass spectrometry (LC-MS). In this study, aspartic acid was reported as a novel non-specific binding inhibitor for phosphopeptide enrichment by titanium dioxide. Firstly, the tryptic peptide mixtures of 3 and 9 standard proteins were used for the comparison of the enrichment efficiency of titanium dioxide. The effects with the presence of aspartic acid, glutamic acid and no-inhibitor in the enrichment systems were compared separately. The results showed that aspartic acid can greatly improve the selectivity of titanium dioxide for phosphopeptide enrichment. Then, aspartic acid was used for the enrichment of tryptic peptide mixture of C57BL/6J mouse liver lysate and good results were also obtained which demonstrated that aspartic acid was a promising non-specific binding inhibitor for complex biological samples. Besides, no contamination in the ion source occurred during the mass spectrometric analysis.

  11. Applying DNA affinity chromatography to specifically screen for sucrose-related DNA-binding transcriptional regulators of Xanthomonas campestris.

    Science.gov (United States)

    Leßmeier, Lennart; Alkhateeb, Rabeaa S; Schulte, Fabian; Steffens, Tim; Loka, Tobias Pascal; Pühler, Alfred; Niehaus, Karsten; Vorhölter, Frank-Jörg

    2016-08-20

    At a molecular level, the regulation of many important cellular processes is still obscure in xanthomonads, a bacterial group of outstanding relevance as world-wide plant pathogens and important for biotechnology as producers of the polysaccharide xanthan. Transcriptome analysis indicated a sucrose-dependent regulation of 18 genes in Xanthomonas campestris pv. campestris (Xcc) B100. The expression of 12 of these genes was clearly increased in the presence of sucrose. Only part of these genes was obviously involved in sucrose utilization. To identify regulatory proteins involved in transcriptional regulation, a DNA fragment-specific pull-down approach was established for Xcc. Putative promoter regions were identified and used to isolate DNA-binding proteins, which were separated by SDS PAGE and identified by MALDI-TOF mass spectrometry. This led to the identification of four transcriptional regulators, among them the global transcriptional regulator Clp and a previously identified regulator of sucrose utilization, SuxR, plus a third DNA-binding transcriptional regulator encoded by xcc-b100_2861 and recently shown to interact with a cyclic di-GMP-binding protein. The fourth regulatory protein was encoded by xcc-b100_2791. These results indicate DNA fragment-specific pull-down experiments as promising approaches to screen for specific DNA-binding regulatory proteins in Xcc. PMID:27060555

  12. Conformational Dynamics and the Binding of Specific and Nonspecific DNA by the Autoinhibited Transcription Factor Ets-1.

    Science.gov (United States)

    Desjardins, Geneviève; Okon, Mark; Graves, Barbara J; McIntosh, Lawrence P

    2016-07-26

    The affinity of the Ets-1 transcription factor for DNA is autoinhibited by an intrinsically disordered serine-rich region (SRR) and a helical inhibitory module (IM) appended to its winged helix-turn-helix ETS domain. Using NMR spectroscopy, we investigated how Ets-1 recognizes specific versus nonspecific DNA, with a focus on the roles of protein dynamics and autoinhibition in these processes. Upon binding either DNA, the two marginally stable N-terminal helices of the IM predominantly unfold, but still sample partially ordered conformations. Also, on the basis of amide chemical shift perturbation mapping, Ets-1 associates with both specific and nonspecific DNA through the same canonical ETS domain interface. These interactions are structurally independent of the SRR, and thus autoinhibition does not impart DNA-binding specificity. However, relative to the pronounced NMR spectroscopic changes in Ets-1 resulting from specific DNA binding, the spectra of the nonspecific DNA complexes showed conformational exchange broadening and lacked several diagnostic amide and indole signals attributable to hydrogen bonding interactions seen in reported X-ray crystallographic structures of this transcription factor with its cognate DNA sequences. Such differences are highlighted by the chemical shift and relaxation properties of several interfacial lysine and arginine side chains. Collectively, these data support a general model in which Ets-1 interacts with nonspecific DNA via dynamic electrostatic interactions, whereas hydrogen bonding drives the formation of well-ordered complexes with specific DNA.

  13. Application of Celluspots peptide arrays for the analysis of the binding specificity of epigenetic reading domains to modified histone tails

    Directory of Open Access Journals (Sweden)

    Dhayalan Arunkumar

    2011-08-01

    Full Text Available Abstract Background Epigenetic reading domains are involved in the regulation of gene expression and chromatin state by interacting with histones in a post-translational modification specific manner. A detailed knowledge of the target modifications of reading domains, including enhancing and inhibiting secondary modifications, will lead to a better understanding of the biological signaling processes mediated by reading domains. Results We describe the application of Celluspots peptide arrays which contain 384 histone peptides carrying 59 post translational modifications in different combinations as an inexpensive, reliable and fast method for initial screening for specific interactions of reading domains with modified histone peptides. To validate the method, we tested the binding specificities of seven known epigenetic reading domains on Celluspots peptide arrays, viz. the HP1ß and MPP8 Chromo domains, JMJD2A and 53BP1 Tudor domains, Dnmt3a PWWP domain, Rag2 PHD domain and BRD2 Bromo domain. In general, the binding results agreed with literature data with respect to the primary specificity of the reading domains, but in almost all cases we obtained additional new information concerning the influence of secondary modifications surrounding the target modification. Conclusions We conclude that Celluspots peptide arrays are powerful screening tools for studying the specificity of putative reading domains binding to modified histone peptides.

  14. Characterization of upstream sequences of the LIM2 gene that bind developmentally regulated and lens-specific proteins

    Institute of Scientific and Technical Information of China (English)

    HSU Heng; Robert L. CHURCH

    2004-01-01

    During lens development, lens epithelial cells differentiate into fiber cells. To date, four major lens fiber cell intrinsic membrane proteins (MIP) ranging in size from 70 kD to 19 kD have been characterized. The second most abundant lens fiber cell intrinsic membrane protein is MP19. This protein probably is involved with lens cell communication and relates with cataractogenesis. The aim of this research is to characterize upstream sequences of the MP19 (also called LIM2) gene that bind developmentally regulated and lens-specific proteins. We have used the gel mobility assays and corresponding competition experiments to identify and characterize cis elements within approximately 500 bases of LIM2 upstream sequences. Our studies locate the positions of some cis elements, including a "CA" repeat, a methylation Hha I island, an FnuD II site, an Ap1 and an Ap2 consensus sequences, and identify some specific cis elements which relate to lens-specific transcription of LIM2. Our experiments also preliminarily identify trans factors which bind to specific cis elements of the LIM2 promoter and/or regulate transcription of LIM2. We conclude that developmental regulation and coordination of the MP 19 gene in ocular lens fiber cells is controlled by the presence of specific cis elements that bind regulatory trans factors that affect LIM2 gene expression. DNA methylation is one mechanism of controlling LIM2 gene expression during lens development.

  15. Lineage-affiliated transcription factors bind the Gata3 Tce1 enhancer to mediate lineage-specific programs

    Science.gov (United States)

    Ohmura, Sakie; Mizuno, Seiya; Oishi, Hisashi; Ku, Chia-Jui; Hermann, Mary; Hosoya, Tomonori; Takahashi, Satoru; Engel, James Douglas

    2016-01-01

    The transcription factor GATA3 is essential for the genesis and maturation of the T cell lineage, and GATA3 dysregulation has pathological consequences. Previous studies have shown that GATA3 function in T cell development is regulated by multiple signaling pathways and that the Notch nuclear effector, RBP-J, binds specifically to the Gata3 promoter. We previously identified a T cell–specific Gata3 enhancer (Tce1) lying 280 kb downstream from the structural gene and demonstrated in transgenic mice that Tce1 promoted T lymphocyte–specific transcription of reporter genes throughout T cell development; however, it was not clear if Tce1 is required for Gata3 transcription in vivo. Here, we determined that the canonical Gata3 promoter is insufficient for Gata3 transcriptional activation in T cells in vivo, precluding the possibility that promoter binding by a host of previously implicated transcription factors alone is responsible for Gata3 expression in T cells. Instead, we demonstrated that multiple lineage-affiliated transcription factors bind to Tce1 and that this enhancer confers T lymphocyte–specific Gata3 activation in vivo, as targeted deletion of Tce1 in a mouse model abrogated critical functions of this T cell–regulatory element. Together, our data show that Tce1 is both necessary and sufficient for critical aspects of Gata3 T cell–specific transcriptional activity. PMID:26808502

  16. Lineage-affiliated transcription factors bind the Gata3 Tce1 enhancer to mediate lineage-specific programs.

    Science.gov (United States)

    Ohmura, Sakie; Mizuno, Seiya; Oishi, Hisashi; Ku, Chia-Jui; Hermann, Mary; Hosoya, Tomonori; Takahashi, Satoru; Engel, James Douglas

    2016-03-01

    The transcription factor GATA3 is essential for the genesis and maturation of the T cell lineage, and GATA3 dysregulation has pathological consequences. Previous studies have shown that GATA3 function in T cell development is regulated by multiple signaling pathways and that the Notch nuclear effector, RBP-J, binds specifically to the Gata3 promoter. We previously identified a T cell-specific Gata3 enhancer (Tce1) lying 280 kb downstream from the structural gene and demonstrated in transgenic mice that Tce1 promoted T lymphocyte-specific transcription of reporter genes throughout T cell development; however, it was not clear if Tce1 is required for Gata3 transcription in vivo. Here, we determined that the canonical Gata3 promoter is insufficient for Gata3 transcriptional activation in T cells in vivo, precluding the possibility that promoter binding by a host of previously implicated transcription factors alone is responsible for Gata3 expression in T cells. Instead, we demonstrated that multiple lineage-affiliated transcription factors bind to Tce1 and that this enhancer confers T lymphocyte-specific Gata3 activation in vivo, as targeted deletion of Tce1 in a mouse model abrogated critical functions of this T cell-regulatory element. Together, our data show that Tce1 is both necessary and sufficient for critical aspects of Gata3 T cell-specific transcriptional activity.

  17. A specific domain in alpha-catenin mediates binding to beta-catenin or plakoglobin.

    Science.gov (United States)

    Huber, O; Krohn, M; Kemler, R

    1997-08-01

    The E-cadherin-catenin adhesion complex has been the subject of many structural and functional studies because of its importance in development, normal tissue function and carcinogenesis. It is well established that the cytoplasmic domain of E-cadherin binds either beta-catenin or plakoglobin, which both can assemble alpha-catenin into the complex. Recently we have identified an alpha-catenin binding site in beta-catenin and plakoglobin and postulated, based on sequence analysis, that these protein-protein interactions are mediated by a hydrophobic interaction mechanism. Here we have now identified the reciprocal complementary binding site in alpha-catenin which mediates its interaction with beta-catenin and plakoglobin. Using in vitro association assays with C-terminal truncations of alpha-catenin expressed as recombinant fusion proteins, we found that the N-terminal 146 amino acids are required for this interaction. We then identified a peptide of 27 amino acids within this sequence (amino acid positions 117-143) which is necessary and sufficient to bind beta-catenin or plakoglobin. As shown by mutational analysis, hydrophobic amino acids within this binding site are important for the interaction. The results described here, together with our previous work, give strong support for the idea that these proteins associate by hydrophobic interactions of two alpha-helices.

  18. p53CP, a putative p53 competing protein that specifically binds to the consensus p53 DNA binding sites: A third member of the p53 family?

    OpenAIRE

    Bian, Junhui; Sun, Yi

    1997-01-01

    p53 tumor suppressor protein negatively regulates cell growth, mainly through the transactivation of its downstream target genes. As a sequence-specific DNA binding transcription factor, p53 specifically binds to a 20-bp consensus motif 5′-PuPuPuC(A/T) (T/A)GPyPyPyPuPuPuC(A/T)(T/A)GPyPyPy-3′. We have now identified, partially purified, and characterized an additional ≈40-kDa nuclear protein, p53CP (p53 competing protein), that specifically binds to the consensus p53 binding sites found in sev...

  19. Binding of cationic peptides (KX)4K to DPPG bilayers. Increasing the hydrophobicity of the uncharged amino acid X drives formation of membrane bound β-sheets: A DSC and FT-IR study.

    Science.gov (United States)

    Hädicke, André; Blume, Alfred

    2016-06-01

    The binding of cationic peptides of the sequence (KX)4K to lipid vesicles of negatively charged dipalmitoyl-phosphatidylglycerol (DPPG) was investigated by differential scanning calorimetry (DSC) and temperature dependent Fourier-transformed infrared (FT-IR) spectroscopy. The hydrophobicity of the uncharged amino acid X was changed from G (glycine) over A (alanine), Abu (α-aminobutyric acid), V (valine) to L (leucine). The binding of the peptides caused an increase of the phase transition temperature (Tm) of DPPG by up to 20°C. The shift depended on the charge ratio and on the hydrophobicity of the amino acid X. Unexpectedly, the upward shift of Tm increased with increasing hydrophobicity of X. FT-IR spectroscopy showed a shift of the CH2 stretching vibrations of DPPG to lower frequency, particularly for bilayers in the liquid-crystalline phase, indicating an ordering of the hydrocarbon chains when the peptides were bound. Changes in the lipid C=O vibrational band indicated a dehydration of the lipid headgroup region after peptide binding. (KG)4K was bound in an unordered structure at all temperatures. All other peptides formed intermolecular antiparallel β-sheets, when bound to gel phase DPPG. However, for (KA)4K and (KAbu)4K, the β-sheets converted into an unordered structure above Tm. In contrast, the β-sheet structures of (KV)4K and (KL)4K remained stable even at 80°C when bound to the liquid-crystalline phase of DPPG. Strong aggregation of DPPG vesicles occurred after peptide binding. For the aggregates, we suggest a structure, where aggregated single β-sheets are sandwiched between opposing DPPG bilayers with a dehydrated interfacial region.

  20. Specificity of the Cyclic GMP-Binding Activity and of a Cyclic GMP-Dependent Cyclic GMP Phosphodiesterase in Dictyostelium discoideum

    NARCIS (Netherlands)

    Haastert, Peter J.M. van; Walsum, Hans van; Meer, Rob C. van der; Bulgakov, Roman; Konijn, Theo M.

    1982-01-01

    The nucleotide specificity of the cyclic GMP-binding activity in a homogenate of Dictyostelium discoideum was determined by competition of cyclic GMP derivatives with [8-3H] cyclic GMP for the binding sites. The results indicate that cyclic GMP is bound to the binding proteins by hydrogen bonds at N

  1. Glucans synthesized in situ in experimental salivary pellicle function as specific binding sites for Streptococcus mutans.

    Science.gov (United States)

    Schilling, K M; Bowen, W H

    1992-01-01

    Many researchers have suggested that the role of glucan-mediated interactions in the adherence of Streptococcus mutans is restricted to accumulation of this cariogenic bacterium following its sucrose (i.e., glucan)-independent binding to saliva-coated tooth surfaces. However, the presence of enzymatically active glucosyltransferase in salivary pellicle suggests that glucans could also promote the initial adherence of S. mutans to the teeth. In the present study, the commonly used hydroxyapatite adherence assay was modified to include the incorporation of glucosyltransferase and the synthesis of glucans in situ on saliva-coated hydroxyapatite beads. Several laboratory strains and clinical isolates of S. mutans were examined for their ability to adhere to experimental pellicles, either with or without the prior formation of glucans in situ. Results showed that most strains of S. mutans bound stereospecifically to glucans synthesized in pellicle. Inhibition studies with various polysaccharides and fungal dextranase indicated that alpha 1,6-linked glucose residues were of primary importance in the glucan binding observed. Scanning electron microscopic analysis showed direct binding of S. mutans to hydroxyapatite surface-associated polysaccharide and revealed no evidence of trapping or cell-to-cell binding. S. mutans strains also attached to host-derived structures in experimental pellicles, and the data suggest that the bacterial adhesins which recognize salivary binding sites were distinct from glucan-binding adhesins. Furthermore, glucans formed in experimental pellicles appeared to mask the host-derived components. These results support the concept that glucans synthesized in salivary pellicle can promote the selective adherence of the cariogenic streptococci which colonize human teeth. Images PMID:1530843

  2. Specific binding of PapI to Lrp-pap DNA complexes.

    OpenAIRE

    Kaltenbach, L S; Braaten, B A; Low, D A

    1995-01-01

    Expression of pyelonephritis-associated pili (Pap) varies between transcriptionally active (ON) and inactive (OFF) phase states. Pap phase variation is controlled by the binding of leucine-responsive regulatory protein (Lrp) to two pap regulatory DNA regions, each containing a deoxyadenosine methylase site and designated GATC-I and GATC-II. Methylation of these GATC sites modulates binding of Lrp and plays an essential role in phase variation. PapI, an 8.8-kDa pap-encoded regulatory protein, ...

  3. Phage display selection of tight specific binding variants from a hyperthermostable Sso7d scaffold protein library.

    Science.gov (United States)

    Zhao, Ning; Schmitt, Margaret A; Fisk, John D

    2016-04-01

    Antibodies, the quintessential biological recognition molecules, are not ideal for many applications because of their large size, complex modifications, and thermal and chemical instability. Identifying alternative scaffolds that may be evolved into tight, specific binding molecules with improved physical properties is of increasing interest, particularly for biomedical applications in resource-limited environments. Hyperthermophilic organisms, such as Sulfolobus solfataricus, are an attractive source of highly stable proteins that may serve as starting points for alternative molecular recognition scaffolds. We describe the first application of phage display to identify binding proteins based on the S. solfataricus protein Sso7d scaffold. Sso7d is a small cysteine-free DNA-binding protein (approximately 7 kDa, 63 amino acids), with a melting temperature of nearly 100 °C. Tight-binding Sso7d variants were selected for a diverse set of protein targets from a 10(10) member library, demonstrating the versatility of the scaffold. These Sso7d variants are able to discriminate among closely related human, bovine and rabbit serum albumins. Equilibrium dissociation constants in the nanomolar to low micromolar range were measured via competitive ELISA. Importantly, the Sso7d variants continue to bind their targets in the absence of the phage context. Furthermore, phage-displayed Sso7d variants retain their binding affinity after exposure to temperatures up to 70 °C. Taken together, our results suggest that the Sso7d scaffold will be a complementary addition to the range of non-antibody scaffold proteins that may be utilized in phage display. Variants of hyperthermostable binding proteins have potential applications in diagnostics and therapeutics for environments with extreme conditions of storage and deployment.

  4. Isolation and characterisation of a 17-kDa staphylococcal heparin-binding protein with broad specificity.

    Science.gov (United States)

    Fallgren, C; Utt, M; Ljungh, A

    2001-06-01

    A previous study reported the ability of staphylococci to bind heparin and heparin-dependent host growth factors. The present study isolated and identified heparin- and basic fibroblast growth factor (bFGF)-binding surface components of S. epidermidis strain RP12 and S. haemolyticus strain SM 131. The staphylococcal heparin-binding component(s) were purified by affinity chromatography on heparin-Sepharose and a major heparin-binding protein, here designated HBP, was identified by immunoblot in these two coagulase-negative staphylococcal (CNS) species. The HBP was shown to be acidic with an approximate pI of 4.6 and a molecular mass around 17 kDa. The binding of heparin to HBP was inhibited by heparin, fucoidan, pentosan polysulphate and various other sulphated polysaccharides, but not by non-sulphated compounds. However, the purified HBP from both S. epidermidis and S. haemolyticus revealed broad specificity, and also bound bFGF, thrombospondin, von Willebrand factor and, weakly, fibrinogen. The N-terminal sequences of the 17-kDa HBP from S. epidermidis and S. haemolyticus showed only limited identity. Comparison of the first 15 amino acid residues derived from either strain with known sequences in the protein databases revealed no close similarities. Taken together, these results suggest that the adhesion of at least some CNS to host sulphated glycosaminoglycans may be mediated by a previously uncharacterised group of surface proteins. PMID:11393292

  5. A SILAC-based screen for Methyl-CpG binding proteins identifies RBP-J as a DNA methylation and sequence-specific binding protein.

    Directory of Open Access Journals (Sweden)

    Stefanie J J Bartels

    Full Text Available BACKGROUND: DNA methylation is an epigenetic modification that plays a crucial role in a variety of biological processes. Methylated DNA is specifically bound by Methyl-CpG Binding Proteins (MBPs. Three different types of MBPs have been identified so far: the Methyl-CpG Binding Domain (MBD family proteins, three BTB/POZ-Zn-finger proteins, and UHRF1. Most of the known MBPs have been identified via homology with the MBD and Zn-finger domains as present in MeCP2 and Kaiso, respectively. It is conceivable that other proteins are capable of recognizing methylated DNA. METHODOLOGY/PRINCIPAL FINDINGS: For the purpose of identifying novel 'readers' we set up a methyl-CpG pull-down assay combined with stable-isotope labeling by amino acids in cell culture (SILAC. In a methyl-CpG pull-down with U937 nuclear extracts, we recovered several known MBPs and almost all subunits of the MBD2/NuRD complex as methylation specific binders, providing proof-of-principle. Interestingly, RBP-J, the transcription factor downstream of Notch receptors, also bound the DNA in a methylation dependent manner. Follow-up pull-downs and electrophoretic mobility shift assays (EMSAs showed that RBP-J binds methylated DNA in the context of a mutated RBP-J consensus motif. CONCLUSIONS/SIGNIFICANCE: The here described SILAC/methyl-CpG pull-down constitutes a new approach to identify potential novel DNAme readers and will advance unraveling of the complete methyl-DNA interactome.

  6. 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.

  7. Theory on the mechanism of rapid binding of transcription factor proteins at specific-sites on DNA

    CERN Document Server

    Murugan, Rajamanickam

    2014-01-01

    We develop revised theoretical ideas on the mechanism by which the transcription factor proteins locate their specific binding sites on DNA faster than the three-dimensional (3D) diffusion controlled rate limit. We demonstrate that the 3D-diffusion controlled rate limit can be enhanced when the protein molecule reads several possible binding stretches of the template DNA via one-dimensional (1D) diffusion upon each 3D-diffusion mediated collision or nonspecific binding event. The overall enhancement of site-specific association rate is directly proportional to the maximum possible sliding length (LA, square root of (6Do/kr) where Do is the 1D-diffusion coefficient and kr is the dissociation rate constant associated with the nonspecific DNA-protein complex) associated with the 1D-diffusion of protein molecule along DNA. Upon considering several possible mechanisms we find that the DNA binding proteins can efficiently locate their cognate sites on DNA by switching across fast-moving, slow-moving and reading sta...

  8. Intermediates in monensin biosynthesis: A late step in biosynthesis of the polyether ionophore monensin is crucial for the integrity of cation binding

    Directory of Open Access Journals (Sweden)

    Wolfgang Hüttel

    2014-02-01

    Full Text Available Polyether antibiotics such as monensin are biosynthesised via a cascade of directed ring expansions operating on a putative polyepoxide precursor. The resulting structures containing fused cyclic ethers and a lipophilic backbone can form strong ionophoric complexes with certain metal cations. In this work, we demonstrate for monensin biosynthesis that, as well as ether formation, a late-stage hydroxylation step is crucial for the correct formation of the sodium monensin complex. We have investigated the last two steps in monensin biosynthesis, namely hydroxylation catalysed by the P450 monooxygenase MonD and O-methylation catalysed by the methyl-transferase MonE. The corresponding genes were deleted in-frame in a monensin-overproducing strain of Streptomyces cinnamonensis. The mutants produced the expected monensin derivatives in excellent yields (ΔmonD: 1.13 g L−1 dehydroxymonensin; ΔmonE: 0.50 g L−1 demethylmonensin; and double mutant ΔmonDΔmonE: 0.34 g L−1 dehydroxydemethylmonensin. Single crystals were obtained from purified fractions of dehydroxymonensin and demethylmonensin. X-ray structure analysis revealed that the conformation of sodium dimethylmonensin is very similar to that of sodium monensin. In contrast, the coordination of the sodium ion is significantly different in the sodium dehydroxymonensin complex. This shows that the final constitution of the sodium monensin complex requires this tailoring step as well as polyether formation.

  9. Intermediates in monensin biosynthesis: A late step in biosynthesis of the polyether ionophore monensin is crucial for the integrity of cation binding.

    Science.gov (United States)

    Hüttel, Wolfgang; Spencer, Jonathan B; Leadlay, Peter F

    2014-01-01

    Polyether antibiotics such as monensin are biosynthesised via a cascade of directed ring expansions operating on a putative polyepoxide precursor. The resulting structures containing fused cyclic ethers and a lipophilic backbone can form strong ionophoric complexes with certain metal cations. In this work, we demonstrate for monensin biosynthesis that, as well as ether formation, a late-stage hydroxylation step is crucial for the correct formation of the sodium monensin complex. We have investigated the last two steps in monensin biosynthesis, namely hydroxylation catalysed by the P450 monooxygenase MonD and O-methylation catalysed by the methyl-transferase MonE. The corresponding genes were deleted in-frame in a monensin-overproducing strain of Streptomyces cinnamonensis. The mutants produced the expected monensin derivatives in excellent yields (ΔmonD: 1.13 g L(-1) dehydroxymonensin; ΔmonE: 0.50 g L(-1) demethylmonensin; and double mutant ΔmonDΔmonE: 0.34 g L(-1) dehydroxydemethylmonensin). Single crystals were obtained from purified fractions of dehydroxymonensin and demethylmonensin. X-ray structure analysis revealed that the conformation of sodium dimethylmonensin is very similar to that of sodium monensin. In contrast, the coordination of the sodium ion is significantly different in the sodium dehydroxymonensin complex. This shows that the final constitution of the sodium monensin complex requires this tailoring step as well as polyether formation. PMID:24605157

  10. Specificity and kinetics of norovirus binding to magnetic bead- conjugated histo-blood group antigens

    Science.gov (United States)

    Histo-blood group antigens (HBGA) have been identified as candidate receptors for human norovirus (NOR). Type A, type H1, and Lewis histo-blood group antigens (HBGAs) in humans have been identified as major targets for NOR binding. Pig HBGA-conjugated magnetic beads have been utilized as a means ...

  11. Recombinant norovirus-specific scFv inhibit virus-like particle binding to cellular ligands

    Directory of Open Access Journals (Sweden)

    Hardy Michele E

    2008-01-01

    Full Text Available Abstract Background Noroviruses cause epidemic outbreaks of gastrointestinal illness in all age-groups. The rapid onset and ease of person-to-person transmission suggest that inhibitors of the initial steps of virus binding to susceptible cells have value in limiting spread and outbreak persistence. We previously generated a monoclonal antibody (mAb 54.6 that blocks binding of recombinant norovirus-like particles (VLP to Caco-2 intestinal cells and inhibits VLP-mediated hemagglutination. In this study, we engineered the antigen binding domains of mAb 54.6 into a single chain variable fragment (scFv and tested whether these scFv could function as cell binding inhibitors, similar to the parent mAb. Results The scFv54.6 construct was engineered to encode the light (VL and heavy (VH variable domains of mAb 54.6 separated by a flexible peptide linker, and this recombinant protein was expressed in Pichia pastoris. Purified scFv54.6 recognized native VLPs by immunoblot, inhibited VLP-mediated hemagglutination, and blocked VLP binding to H carbohydrate antigen expressed on the surface of a CHO cell line stably transfected to express α 1,2-fucosyltransferase. Conclusion scFv54.6 retained the functional properties of the parent mAb with respect to inhibiting norovirus particle interactions with cells. With further engineering into a form deliverable to the gut mucosa, norovirus neutralizing antibodies represent a prophylactic strategy that would be valuable in outbreak settings.

  12. The Caenorhabditis elegans Kinesin-3 motor UNC-104/KIF1A is degraded upon loss of specific binding to cargo.

    Directory of Open Access Journals (Sweden)

    Jitendra Kumar

    2010-11-01

    Full Text Available UNC-104/KIF1A is a Kinesin-3 motor that transports synaptic vesicles from the cell body towards the synapse by binding to PI(4,5P(2 through its PH domain. The fate of the motor upon reaching the synapse is not known. We found that wild-type UNC-104 is degraded at synaptic regions through the ubiquitin pathway and is not retrogradely transported back to the cell body. As a possible means to regulate the motor, we tested the effect of cargo binding on UNC-104 levels. The unc-104(e1265 allele carries a point mutation (D1497N in the PI(4,5P(2 binding pocket of the PH domain, resulting in greatly reduced preferential binding to PI(4,5P(2in vitro and presence of very few motors on pre-synaptic vesicles in vivo. unc-104(e1265 animals have poor locomotion irrespective of in vivo PI(4,5P(2 levels due to reduced anterograde transport. Moreover, they show highly reduced levels of UNC-104 in vivo. To confirm that loss of cargo binding specificity reduces motor levels, we isolated two intragenic suppressors with compensatory mutations within the PH domain. These show partial restoration of in vitro preferential PI(4,5P(2 binding and presence of more motors on pre-synaptic vesicles in vivo. These animals show improved locomotion dependent on in vivo PI(4,5P(2 levels, increased anterograde transport, and partial restoration of UNC-104 protein levels in vivo. For further proof, we mutated a conserved residue in one suppressor background. The PH domain in this triple mutant lacked in vitro PI(4,5P(2 binding specificity, and the animals again showed locomotory defects and reduced motor levels. All allelic variants show increased UNC-104 levels upon blocking the ubiquitin pathway. These data show that inability to bind cargo can target motors for degradation. In view of the observed degradation of the motor in synaptic regions, this further suggests that UNC-104 may get degraded at synapses upon release of cargo.

  13. Binding and Clustering of Glycosaminoglycans: A Common Property of Mono- and Multivalent Cell-Penetrating Compounds

    OpenAIRE

    Ziegler, André; Seelig, Joachim

    2007-01-01

    Recent observations in cell culture provide evidence that negatively charged glycosaminoglycans (GAGs) at the surface of biological cells bind cationic cell-penetrating compounds (CPCs) and cluster during CPC binding, thereby contributing to their endocytotic uptake. The GAG binding and clustering occur in the low-micromolar concentration range and suggest a tight interaction between GAGs and CPCs, although the relation between binding affinity and specificity of this interaction remains to b...

  14. The intriguing Cyclophilin A-HIV-1 Vpr interaction: prolyl cis/trans isomerisation catalysis and specific binding

    Directory of Open Access Journals (Sweden)

    Henklein Petra

    2010-10-01

    Full Text Available Abstract Background Cyclophilin A (CypA represents a potential target for antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1 replication, although the mechanism through which CypA modulates HIV-1 infectivity still remains unclear. The interaction of HIV-1 viral protein R (Vpr with the human peptidyl prolyl isomerase CypA is known to occur in vitro and in vivo. However, the nature of the interaction of CypA with Pro-35 of N-terminal Vpr has remained undefined. Results Characterization of the interactions of human CypA with N-terminal peptides of HIV-1 Vpr has been achieved using a combination of nuclear magnetic resonace (NMR exchange spectroscopy and surface plasmon resonance spectroscopy (SPR. NMR data at atomic resolution indicate prolyl cis/trans isomerisation of the highly conserved proline residues Pro-5, -10, -14 and -35 of Vpr are catalyzed by human CypA and require only very low concentrations of the isomerase relative to that of the peptide substrates. Of the N-terminal peptides of Vpr only those containing Pro-35 bind to CypA in a biosensor assay. SPR studies of specific N-terminal peptides with decreasing numbers of residues revealed that a seven-residue motif centred at Pro-35 consisting of RHFPRIW, which under membrane-like solution conditions comprises the loop region connecting helix 1 and 2 of Vpr and the two terminal residues of helix 1, is sufficient to maintain strong specific binding. Conclusions Only N-terminal peptides of Vpr containing Pro-35, which appears to be vital for manifold functions of Vpr, bind to CypA in a biosensor assay. This indicates that Pro-35 is essential for a specific CypA-Vpr binding interaction, in contrast to the general prolyl cis/trans isomerisation observed for all proline residues of Vpr, which only involve transient enzyme-substrate interactions. Previously suggested models depicting CypA as a chaperone that plays a role in HIV-1 virulence are

  15. Sequence-specific binding and cleavage of duplex DNA by a radioiodinated, intercalator-linked, triplex-forming oligonucleotide

    Energy Technology Data Exchange (ETDEWEB)

    Orson, Frank M.; McShan, W. Michael; Kinsey, Berma M

    1996-05-01

    Applications of oligodeoxynucleotides to modulate gene expression have been the subject of much recent research. We have sought to develop a method to permanently inactivate a gene, or potentially kill cells containing abnormal genes. In this report, we show that a DNA intercalator conjugated to a triplex-forming oligonucleotide can be labeled with an Auger electron emitting radioisotope, can cleave its duplex DNA target, and can specifically bind the target sequence contained in a total of 10 kilobases of irrelevant DNA.

  16. Sequence-specific binding of a chloroplast pentatricopeptide repeat protein to its native group II intron ligand

    OpenAIRE

    Williams-Carrier, Rosalind; Kroeger, Tiffany; Barkan, Alice

    2008-01-01

    Pentatricopeptide repeat (PPR) proteins are defined by degenerate 35-amino acid repeats that are related to the tetratricopeptide repeat (TPR). Most characterized PPR proteins mediate specific post-transcriptional steps in gene expression in mitochondria or chloroplasts. However, little is known about the structure of PPR proteins or the biochemical mechanisms through which they act. Here we establish features of PPR protein structure and nucleic acid binding activity through in vitro experim...

  17. Sequence-specific binding and cleavage of duplex DNA by a radioiodinated, intercalator-linked, triplex-forming oligonucleotide

    International Nuclear Information System (INIS)

    Applications of oligodeoxynucleotides to modulate gene expression have been the subject of much recent research. We have sought to develop a method to permanently inactivate a gene, or potentially kill cells containing abnormal genes. In this report, we show that a DNA intercalator conjugated to a triplex-forming oligonucleotide can be labeled with an Auger electron emitting radioisotope, can cleave its duplex DNA target, and can specifically bind the target sequence contained in a total of 10 kilobases of irrelevant DNA

  18. Structural Comparison, Substrate Specificity, and Inhibitor Binding of AGPase Small Subunit from Monocot and Dicot: Present Insight and Future Potential

    Directory of Open Access Journals (Sweden)

    Kishore Sarma

    2014-01-01

    Full Text Available ADP-glucose pyrophosphorylase (AGPase is the first rate limiting enzyme of starch biosynthesis pathway and has been exploited as the target for greater starch yield in several plants. The structure-function analysis and substrate binding specificity of AGPase have provided enormous potential for understanding the role of specific amino acid or motifs responsible for allosteric regulation and catalytic mechanisms, which facilitate the engineering of AGPases. We report the three-dimensional structure, substrate, and inhibitor binding specificity of AGPase small subunit from different monocot and dicot crop plants. Both monocot and dicot subunits were found to exploit similar interactions with the substrate and inhibitor molecule as in the case of their closest homologue potato tuber AGPase small subunit. Comparative sequence and structural analysis followed by molecular docking and electrostatic surface potential analysis reveal that rearrangements of secondary structure elements, substrate, and inhibitor binding residues are strongly conserved and follow common folding pattern and orientation within monocot and dicot displaying a similar mode of allosteric regulation and catalytic mechanism. The results from this study along with site-directed mutagenesis complemented by molecular dynamics simulation will shed more light on increasing the starch content of crop plants to ensure the food security worldwide.

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

    Science.gov (United States)

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

    2002-11-01

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

  20. Ligand-specific conformational changes in the alpha1 glycine receptor ligand-binding domain

    DEFF Research Database (Denmark)

    Pless, Stephan Alexander; Lynch, Joseph W

    2009-01-01

    indicate that channel opening is accompanied by conformational rearrangements in both beta-sheets. In an attempt to resolve ligand-dependent movements in the ligand-binding domain, we employed voltage-clamp fluorometry on alpha1 glycine receptors to compare changes mediated by the agonist, glycine...... in the inner beta-sheet and pre-M1 domain that may be important for activation, desensitization, or both. In contrast, most labeled residues in loops C and F yielded fluorescence changes identical in magnitude for glycine and strychnine. A notable exception was H201C in loop C. This labeled residue responded...... differently to glycine and strychnine, thus underlining the importance of loop C in ligand discrimination. These results provide an important step toward mapping the domains crucial for ligand discrimination in the ligand-binding domain of glycine receptors and possibly other Cys loop receptors....

  1. Biological activities of binding site specific monoclonal antibodies to prolactin receptors of rabbit mammary gland

    International Nuclear Information System (INIS)

    The biological activity of three monoclonal antibodies (mAbs) against the rabbit mammary prolactin (PRL) receptor (M110, A82, and A917) were investigated using explants of rabbit mammary gland. The three mAbs which were all able to inhibit the binding of 125I-ovine prolactin to its receptor had different biological activities. Two mAbs (M110 and A82) were able to prevent the stimulating effect of PRL on casein synthesis when the molar ratio between the mAb and PRL was 100. One mAb (A917) was able to mimic the action of PRL on both casein and DNA ([3H]thymidine incorporation) synthesis, whereas the other two mAbs were without any stimulatory effect. For this stimulatory effect to be observed, bivalency of the antibody was essential, since monovalent fragments, which were able to inhibit PRL binding, had no agonistic activity. The ability of the mAbs to induce a down-regulation of receptors was also studied. These studies suggest that the binding domain of the receptor might be relatively complex, since only a part of this domain recognized by the antibody with PRL-like activity was able to induce hormonal action. Alternatively, only those antibodies able to microaggregate the receptors may possess PRL-like activity

  2. Analysis of the PDZ binding specificities of Influenza A Virus NS1 proteins

    Directory of Open Access Journals (Sweden)

    Nagasaka Kazunori

    2011-01-01

    Full Text Available Abstract The Influenza A virus non-structural protein 1 (NS1 is a multifunctional virulence factor with several protein-protein interaction domains, involved in preventing apoptosis of the infected cell and in evading the interferon response. In addition, the majority of influenza A virus NS1 proteins have a class I PDZ-binding motif at the C-terminus, and this itself has been shown to be a virulence determinant. In the majority of human influenza NS1 proteins the consensus motif is RSxV: in avian NS1 it is ESxV. Of the few human strains that have the avian motif, all were from very high mortality outbreaks of the disease. Previous work has shown that minor differences in PDZ-binding motifs can have major effects on the spectrum of cellular proteins targeted. In this study we analyse the effect of these differences upon the binding of Influenza A virus NS1 protein to a range of cellular proteins involved in polarity and signal transduction.

  3. Binding of Folic Acid Induces Specific Self-Aggregation of Lactoferrin: Thermodynamic Characterization.

    Science.gov (United States)

    Tavares, Guilherme M; Croguennec, Thomas; Lê, Sébastien; Lerideau, Olivia; Hamon, Pascaline; Carvalho, Antônio F; Bouhallab, Saïd

    2015-11-17

    In the study presented here, we investigated the interaction at pH 5.5 between folic acid (FA) and lactoferrin (LF), a positively charged protein. We found a binding constant Ka of 10(5) M(-1) and a high stoichiometry of 10 mol of FA/mol of LF. The size and charge of the complexes formed evolved during titration experiments. Increasing the ionic strength to 50 mM completely abolished the isothermal titration calorimetry (ITC) signal, suggesting the predominance of electrostatic interactions in the exothermic binding obtained. We developed a theoretical model that explains the complex triphasic ITC profile. Our results revealed a two-step mechanism: FA/LF interaction followed by self-association of the complexes thus formed. We suggest that 10 FA molecules bind to LF to form saturated reactive complexes (FA10/LF) that further self-associate into aggregates with a finite size of around 15 nm. There is thus a critical saturation degree of the protein, above which the self-association can take place. We present here the first results that provide comprehensive details of the thermodynamics of FA/LF complexation-association. Given the high stoichiometry, allowing a load of 55 mg of FA/g of LF, we suggest that FA/LF aggregates would be an effective vehicle for FA in fortified drinks. PMID:26488446

  4. Structure elucidation and DNA binding specificity of natural compounds from Cassia siamea leaves: A biophysical approach.

    Science.gov (United States)

    Parveen, Mehtab; Ahmad, Faheem; Malla, Ali Mohammed; Khan, Mohd Sohrab; Rehman, Sayeed Ur; Tabish, Mohammad; Silva, Manuela Ramos; Silva, P S Pereira

    2016-06-01

    A novel isoflavone, 5,6,7-trimethoxy-3-(3',4',5'-trimethoxyphenyl)-4H-chromen-4-one (1) along with a known pyranocoumarin, Seselin (2) have been isolated from the ethanolic extract of the leaves of Cassia siamea (Family: Fabaceae). Compound 1 has been reported for the first time from any natural source and has not been synthesized so far. Their structures were elucidated on the basis of chemical and physical evidences viz. elemental analysis, UV, FT-IR, (1)H-NMR, (13)C-NMR and mass spectral analysis. Structure of compound (1) was further authenticated by single-crystal X-ray analysis and density functional theory (DFT) calculations. A multi-technique approach employing UV-Visible spectroscopy, fluorescence, KI quenching studies, competitive displacement assay, circular dichroism and viscosity studies have been utilized to probe the extent of interaction and possible binding modes of isolated compounds (1-2) with calf thymus DNA (CT-DNA). Both the compounds were found to interact with DNA via non-intercalative binding mode with moderate proficiencies. Groove binding was the major interaction mode in the case of compound 2 while compound 1 probably interacts with DNA through electrostatic interactions. These studies provide deeper insight in understanding of DNA-drug (natural products) interaction which could be helpful to improve their bioavailability for therapeutic purposes. PMID:27085054

  5. Structure and DNA-binding of meiosis-specific protein Hop2

    Science.gov (United States)

    Zhou, Donghua; Moktan, Hem; Pezza, Roberto

    2014-03-01

    Here we report structure elucidation of the DNA binding domain of homologous pairing protein 2 (Hop2), which is important to gene diversity when sperms and eggs are produced. Together with another protein Mnd1, Hop2 enhances the strand invasion activity of recombinase Dmc1 by over 30 times, facilitating proper synapsis of homologous chromosomes. However, the structural and biochemical bases for the function of Hop2 and Mnd1 have not been well understood. As a first step toward such understanding, we recently solved the structure for the N-terminus of Hop2 (1-84) using solution NMR. This fragment shows a typical winged-head conformation with recognized DNA binding activity. DNA interacting sites were then investigated by chemical shift perturbations in a titration experiment. Information of these sites was used to guide protein-DNA docking with MD simulation, revealing that helix 3 is stably lodged in the DNA major groove and that wing 1 (connecting strands 2 and 3) transiently comes in contact with the minor groove in nanosecond time scale. Mutagenesis analysis further confirmed the DNA binding sites in this fragment of the protein.

  6. Detection and identification of tissue specific lectins of the tsetse fly, Glossina tachinoides: Midgut lectin activity with lipopolysaccharide binding specificity

    International Nuclear Information System (INIS)

    Lectin that agglutinates human and animal red blood cells (RBCs) was demonstrated in midgut extracts of Glossina tachinoides. The highest haemagglutination titres were against pig and rabbit RBCs. Treatment of rabbit RBCs with pronase, trypsin, neuraminidase, bromelain, glutaraldehyde and periodate reduced the agglutination titres. The lectin is specific for amino, methyl and deoxy derivates of glucose, amino and methyl derivates of mannose, D-galactosamine, N-acetylneuraminic acid and trehalose. In addition, very high reactivity against the lipopolysaccharide of E. coli K 235 was found. Lectin is secreted to the midgut lumen. It consists of a 27 kilodalton protein component that is not glycosylated. Sandwich ELISA permits quantification of lectin in tissue samples. (author). 15 refs, 3 figs, 1 tab

  7. The role of antigen specificity in the binding of murine monoclonal anti-DNA antibodies to microparticles from apoptotic cells.

    Science.gov (United States)

    Ullal, Anirudh J; Marion, Tony N; Pisetsky, David S

    2014-10-01

    Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus and markers of underlying immune system disturbances. These antibodies bind to both single-stranded and double-stranded DNA, mediating pathogenesis by forming immune complexes. As shown recently, DNA in blood exists in both free and particulate forms, with DNA representing an important component of microparticles. Microparticles are membrane-bound vesicles containing nuclear molecules, released by membrane blebbing during cell death and activation. A panel of monoclonal NZB/NZW F1 anti-DNA antibodies was tested for binding to microparticles generated from apoptotic THP-1 and Jurkat cells. These studies showed that only certain anti-DNA antibodies in the panel, specific for double-stranded DNA, bound to microparticles. Binding to particles was reduced by soluble DNA or DNase treatment. Together, these results indicate that particle binding is a feature of only certain anti-DNA antibodies, reflecting immunochemical properties of the antibodies and the nature of the exposed DNA antigens.

  8. Sequence-specific DNA binding of individual cut repeats of the human CCAAT displacement/cut homeodomain protein.

    Science.gov (United States)

    Aufiero, B; Neufeld, E J; Orkin, S H

    1994-08-01

    CCAAT displacement protein (CDP), a nuclear protein of 180-190 kDa, contains a triplicated motif, the cut domain, similar (80-90% conserved) to three repeats of 60-65 amino acids first identified in Drosophila cut, a homeo-domain protein involved in cell-fate decisions in development. Cut repeats bind DNA and exhibit subtle differences in target-site recognition. DNA sequences specifically bound by cut repeats were isolated by PCR-mediated DNA target-site selection. Sequences selected for cut repeat 2 and 3 (CR2 and CR3) binding are A+T-rich and favor an ATA motif with similar, but not identical, flanking base preferences. CR2 and CR3 discriminate among similar target sequences. CR1, which is more divergent from CR2 and CR3, displays the most restricted pattern of DNA sequence recognition. Methylation interference analysis demonstrates different protein-DNA contacts for CR1 and CR3 binding to a target sequence. Thus, CDP/cut is a complex protein whose DNA-binding properties reflect the combinatorial interaction of four domains (three cut repeats and one homeodomain) with target DNA sequences. PMID:7914370

  9. The peptide-binding specificity of HLA-A*3001 demonstrates membership of the HLA-A3 supertype

    DEFF Research Database (Denmark)

    Lamberth, K; Røder, G; Harndahl, M;

    2008-01-01

    Human leukocyte antigen class I (HLA-I) molecules are highly polymorphic peptide receptors, which select and present endogenously derived peptide epitopes to CD8+ cytotoxic T cells (CTL). The specificity of the HLA-I system is an important component of the overall specificity of the CTL immune...... system. Unfortunately, the large and rapidly increasing number of known HLA-I molecules seriously complicates a comprehensive analysis of the specificities of the entire HLA-I system (as of June 2008, the international HLA registry holds >1,650 unique HLA-I protein entries). In an attempt to reduce this...... complexity, it has been suggested to cluster the different HLA-I molecules into "supertypes" of largely overlapping peptide-binding specificities. Obviously, the HLA supertype concept is only valuable if membership can be assigned with reasonable accuracy. The supertype assignment of HLA-A*3001, a common HLA...

  10. Structure-specific tRNA-binding protein from the extreme thermophile Aquifex aeolicus.

    OpenAIRE

    Morales, A. J; Swairjo, M A; Schimmel, P

    1999-01-01

    The genome of the bacterium Aquifex aeolicus encodes a polypeptide which is related to a small portion of a sequence found in one prokaryotic and two eukaryotic tRNA synthetases. It also is related to a portion of Arc1p, a tRNA-binding protein believed to be important for nuclear trafficking of tRNAs. Here we cloned, expressed and purified the 111 amino acid polypeptide (designated Trbp111) and showed by ultracentrifugation analysis that it is a stable dimer in solution. The protein was also ...

  11. DNA binding by the plant-specific NAC transcription factors in crystal and solution

    DEFF Research Database (Denmark)

    Welner, Ditte Hededam; Lindemose, Søren; Grossmann, J. Günter;

    2012-01-01

    NAC (NAM/ATAF/CUC) plant transcription factors regulate essential processes in development, stress responses and nutrient distribution in important crop and model plants (rice, Populus, Arabidopsis), which makes them highly relevant in the context of crop optimization and bioenergy production...... transcription factors and the mammalian GCM (Glial cell missing) transcription factors, which all use a ß-strand motif for DNA-binding. The structure shows that the NAC domain inserts the edge of its core ß-sheet into the major groove, while leaving the DNA largely undistorted. The structure of the NAC...

  12. Protein-specific force field derived from the fragment molecular orbital method can improve protein-ligand binding interactions.

    Science.gov (United States)

    Chang, Le; Ishikawa, Takeshi; Kuwata, Kazuo; Takada, Shoji

    2013-05-30

    Accurate computational estimate of the protein-ligand binding affinity is of central importance in rational drug design. To improve accuracy of the molecular mechanics (MM) force field (FF) for protein-ligand simulations, we use a protein-specific FF derived by the fragment molecular orbital (FMO) method and by the restrained electrostatic potential (RESP) method. Applying this FMO-RESP method to two proteins, dodecin, and lysozyme, we found that protein-specific partial charges tend to differ more significantly from the standard AMBER charges for isolated charged atoms. We did not see the dependence of partial charges on the secondary structure. Computing the binding affinities of dodecin with five ligands by MM PBSA protocol with the FMO-RESP charge set as well as with the standard AMBER charges, we found that the former gives better correlation with experimental affinities than the latter. While, for lysozyme with five ligands, both charge sets gave similar and relatively accurate estimates of binding affinities. PMID:23420697

  13. Transcription of the human beta enolase gene (ENO-3) is regulated by an intronic muscle-specific enhancer that binds myocyte-specific enhancer factor 2 proteins and ubiquitous G-rich-box binding factors.

    Science.gov (United States)

    Feo, S; Antona, V; Barbieri, G; Passantino, R; Calì, L; Giallongo, A

    1995-01-01

    To provide evidence for the cis-regulatory DNA sequences and trans-acting factors involved in the complex pattern of tissue- and stage-specific expression of the beta enolase gene, constructs containing fragments of the gene fused to the chloramphenicol acetyltransferase gene were used in transient-transfection assays of C2C12 myogenic cells. Deletion analysis revealed the presence of four major regions: two negative regions in the 5'-flanking sequence, a basal promoter region which directs expression at low levels in proliferating and differentiated muscle cells, and a positive region within the first intron that confers cell-type-specific and differentiation-induced expression. This positive regulatory element is located in the 3'-proximal portion of the first intron (nucleotides +504 to +637) and acts as an enhancer irrespective of orientation and position from the homologous beta enolase promoter or the heterologous thymidine kinase promoter, conferring in both cases muscle-specific expression to the linked reporter gene. Deletion of a putative myocyte-specific enhancer factor 1 (MEF-1) binding site, containing a canonical E-box motif, had no effects on muscle-specific transcription, indicating that this site is not required for the activity of the enhancer. Gel mobility shift assays, competition analysis, DNase I footprinting, and mutagenesis studies indicated that this element interacts through an A/T-rich box with a MEF-2 protein(s) and through a G-rich box with a novel ubiquitous factor(s). Mutation of either the G-rich box or the A/T-rich box resulted in a significantly reduced activity of the enhancer in transient-transfection assays. These data indicate that MEF-2 and G-rich-box binding factors are each necessary for tissue-specific expression of the beta enolase gene in skeletal muscle cells. PMID:7565752

  14. Measuring specific receptor binding of a PET radioligand in human brain without pharmacological blockade: The genomic plot.

    Science.gov (United States)

    Veronese, Mattia; Zanotti-Fregonara, Paolo; Rizzo, Gaia; Bertoldo, Alessandra; Innis, Robert B; Turkheimer, Federico E

    2016-04-15

    PET studies allow in vivo imaging of the density of brain receptor species. The PET signal, however, is the sum of the fraction of radioligand that is specifically bound to the target receptor and the non-displaceable fraction (i.e. the non-specifically bound radioligand plus the free ligand in tissue). Therefore, measuring the non-displaceable fraction, which is generally assumed to be constant across the brain, is a necessary step to obtain regional estimates of the specific fractions. The nondisplaceable binding can be directly measured if a reference region, i.e. a region devoid of any specific binding, is available. Many receptors are however widely expressed across the brain, and a true reference region is rarely available. In these cases, the nonspecific binding can be obtained after competitive pharmacological blockade, which is often contraindicated in humans. In this work we introduce the genomic plot for estimating the nondisplaceable fraction using baseline scans only. The genomic plot is a transformation of the Lassen graphical method in which the brain maps of mRNA transcripts of the target receptor obtained from the Allen brain atlas are used as a surrogate measure of the specific binding. Thus, the genomic plot allows the calculation of the specific and nondisplaceable components of radioligand uptake without the need of pharmacological blockade. We first assessed the statistical properties of the method with computer simulations. Then we sought ground-truth validation using human PET datasets of seven different neuroreceptor radioligands, where nonspecific fractions were either obtained separately using drug displacement or available from a true reference region. The population nondisplaceable fractions estimated by the genomic plot were very close to those measured by actual human blocking studies (mean relative difference between 2% and 7%). However, these estimates were valid only when mRNA expressions were predictive of protein levels (i

  15. Measuring specific receptor binding of a PET radioligand in human brain without pharmacological blockade: The genomic plot

    Science.gov (United States)

    Veronese, Mattia; Zanotti-Fregonara, Paolo; Rizzo, Gaia; Bertoldo, Alessandra; Innis, Robert B.; Turkheimer, Federico E.

    2016-01-01

    PET studies allow in vivo imaging of the density of brain receptor species. The PET signal, however, is the sum of the fraction of radioligand that is specifically bound to the target receptor and the non-displaceable fraction (i.e. the non-specifically bound radioligand plus the free ligand in tissue). Therefore, measuring the non-displaceable fraction, which is generally assumed to be constant across the brain, is a necessary step to obtain regional estimates of the specific fractions. The nondisplaceable binding can be directly measured if a reference region, i.e. a region devoid of any specific binding, is available. Many receptors are however widely expressed across the brain, and a true reference region is rarely available. In these cases, the nonspecific binding can be obtained after competitive pharmacological blockade, which is often contraindicated in humans. In this work we introduce the genomic plot for estimating the nondisplaceable fraction using baseline scans only. The genomic plot is a transformation of the Lassen graphical method in which the brain maps of mRNA transcripts of the target receptor obtained from the Allen brain atlas are used as a surrogate measure of the specific binding. Thus, the genomic plot allows the calculation of the specific and nondisplaceable components of radioligand uptake without the need of pharmacological blockade. We first assessed the statistical properties of the method with computer simulations. Then we sought ground-truth validation using human PET datasets of seven different neuroreceptor radioligands, where nonspecific fractions were either obtained separately using drug displacement or available from a true reference region. The population nondisplaceable fractions estimated by the genomic plot were very close to those measured by actual human blocking studies (mean relative difference between 2% and 7%). However, these estimates were valid only when mRNA expressions were predictive of protein levels (i

  16. Allele-specific transcription factor binding to common and rare variants associated with disease and gene expression.

    Science.gov (United States)

    Cavalli, Marco; Pan, Gang; Nord, Helena; Wallerman, Ola; Wallén Arzt, Emelie; Berggren, Olof; Elvers, Ingegerd; Eloranta, Maija-Leena; Rönnblom, Lars; Lindblad Toh, Kerstin; Wadelius, Claes

    2016-05-01

    Genome-wide association studies (GWAS) have identified a large number of disease-associated SNPs, but in few cases the functional variant and the gene it controls have been identified. To systematically identify candidate regulatory variants, we sequenced ENCODE cell lines and used public ChIP-seq data to look for transcription factors binding preferentially to one allele. We found 9962 candidate regulatory SNPs, of which 16 % were rare and showed evidence of larger functional effect than common ones. Functionally rare variants may explain divergent GWAS results between populations and are candidates for a partial explanation of the missing heritability. The majority of allele-specific variants (96 %) were specific to a cell type. Furthermore, by examining GWAS loci we found >400 allele-specific candidate SNPs, 141 of which were highly relevant in our cell types. Functionally validated SNPs support identification of an SNP in SYNGR1 which may expose to the risk of rheumatoid arthritis and primary biliary cirrhosis, as well as an SNP in the last intron of COG6 exposing to the risk of psoriasis. We propose that by repeating the ChIP-seq experiments of 20 selected transcription factors in three to ten people, the most common polymorphisms can be interrogated for allele-specific binding. Our strategy may help to remove the current bottleneck in functional annotation of the genome. PMID:26993500

  17. Binding properties of a mannose-specific lectin from the snowdrop (Galanthus nivalis) bulb.

    Science.gov (United States)

    Shibuya, N; Goldstein, I J; Van Damme, E J; Peumans, W J

    1988-01-15

    Carbohydrate binding properties of a new plant lectin (GNA) isolated from snowdrop bulbs were studied using the technique of quantitative precipitation, hapten inhibition, and affinity chromatography on immobilized lectin. Purified GNA precipitated highly branched yeast mannans but did not react with most glucans. Hapten inhibition experiments showed that D-mannose is an inhibitor of GNA-mannan interaction but neither N-acetyl-D-mannosamine nor D-glucose is an inhibitor. Hapten inhibition with various sugars showed that GNA requires the presence of equatorial hydroxyl groups at the C-3 and C-4 positions and an axial group at the C-2 position of the D-pyranose ring. A nonreducing terminal D-mannose residue is necessary for the interaction of oligosaccharides, and oligosaccharides with terminal Man(alpha-1-3)Man units showed the highest inhibitory potency (10-30 times greater than D-mannose) among the manno-oligosaccharides tested. The presence of the hydrophobic p-nitrophenyl aglycone increased the affinity of D-mannose only slightly. Immobilized GNA bound yeast mannan but did not bind glycogen. The behavior of glycoproteins with high mannose type glycan chains depended on the density and the structure of their glycan chains. Glycopeptides which carry Man(alpha 1-3)Man units were retarded on the immobilized GNA column whereas those lacking this unit or with hybrid type glycan chains were not retarded on the column.

  18. Mutagen-specific mutation signature determines global microRNA binding.

    Directory of Open Access Journals (Sweden)

    Eyal Greenberg

    Full Text Available Micro-RNAs (miRNAs are small non-coding RNAs that regulate gene products at the post-transcriptional level. It is thought that loss of cell regulation by miRNAs supports cancer development. Based on whole genome sequencing of a melanoma tumor, we predict, using three different computational algorithms, that the melanoma somatic mutations globally reduce binding of miRNAs to the mutated 3'UTRs. This phenomenon reflects the nature of the characteristic UV-induced mutation, C-to-T. Furthermore, we show that seed regions are enriched with Guanine, thus rendering miRNAs prone to reduced binding to UV-mutated 3'UTRs. Accordingly, mutation patterns in non UV-induced malignancies e.g. lung cancer and leukemia do not yield similar predictions. It is suggested that UV-induced disruption of miRNA-mediated gene regulation plays a carcinogenic role. Remarkably, dark-skinned populations have significantly higher GC content in 3'UTR SNPs than light-skinned populations, which implies on evolutionary pressure to preserve regulation by trans-acting oligonucleotides under conditions with excess UV radiation.

  19. MetaMHCpan, A Meta Approach for Pan-Specific MHC Peptide Binding Prediction.

    Science.gov (United States)

    Xu, Yichang; Luo, Cheng; Mamitsuka, Hiroshi; Zhu, Shanfeng

    2016-01-01

    Recent computational approaches in bioinformatics can achieve high performance, by which they can be a powerful support for performing real biological experiments, making biologists pay more attention to bioinformatics than before. In immunology, predicting peptides which can bind to MHC alleles is an important task, being tackled by many computational approaches. However, this situation causes a serious problem for immunologists to select the appropriate method to be used in bioinformatics. To overcome this problem, we develop an ensemble prediction-based Web server, which we call MetaMHCpan, consisting of two parts: MetaMHCIpan and MetaMHCIIpan, for predicting peptides which can bind MHC-I and MHC-II, respectively. MetaMHCIpan and MetaMHCIIpan use two (MHC2SKpan and LApan) and four (TEPITOPEpan, MHC2SKpan, LApan, and MHC2MIL) existing predictors, respectively. MetaMHCpan is available at http://datamining-iip.fudan.edu.cn/MetaMHCpan/index.php/pages/view/info . PMID:27076335

  20. Sequence-specific, nanomolar peptide binding via cucurbit[8]uril-induced folding and inclusion of neighboring side chains.

    Science.gov (United States)

    Smith, Lauren C; Leach, David G; Blaylock, Brittney E; Ali, Omar A; Urbach, Adam R

    2015-03-18

    This paper describes the molecular recognition of the tripeptide Tyr-Leu-Ala by the synthetic receptor cucurbit[8]uril (Q8) in aqueous buffer with nanomolar affinity and exceptional specificity. This combination of characteristics, which also applies to antibodies, is desirable for applications in biochemistry and biotechnology but has eluded supramolecular chemists for decades. Building on prior knowledge that Q8 binds to peptides with N-terminal aromatic residues, a library screen of 105 peptides was designed to test the effects of residues adjacent to N-terminal Trp, Phe, or Tyr. The screen used tetramethylbenzobis(imidazolium) (MBBI) as a fluorescent indicator and resulted in the unexpected discovery that MBBI can serve not only as a turn-off sensor via the simultaneous inclusion of a Trp residue but also as a turn-on sensor via the competitive displacement of MBBI upon binding of Phe- or Tyr-terminated peptides. The unusual fluorescence response of the Tyr series prompted further investigation by (1)H NMR spectroscopy, electrospray ionization mass spectrometry, and isothermal titration calorimetry. From these studies, a novel binding motif was discovered in which only 1 equiv of peptide binds to Q8, and the side chains of both the N-terminal Tyr residue and its immediate neighbor bind within the Q8 cavity. For the peptide Tyr-Leu-Ala, the equilibrium dissociation constant value is 7.2 nM, whereas that of its sequence isomer Tyr-Ala-Leu is 34 μM. The high stability, recyclability, and low cost of Q8 combined with the straightforward incorporation of Tyr-Leu-Ala into recombinant proteins should make this system attractive for the development of biological applications.

  1. Kinetic study of the effects of calcium ions on cationic artichoke (Cynara scolymus L.) peroxidase: calcium binding, steady-state kinetics and reactions with hydrogen peroxide.

    Science.gov (United States)

    Hiner, Alexander N P; Sidrach, Lara; Chazarra, Soledad; Varón, Ramón; Tudela, José; García-Cánovas, Francisco; Rodríguez-López, José Neptuno

    2004-01-01

    The apparent catalytic constant (k(cat)) of artichoke (Cynara scolymus L.) peroxidase (AKPC) with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) increased 130-fold in the presence of calcium ions (Ca2+) but the affinity (K(m)) of the enzyme for ABTS was 500 times lower than for Ca2+-free AKPC. AKPC is known to exhibit an equilibrium between 6-aquo hexa-coordinate and penta-coordinate forms of the haem iron that is modulated by Ca2+ and affects compound I formation. Measurements of the Ca2+ dissociation constant (K(D)) were complicated by the water-association/dissociation equilibrium yielding a global value more than 1000 times too high. The value for the Ca2+ binding step alone has now been determined to be K(D) approximately 10 nM. AKPC-Ca2+ was more resistant to inactivation by hydrogen peroxide (H(2)O(2)) and exhibited increased catalase activity. An analysis of the complex H(2)O(2) concentration dependent kinetics of Ca2+-free AKPC is presented. PMID:15556277

  2. Uncovering the Peptide-Binding Specificities of HLA-C: A General Strategy To Determine the Specificity of Any MHC Class I Molecule

    DEFF Research Database (Denmark)

    Rasmussen, Michael; Harndahl, Mikkel; Stryhn, Anette;

    2014-01-01

    library approach with a peptide-HLA-I dissociation assay, in this study we present a general strategy to determine the peptide-binding specificity of any MHC class I molecule. We applied this novel strategy to 17 of the most common HLA-C molecules, and for 16 of these we successfully generated matrices...... representing their peptide-binding motifs. The motifs prominently shared a conserved C-terminal primary anchor with hydrophobic amino acid residues, as well as one or more diverse primary and auxiliary anchors at P1, P2, P3, and/or P7. Matrices were used to generate a large panel of HLA-C-specific peptide...... molecules. Assessing the functional significance of these new tools, HLA-C*07:01 transgenic mice were immunized with stable HLA-C*07:01 binders; six of six tested stable peptide binders were immunogenic. Finally, we generated HLA-C tetramers and labeled human CD8(+) T cells and NK cells. These new resources...

  3. Lectin Domains of Polypeptide GalNAc Transferases Exhibit Glycopeptide Binding Specificity

    DEFF Research Database (Denmark)

    Pedersen, Johannes W; Bennett, Eric P; Schjoldager, Katrine T-B G;

    2011-01-01

    UDP-GalNAc:polypeptide a-N-acetylgalactosaminyltransferases (GalNAc-Ts) constitute a family of up to 20 transferases that initiate mucin-type O-glycosylation. The transferases are structurally composed of catalytic and lectin domains. Two modes have been identified for the selection...... of glycosylation sites by GalNAc-Ts: confined sequence recognition by the catalytic domain alone, and concerted recognition of acceptor sites and adjacent GalNAc-glycosylated sites by the catalytic and lectin domains, respectively. Thus far, only the catalytic domain has been shown to have peptide sequence...... on sequences of mucins MUC1, MUC2, MUC4, MUC5AC, MUC6, and MUC7 as well as a random glycopeptide bead library, we examined the binding properties of four different lectin domains. The lectin domains of GalNAc-T1, -T2, -T3, and -T4 bound different subsets of small glycopeptides. These results indicate...

  4. Energy Landscape Topography Reveals the Underlying Link Between Binding Specificity and Activity of Enzymes

    Science.gov (United States)

    Chu, Wen-Ting; Wang, Jin

    2016-06-01

    Enzyme activity (often quantified by kcat/Km) is the main function of enzyme when it is active against the specific substrate. Higher or lower activities are highly desired for the design of novel enzyme and drug resistance. However, it is difficult to measure the activities of all possible variants and find the “hot-spot” within the limit of experimental time. In this study, we explore the underlying energy landscape of enzyme-substrate interactions and introduce the intrinsic specificity ratio (ISR), which reflects the landscape topography. By studying two concrete systems, we uncover the statistical correlation between the intrinsic specificity and the enzyme activity kcat/Km. This physics-based concept and method show that the energy landscape topography is valuable for understanding the relationship between enzyme specificity and activity. In addition, it can reveal the underlying mechanism of enzyme-substrate actions and has potential applications on enzyme design.

  5. Cationic lipids and cationic ligands induce DNA helix denaturation: detection of single stranded regions by KMnO4 probing.

    Science.gov (United States)

    Prasad, T K; Gopal, Vijaya; Rao, N Madhusudhana

    2003-09-25

    Cationic lipids and cationic polymers are widely used in gene delivery. Using 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) as a cationic lipid, we have investigated the stability of the DNA in DOTAP:DNA complexes by probing with potassium permanganate (KMnO4). Interestingly, thymidines followed by a purine showed higher susceptibility to cationic ligand-mediated melting. Similar studies performed with other water-soluble cationic ligands such as polylysine, protamine sulfate and polyethyleneimine also demonstrated melting of the DNA but with variations. Small cations such as spermine and spermidine and a cationic detergent, cetyl trimethylammonium bromide, also rendered the DNA susceptible to modification by KMnO4. The data presented here provide direct proof for melting of DNA upon interaction with cationic lipids. Structural changes subsequent to binding of cationic lipids/ligands to DNA may lead to instability and formation of DNA bubbles in double-stranded DNA.

  6. DNA-binding preferences of bisantrene analogues: relevance to the sequence specificity of drug-mediated topoisomerase II poisoning.

    Science.gov (United States)

    Sissi, C; Bolgan, L; Moro, S; Zagotto, G; Bailly, C; Menta, E; Capranico, G; Palumbo, M

    1998-12-01

    To elucidate structure-activity relationships for drugs that are able to poison or inhibit topoisomerase II, we investigated the thermodynamics and stereochemistry of the DNA binding of a number of anthracene derivatives bearing one or two 4, 5-dihydro-1H-imidazol-2-yl-hydrazone side chains (characteristic of bisantrene) at different positions of the planar aromatic system. An aza-bioisostere, which can be considered a bisantrene-amsacrine hybrid, was also tested. The affinity for nucleic acids in different sequence contexts was evaluated by spectroscopic techniques, using various experimental conditions. DNA-melting and DNase I footprinting experiments were also performed. The location and number of the otherwise identical side chains dramatically affected the affinity of the test compounds for the nucleic acid. In addition, the new compounds exhibited different DNA sequence preferences, depending on the locations of the dihydroimidazolyl-hydrazone groups, which indicates a major role for the side-chain position in generating specific contacts with the nucleic acid. Molecular modeling studies of the intercalative binding of the 1- or 9-substituted isomers to DNA fully supported the experimental data, because a substantially more favorable recognition of A-T steps, compared with G-C steps, was found for the 9-substituted derivative, whereas a much closer energy balance was found for the 1-substituted isomer. These results compare well with the alteration of base specificity found for the topoisomerase II-mediated DNA cleavage stimulated by the isomeric drugs. Therefore, DNA-binding specificity appears to represent an important determinant for the recognition of the topoisomerase-DNA cleavable complex by the drug, at least for poisons belonging to the amsacrine-bisantrene family. PMID:9855632

  7. Protein-DNA binding specificity: a grid-enabled computational approach applied to single and multiple protein assemblies.

    Science.gov (United States)

    Zakrzewska, Krystyna; Bouvier, Benjamin; Michon, Alexis; Blanchet, Christophe; Lavery, Richard

    2009-12-01

    We use a physics-based approach termed ADAPT to analyse the sequence-specific interactions of three proteins which bind to DNA on the side of the minor groove. The analysis is able to estimate the binding energy for all potential sequences, overcoming the combinatorial problem via a divide-and-conquer approach which breaks the protein-DNA interface down into a series of overlapping oligomeric fragments. All possible base sequences are studied for each fragment. Energy minimisation with an all-atom representation and a conventional force field allows for conformational adaptation of the DNA and of the protein side chains for each new sequence. As a result, the analysis depends linearly on the length of the binding site and complexes as large as the nucleosome can be treated, although this requires access to grid computing facilities. The results on the three complexes studied are in good agreement with experiment. Although they all involve significant DNA deformation, it is found that this does not necessarily imply that the recognition will be dominated by the sequence-dependent mechanical properties of DNA.

  8. Hemagglutinin of influenza A virus binds specifically to cell surface nucleolin and plays a role in virus internalization.

    Science.gov (United States)

    Chan, Che-Man; Chu, Hin; Zhang, Anna Jinxia; Leung, Lai-Han; Sze, Kong-Hung; Kao, Richard Yi-Tsun; Chik, Kenn Ka-Heng; To, Kelvin Kai-Wang; Chan, Jasper Fuk-Woo; Chen, Honglin; Jin, Dong-Yan; Liu, Liang; Yuen, Kwok-Yung

    2016-07-01

    The hemagglutinin (HA) protein of influenza A virus initiates cell entry by binding to sialic acids on target cells. In the current study, we demonstrated that in addition to sialic acids, influenza A/Puerto Rico/8/34 H1N1 (PR8) virus HA specifically binds to cell surface nucleolin (NCL). The interaction between HA and NCL was initially revealed with virus overlay protein binding assay (VOPBA) and subsequently verified with co-immunoprecipitation. Importantly, inhibiting cell surface NCL with NCL antibody, blocking PR8 viruses with purified NCL protein, or depleting endogenous NCL with siRNA all substantially reduced influenza virus internalization. We further demonstrated that NCL was a conserved cellular factor required for the entry of multiple influenza A viruses, including H1N1, H3N2, H5N1, and H7N9. Overall, our findings identified a novel role of NCL in influenza virus life cycle and established NCL as one of the host cell surface proteins for the entry of influenza A virus. PMID:27085069

  9. 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.

  10. Identification of tissue-specific DNA-protein binding sites by means of two-dimensional electrophoretic mobility shift assay display.

    Science.gov (United States)

    Chernov, Igor P; Timchenko, Kira A; Akopov, Sergey B; Nikolaev, Lev G; Sverdlov, Eugene D

    2007-05-01

    We developed a technique of differential electrophoretic mobility shift assay (EMSA) display allowing identification of tissue-specific protein-binding sites within long genomic sequences. Using this approach, we identified 10 cell type-specific protein-binding sites (protein target sites [PTSs]) within a 137-kb human chromosome 19 region. In general, tissue-specific binding of proteins from different nuclear extracts by individual PTSs did not follow the all-or-nothing principle. Most often, PTS-protein complexes were formed in all cases, but they were different for different nuclear extracts used. PMID:17359930

  11. Oriented Immobilization of Fab Fragments by Site-Specific Biotinylation at the Conserved Nucleotide Binding Site for Enhanced Antigen Detection.

    Science.gov (United States)

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

    2015-09-01

    Oriented immobilization of antibodies and antibody fragments has become increasingly important as a result of the efforts to reduce the size of diagnostic and sensor devices to miniaturized dimensions for improved accessibility to the end-user. Reduced dimensions of sensor devices necessitate the immobilized antibodies to conserve their antigen binding activity for proper operation. Fab fragments are becoming more commonly used in small-scaled diagnostic devices due to their small size and ease of manufacture. In this study, we used the previously described UV-NBS(Biotin) method to functionalize Fab fragments with IBA-EG11-Biotin linker utilizing UV energy to initiate a photo-cross-linking reaction between the nucleotide binding site (NBS) on the Fab fragment and IBA-Biotin molecule. Our results demonstrate that immobilization of biotinylated Fab fragments via UV-NBS(Biotin) method generated the highest level of immobilized Fab on surfaces when compared to other typical immobilization methods while preserving antigen binding activity. UV-NBS(Biotin) method provided 432-fold, 114-fold, and 29-fold improved antigen detection sensitivity than physical adsorption, NHS-Biotin, and ε-NH3(+), methods, respectively. Additionally, the limit of detection (LOD) for PSA utilizing Fab fragments immobilized via UV-NBS(Biotin) method was significantly lower than that of the other immobilization methods, with an LOD of 0.4 pM PSA. In summary, site-specific biotinylation of Fab fragments without structural damage or loss in antigen binding activity provides a wide range of application potential for UV-NBS immobilization technique across numerous diagnostic devices and nanotechnologies.

  12. Human proteins that specifically bind to 8-oxoguanine-containing RNA and their responses to oxidative stress

    Energy Technology Data Exchange (ETDEWEB)

    Hayakawa, Hiroshi, E-mail: hiroshi@college.fdcnet.ac.jp [Department of Functional Bioscience and Advanced Science Research Center, Fukuoka Dental College, Fukuoka 814-0193 (Japan); Fujikane, Aya; Ito, Riyoko [Department of Functional Bioscience and Advanced Science Research Center, Fukuoka Dental College, Fukuoka 814-0193 (Japan); Matsumoto, Masaki; Nakayama, Keiichi I. [Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582 (Japan); Sekiguchi, Mutsuo [Department of Functional Bioscience and Advanced Science Research Center, Fukuoka Dental College, Fukuoka 814-0193 (Japan)

    2010-12-10

    Research highlights: {yields} We performed comprehensive survey for proteins that bind to oxidized RNA. {yields} HNRNPD and HNRNPC proteins were identified as oxidized RNA binding proteins. {yields} Knockdown of HNRNPD/C expression caused increased sensitivity to H{sub 2}O{sub 2}. {yields} Amounts of HNRNPD protein rapidly decreased when cells were exposed to H{sub 2}O{sub 2}. -- Abstract: Exposure of cells to oxygen radicals damage various biologically important molecules. Among the oxidized bases produced in nucleic acids, 8-oxo-7,8-dihydroguanine (8-oxoguanine) is particularly important since it causes base mispairing. To ensure accurate gene expression, organisms must have a mechanism to discriminate 8-oxoguanine-containing RNA from normal transcripts. We searched for proteins that specifically bind to 8-oxoguanine-containing RNA from human HeLa cell extracts, and the candidate proteins were identified using mass spectrometry. Among the identified candidates, splicing isoform 1 of heterogeneous nuclear ribonucleoprotein D0 (HNRNPD) and splicing isoform C1 of heterogeneous nuclear ribonucleoprotein C1/C2 (HNRNPC) exhibited strong abilities to bind to oxidized RNA. The amount of HNRNPD protein rapidly decreased when cells were exposed to hydrogen peroxide, an agent that enhances oxidative stress. Moreover, the suppression of HNRNPD expression by siRNA caused cells to exhibit an increased sensitivity to hydrogen peroxide. The application of siRNA against HNRNPC also caused an increase in sensitivity to hydrogen peroxide. Since no additive effect was observed with a combined addition of siRNAs for HNRNPD and HNRNPC, we concluded that the two proteins may function in the same mechanism for the accurate gene expression.

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

    Directory of Open Access Journals (Sweden)

    Helene J Bustad

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

  14. Characterization of binding specificities of bovine leucocyte class I molecules: impacts for rational epitope discovery

    DEFF Research Database (Denmark)

    Hansen, Andreas M.; Rasmussen, Michael; Svitek, Nicholas;

    2014-01-01

    . Using this strategy, we characterized eight BoLA-I molecules, and found the peptide specificity to resemble that of human MHC-I molecules with primary anchors most often at P2 and P9, and occasional auxiliary P1/P3/P5/P6 anchors. We analyzed nine reported CTL epitopes from Theileria parva, and in eight...

  15. Natamycin blocks fungal growth by binding specifically to ergosterol without permeabilizing the membrane

    NARCIS (Netherlands)

    te Welscher, Y.M.; ten Napel, H.H.; Masià Balagué, M.; Souza, C.M.; Riezman, H.; de Kruijff, B.; Breukink, E.J.

    2008-01-01

    Natamycin is a polyene antibiotic that is commonly used as an antifungal agent because of its broad spectrum of activity and the lack of development of resistance. Other polyene antibiotics, like nystatin and filipin are known to interact with sterols, with some specificity for ergosterol thereby ca

  16. Liquid-solid extraction of cationic metals by cationic amphiphiles

    International Nuclear Information System (INIS)

    In the field of selective separation for recycling of spent nuclear fuel, liquid-liquid extraction processes are widely used (PUREX, DIAMEX..) in industrial scale. In order to guarantee a sustainable nuclear energy for the forthcoming generations, alternative reprocessing techniques are under development. One of them bases on the studies from Heckmann et al in the 80's and consists in selectively precipitating actinides from aqueous waste solutions by cationic surfactants (liquid-solid extraction). This technique has some interesting advantages over liquid-liquid extraction techniques, because several steps are omitted like stripping or solvent washing. Moreover, the amount of waste is decreased considerably, since no contaminated organic solvent is produced. In this thesis, we have carried out a physico-chemical study to understand the specific interactions between the metallic cations with the cationic surfactant. First, we have analysed the specific effect of the different counter-ions (Cl-, NO3-, C2O42-) and then the effect of alkaline cations on the structural properties of the surfactant aggregation in varying thermodynamical conditions. Finally, different multivalent cations (Cu2+, Zn2+, UO22+, Fe3+, Nd3+, Eu3+, Th4+) were considered; we have concluded that depending on the anionic complex of these metals formed in acidic media, we can observe either an adsorption at the micellar interface or not. This adsorption has a large influence of the surfactant aggregation properties and determines the limits of the application in term of ionic strength, temperature and surfactant concentration. (author)

  17. Specific Na+ and K+ Cation Effects on the Interfacial Water Molecules at the Air/Aqueous Salt Solution Interfaces Probed with Non-resonant Second Harmonic Generation (SHG)

    CERN Document Server

    Bian, Hong-tao; Guo, Yuan; Wang, Hong-fei

    2008-01-01

    Here we report the polarization dependent non-resonant second harmonic generation (SHG) measurement of the interfacial water molecules at the aqueous solution of the following salts: NaF, NaCl, NaBr, KF, KCl, and KBr. Through quantitative polarization analysis of the SHG data,the orientational parameter D value and the relative surface density of the interfacial water molecules at these aqueous solution surfaces were determined. From these results we found that addition of each of the six salts caused increase of the thickness of the interfacial water layer at the surfaces to a certain extent. Noticeably, both the cations and the anions contributed to the changes, and the abilities to increase the thickness of the interfacial water layer were in the following order: KBr > NaBr > KCl > NaCl ~ NaF > KF. Since these changes can not be factorized into individual anion and cation contributions, there are possible ion pairing or association effects, especially for the NaF case. We also found that the orientational ...

  18. Prediction of Surface and pH-Specific Binding of Peptides to Metal and Oxide Nanoparticles

    Science.gov (United States)

    Heinz, Hendrik; Lin, Tzu-Jen; Emami, Fateme Sadat; Ramezani-Dakhel, Hadi; Naik, Rajesh; Knecht, Marc; Perry, Carole C.; Huang, Yu

    2015-03-01

    The mechanism of specific peptide adsorption onto metallic and oxidic nanostructures has been elucidated in atomic resolution using novel force fields and surface models in comparison to measurements. As an example, variations in peptide adsorption on Pd and Pt nanoparticles depending on shape, size, and location of peptides on specific bounding facets are explained. Accurate computational predictions of reaction rates in C-C coupling reactions using particle models derived from HE-XRD and PDF data illustrate the utility of computational methods for the rational design of new catalysts. On oxidic nanoparticles such as silica and apatites, it is revealed how changes in pH lead to similarity scores of attracted peptides lower than 20%, supported by appropriate model surfaces and data from adsorption isotherms. The results demonstrate how new computational methods can support the design of nanoparticle carriers for drug release and the understanding of calcification mechanisms in the human body.

  19. GA-Binding Protein Is Dispensable for Neuromuscular Synapse Formation and Synapse-Specific Gene Expression▿

    OpenAIRE

    Jaworski, Alexander; Smith, Cynthia L.; Burden, Steven J.

    2007-01-01

    The mRNAs encoding postsynaptic components at the neuromuscular junction are concentrated in the synaptic region of muscle fibers. Accumulation of these RNAs in the synaptic region is mediated, at least in part, by selective transcription of the corresponding genes in synaptic myofiber nuclei. The transcriptional mechanisms that are responsible for synapse-specific gene expression are largely unknown, but an Ets site in the promoter regions of acetylcholine receptor (AChR) subunit genes and o...

  20. Ultrasensitive human thyrotropin (h TSH) immunoradiometric assay (IRMA) set up, through identification and minimization of non specific bindings

    International Nuclear Information System (INIS)

    An IRMA of h TSH, based on magnetic solid phase separation, was studied especially for what concerns its non specific bindings. These were identified as a product of the interaction between an altered form of radioiodinated anti-h TSH monoclonal antibody (125 I-m AB) and the uncoupled magnetizable cellulose particle (matrix). Apparently this form of 125 I-m AB is a type of aggregate that can be partly resolved from the main peak on Sephadex G-200 and further minimized via a single pre-incubation with the same matrix. Solid phase saturation with milk proteins, tracer storage at 40 C and serum addition during incubation were also found particularly effective is preventing its formation. These findings were used in order to reproducibly decrease non specific bindings to values 60/BO) up to values of 300-500. This way we obtained h TSH radio assays with functional sensitivities of about 0.05 m IU/L and analytical sensitivities of the order of 0.02 m IU/L, which classify them at least as among the best second generation assays and that are excellent indeed for magnetic IRMA s. A more optimistic sensitivity calculation, based on Rodbard's definition, provided values down to 0.008 m IU/L. Such sensitivities, moreover, were obtained in a very reproducible way and all over the useful tracer life. (author). 83 refs, 13 figs, 25 tabs

  1. Liver-specific gene expression: A-activator-binding site, a promoter module present in vitellogenin and acute-phase genes.

    OpenAIRE

    Kaling, M; Kugler, W.; Ross, K.; Zoidl, C.; Ryffel, G U

    1991-01-01

    The A2 vitellogenin gene of Xenopus laevis, which is expressed liver specifically, contains an A-activator-binding site (AABS) that mediates high in vitro transcriptional activity in rat liver nuclear extracts. Footprint experiments with DNase I and gel retardation assays revealed the binding of several proteins to AABS. Using binding sites of known DNA-binding proteins as competitors in the gel retardation assay, we found that the transcription factor C/EBP and/or one of its "iso-binders" as...

  2. Substrate Specificity and Ionic Regulation of GlnPQ from Lactococcus lactis. An ATP-Binding Cassette Transporter with Four Extracytoplasmic Substrate-Binding Domains

    NARCIS (Netherlands)

    Schuurman-Wolters, Gea K.; Poolman, Bert

    2005-01-01

    We report on the functional characterization of GlnPQ, an ATP-binding cassette transporter with four extracytoplasmic substrate-binding domains. The first predicted transmembrane helix of GlnP was cleaved off in the mature protein and most likely serves as the signal sequence for the extracytoplasmi

  3. Quantification of Non-Specific Binding of Magnetic Micro and Nano particles using Cell Tracking Velocimetry: Implication for magnetic cell separation and detection

    OpenAIRE

    Chalmers, J. J.; Xiong, Y; X. Jin; Shao, M.; Tong, X; Farag, S.; Zborowski, M.

    2010-01-01

    The maturation of magnetic cell separation technology places increasing demands on magnetic cell separation performance. While a number of factors can cause suboptimal performance, one of the major challenges can be non-specific binding of magnetic nano or micro particles to non-targeted cells. Depending on the type of separation, this non-specific binding can have a negative effect on the final purity, the recovery of the targeted cells, or both. In this work, we quantitatively demonstrate t...

  4. The binding of NCAM to FGFR1 induces a specific cellular response mediated by receptor trafficking

    DEFF Research Database (Denmark)

    Francavilla, Chiara; Cattaneo, Paola; Berezin, Vladimir;

    2009-01-01

    different from that elicited by FGF-2. In contrast to FGF-induced degradation of endocytic FGFR1, NCAM promotes the stabilization of the receptor, which is recycled to the cell surface in a Rab11- and Src-dependent manner. In turn, FGFR1 recycling is required for NCAM-induced sustained activation of various...... effectors. Furthermore, NCAM, but not FGF-2, promotes cell migration, and this response depends on FGFR1 recycling and sustained Src activation. Our results implicate NCAM as a nonconventional ligand for FGFR1 that exerts a peculiar control on the intracellular trafficking of the receptor, resulting...... in a specific cellular response. Besides introducing a further level of complexity in the regulation of FGFR1 function, our findings highlight the link of FGFR recycling with sustained signaling and cell migration and the critical role of these events in dictating the cellular response evoked by receptor...

  5. A urokinase receptor-associated protein with specific collagen binding properties

    DEFF Research Database (Denmark)

    Behrendt, N; Jensen, Ole Nørregaard; Engelholm, L H;

    2000-01-01

    The plasminogen activation cascade system, directed by urokinase and the urokinase receptor, plays a key role in extracellular proteolysis during tissue remodeling. To identify molecular interaction partners of these trigger proteins on the cell, we combined covalent protein cross-linking with mass...... spectrometry based methods for peptide mapping and primary structure analysis of electrophoretically isolated protein conjugates. A specific tri-molecular complex was observed upon addition of pro-urokinase to human U937 cells. This complex included the urokinase receptor, pro-urokinase, and an unknown, high...... molecular weight urokinase receptor-associated protein. The tryptic peptide mixture derived from a cross-linked complex of pro-urokinase and the latter protein was analyzed by nanoelectrospray tandem mass spectrometric sequencing. This analysis identified the novel protein as the human homologue of a murine...

  6. 125I-Clq-binding and specific antibodies as indicators of pulmonary disease activity in cystic fibrosis

    International Nuclear Information System (INIS)

    We studied the incidence and levels of circulating immune complexes by the 125I-Clq-binding assay in patients with cystic fibrosis in relation to clinical respiratory status and specific IgG and IgE antibodies to Pseudomonas aeruginosa. Staphylococcus aureus, Aspergillus fumigatus, and Candida albicans. Overall prevalence of CIC was 43%, but 86% of serially studied patients had evidence of CIC at some time. Patients with acute respiratory exacerbations and deteriorating pulmonary function had a higher incidence of CIC (76%) as compared to stable patients (36%, P less than 0.01), as well as significantly higher levels of CIC. Acute exacerbations were also associated with significant increases in IgG antibody to Pseudomonas (P less than 0.005) but not in other antibodies. CIC did not correlate with Pseudomonas-specific IgG nor with any other specific antibody studied. A variety of age-related differences in specific antibody levels were seen. The episodic appearance of CIC is common in CF and is usually associated with exacerbation of lung disease

  7. Crystal Structure of the Fab Fragment of an Anti-ofloxacin Antibody and Exploration of Its Specific Binding.

    Science.gov (United States)

    He, Kuo; Du, Xinjun; Sheng, Wei; Zhou, Xiaonan; Wang, Junping; Wang, Shuo

    2016-03-30

    The limited knowledge on the mechanism of interactions between small contaminants and the corresponding antibodies greatly inhibits the development of enzyme-linked immunosorbent assay methods. In this study, the crystal structure of a Fab fragment specific for ofloxacin was obtained. On the basis of the crystal characteristics, the modeling of the interactions between ofloxacin and the Fab revealed that TYR31 and HIS99 of the heavy chain and MET20 and GLN79 of the light chain formed a hydrophobic region and that SER52 and ALA97 of the heavy chain and TYR35 of the light chain formed a salt bridge and two hydrogen bonds for specific binding. The key roles of SER52 and ALA97 were further confirmed by site-directed mutation. A specificity analysis using 14 ofloxacin analogues indicates that the length of the bond formed between the piperazine ring and the antibody plays key roles in specific recognition. This work helps to clarify the mechanisms through which antibodies recognize small molecules and improve immune detection methods.

  8. 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...

  9. Formation of target-specific binding sites in enzymes: solid-phase molecular imprinting of HRP

    Science.gov (United States)

    Czulak, J.; Guerreiro, A.; Metran, K.; Canfarotta, F.; Goddard, A.; Cowan, R. H.; Trochimczuk, A. W.; Piletsky, S.

    2016-05-01

    Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates.Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike

  10. Oligomeric properties and DNA binding specificities of repressor isoforms from the Streptomyces bacteriophage phiC31.

    Science.gov (United States)

    Wilson, S E; Smith, M C

    1998-05-15

    Three protein isoforms (74, 54 and 42 kDa) are expressed from repressor gene c in the Streptomyces temperate bacteriophage phiC31. Because expression of the two smaller isoforms, 54 and 42 kDa, is sufficient for superinfection immunity, the interaction between these isoforms was studied. The native 42 kDa repressor (Nat42) and an N-terminally 6x histidine-tagged 54 kDa isoform (His54) were shown by co-purification on a Ni-NTA column to interact in Streptomyces lividans . In vitro three repressor preparations, containing Nat42, His54 and the native 54 and 42 kDa isoforms expressed together (Nat54&42), were subjected to chemical crosslinking and gel filtration analysis. Homo- and hetero-tetramers were observed. Previous work showed that the smallest isoform bound to 17 bp operators containing aconservedinvertedrepeat (CIR) and that the CIRs were located at 16 loci throughout the phiC31 genome. One of the CIRs (CIR6) is believed to be critical for regulating the lytic pathway. The DNA binding activities of the three repressor preparations were studied using fragments containing CIRs (CIR3-CIR6) from the essential early region as templates for DNase I footprinting. Whereas Nat42 bound to CIR6, poorly to CIR5 but undetectably to CIR3 or CIR4, the Nat54&42 preparation could bind to all CIRs tested, albeit poorly to CIR3 and CIR4. The His54 isoform bound all CIRs tested. Isoforms expressed from the phiC31 repressor gene, like those which are expressed from many eukaryotic transcription factor genes, apparently have different binding specificities.

  11. Dengue virus specific dual HLA binding T cell epitopes induce CD8+ T cell responses in seropositive individuals.

    Science.gov (United States)

    Comber, Joseph D; Karabudak, Aykan; Huang, Xiaofang; Piazza, Paolo A; Marques, Ernesto T A; Philip, Ramila

    2014-01-01

    Dengue virus infects an estimated 300 million people each year and even more are at risk of becoming infected as the virus continues to spread into new areas. Despite the increase in viral prevalence, no anti-viral medications or vaccines are approved for treating or preventing infection. CD8+ T cell responses play a major role in viral clearance. Therefore, effective vaccines that induce a broad, multi-functional T cell response with substantial cross-reactivity between all virus serotypes can have major impacts on reducing infection rates and infection related complications. Here, we took an immunoproteomic approach to identify novel MHC class I restricted T cell epitopes presented by dengue virus infected cells, representing the natural and authentic targets of the T cell response. Using this approach we identified 4 novel MHC-I restricted epitopes: 2 with the binding motif for HLA-A24 molecules and 2 with both HLA-A2 and HLA-A24 binding motifs. These peptides were able to activate CD8+ T cell responses in both healthy, seronegative individuals and in seropositive individuals who have previously been infected with dengue virus. Importantly, the dual binding epitopes activated pre-existing T cell precursors in PBMCs obtained from both HLA-A2+ and HLA-A24+ seropositive individuals. Together, the data indicate that these epitopes are immunologically relevant T cell activating peptides presented on infected cells during a natural infection and therefore may serve as candidate antigens for the development of effective multi-serotype specific dengue virus vaccines. PMID:25668665

  12. Pregnancy-specific glycoproteins bind integrin αIIbβ3 and inhibit the platelet-fibrinogen interaction.

    Directory of Open Access Journals (Sweden)

    Daniel K Shanley

    Full Text Available Pregnancy-specific glycoproteins (PSGs are immunoglobulin superfamily members encoded by multigene families in rodents and primates. In human pregnancy, PSGs are secreted by the syncytiotrophoblast, a fetal tissue, and reach a concentration of up to 400 ug/ml in the maternal bloodstream at term. Human and mouse PSGs induce release of anti-inflammatory cytokines such as IL-10 and TGFβ1 from monocytes, macrophages, and other cell types, suggesting an immunoregulatory function. RGD tri-peptide motifs in the majority of human PSGs suggest that they may function like snake venom disintegrins, which bind integrins and inhibit interactions with ligands. We noted that human PSG1 has a KGD, rather than an RGD motif. The presence of a KGD in barbourin, a platelet integrin αIIbβ3 antagonist found in snake venom, suggested that PSG1 may be a selective αIIbβ3 ligand. Here we show that human PSG1 binds αIIbβ3 and inhibits the platelet - fibrinogen interaction. Unexpectedly, however, the KGD is not critical as multiple PSG1 domains independently bind and inhibit αIIbβ3 function. Human PSG9 and mouse Psg23 are also inhibitory suggesting conservation of this function across primate and rodent PSG families. Our results suggest that in species with haemochorial placentation, in which maternal blood is in direct contact with fetal trophoblast, the high expression level of PSGs reflects a requirement to antagonise abundant (3 mg/ml fibrinogen in the maternal circulation, which may be necessary to prevent platelet aggregation and thrombosis in the prothrombotic maternal environment of pregnancy.

  13. Characterization of hydrophobic-ligand-binding proteins of Taenia solium that are expressed specifically in the adult stage.

    Science.gov (United States)

    Rahman, M; Lee, E-G; Kim, S-H; Bae, Y-A; Wang, H; Yang, Y; Kong, Y

    2012-09-01

    Taenia solium, a causative agent of taeniasis and cysticercosis, has evolved a repertoire of lipid uptake mechanisms. Proteome analysis of T. solium excretory-secretory products (TsESP) identified 10 kDa proteins displaying significant sequence identity with cestode hydrophobic-ligand-binding-proteins (HLBPs). Two distinct 362- and 352-bp-long cDNAs encoding 264- and 258-bp-long open reading frames (87 and 85 amino acid polypeptides) were isolated by mining the T. solium expressed sequence tags and a cDNA library screening (TsHLBP1 and TsHLBP2; 94% sequence identity). They clustered into the same clade with those found in Moniezia expansa and Hymenolepis diminuta. Genomic structure analysis revealed that these genes might have originated from a common ancestor. Both the crude TsESP and bacterially expressed recombinant proteins exhibited binding activity toward 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS), which was competitively inhibited by oleic acid. The proteins also bound to cis-parinaric acid (cPnA) and 16-(9-anthroyloxy) palmitic acid (16-AP), but showed no binding activity against 11-[(5-dimethylaminonaphthalene-1-sulfonyl) amino] undecanoic acid (DAUDA) and dansyl-DL-α-aminocaprylic acid (DACA). Unsaturated fatty acids (FAs) showed greater affinity than saturated FAs. The proteins were specifically expressed in adult worms throughout the strobila. The TsHLBPs might be involved in uptake and/or sequestration of hydrophobic molecules provided by their hosts, thus contributing to host-parasite interface interrelationships.

  14. Production of a soluble single-chain variable fragment antibody against okadaic acid and exploration of its specific binding.

    Science.gov (United States)

    He, Kuo; Zhang, Xiuyuan; Wang, Lixia; Du, Xinjun; Wei, Dong

    2016-06-15

    Okadaic acid is a lipophilic marine algal toxin commonly responsible for diarrhetic shellfish poisoning (DSP). Outbreaks of DSP have been increasing and are of worldwide public health concern; therefore, there is a growing demand for more rapid, reliable, and economical analytical methods for the detection of this toxin. In this study, anti-okadaic acid single-chain variable fragment (scFv) genes were prepared by cloning heavy and light chain genes from hybridoma cells, followed by fusion of the chains via a linker peptide. An scFv-pLIP6/GN recombinant plasmid was constructed and transformed into Escherichia coli for expression, and the target scFv was identified with IC-CLEIA (chemiluminescent enzyme immunoassay). The IC15 was 0.012 ± 0.02 μg/L, and the IC50 was 0.25 ± 0.03 μg/L. The three-dimensional structure of the scFv was simulated with computer modeling, and okadaic acid was docked to the scFv model to obtain a putative structure of the binding complex. Two predicted critical amino acids, Ser32 and Thr187, were then mutated to verify this theoretical model. Both mutants exhibited significant loss of binding activity. These results help us to understand this specific scFv-antigen binding mechanism and provide guidance for affinity maturation of the antibody in vitro. The high-affinity scFv developed here also has potential for okadaic acid toxin detection. PMID:26772159

  15. Non-specific binding and steric hindrance thresholds for penetration of particulate drug carriers within tumor tissue.

    Science.gov (United States)

    Dancy, Jimena G; Wadajkar, Aniket S; Schneider, Craig S; Mauban, Joseph R H; Goloubeva, Olga G; Woodworth, Graeme F; Winkles, Jeffrey A; Kim, Anthony J

    2016-09-28

    Therapeutic nanoparticles (NPs) approved for clinical use in solid tumor therapy provide only modest improvements in patient survival, in part due to physiological barriers that limit delivery of the particles throughout the entire tumor. Here, we explore the thresholds for NP size and surface poly(ethylene glycol) (PEG) density for penetration within tumor tissue extracellular matrix (ECM). We found that NPs as large as 62nm, but less than 110nm in diameter, diffused rapidly within a tumor ECM preparation (Matrigel) and breast tumor xenograft slices ex vivo. Studies of PEG-density revealed that increasing PEG density enhanced NP diffusion and that PEG density below a critical value led to adhesion of NP to ECM. Non-specific binding of NPs to tumor ECM components was assessed by surface plasmon resonance (SPR), which revealed excellent correlation with the particle diffusion results. Intravital microscopy of NP spread in breast tumor tissue confirmed a significant difference in tumor tissue penetration between the 62 and 110nm PEG-coated NPs, as well as between PEG-coated and uncoated NPs. SPR assays also revealed that Abraxane, an FDA-approved non-PEGylated NP formulation used for cancer therapy, binds to tumor ECM. Our results establish limitations on the size and surface PEG density parameters required to achieve uniform and broad dispersion within tumor tissue and highlight the utility of SPR as a high throughput method to screen NPs for tumor penetration. PMID:27460683

  16. Region-specific up-regulation of oxytocin receptor binding in the brain of mice following chronic nicotine administration.

    Science.gov (United States)

    Zanos, Panos; Georgiou, Polymnia; Metaxas, Athanasios; Kitchen, Ian; Winsky-Sommerer, Raphaelle; Bailey, Alexis

    2015-07-23

    Nicotine addiction is considered to be the main preventable cause of death worldwide. While growing evidence indicates that the neurohypophysial peptide oxytocin can modulate the addictive properties of several abused drugs, the regulation of the oxytocinergic system following nicotine administration has so far received little attention. Here, we examined the effects of long-term nicotine or saline administration on the central oxytocinergic system using [(125)I]OVTA autoradiographic binding in mouse brain. Male, 7-week old C57BL6J mice were treated with either nicotine (7.8 mg/kg daily; rate of 0.5 μl per hour) or saline for a period of 14-days via osmotic minipumps. Chronic nicotine administration induced a marked region-specific upregulation of the oxytocin receptor binding in the amygdala, a brain region involved in stress and emotional regulation. These results provide direct evidence for nicotine-induced neuroadaptations in the oxytocinergic system, which may be involved in the modulation of nicotine-seeking as well as emotional consequence of chronic drug use. PMID:26037668

  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. Cardiac-specific deletion of the microtubule-binding protein CENP-F causes dilated cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Ellen Dees

    2012-07-01

    CENP-F is a large multifunctional protein with demonstrated regulatory roles in cell proliferation, vesicular transport and cell shape through its association with the microtubule (MT network. Until now, analysis of CENP-F has been limited to in vitro analysis. Here, using a Cre-loxP system, we report the in vivo disruption of CENP-F gene function in murine cardiomyocytes, a cell type displaying high levels of CENP-F expression. Loss of CENP-F function in developing myocytes leads to decreased cell division, blunting of trabeculation and an initially smaller, thin-walled heart. Still, embryos are born at predicted mendelian ratios on an outbred background. After birth, hearts lacking CENP-F display disruption of their intercalated discs and loss of MT integrity particularly at the costamere; these two structures are essential for cell coupling/electrical conduction and force transduction in the heart. Inhibition of myocyte proliferation and cell coupling as well as loss of MT maintenance is consistent with previous reports of generalized CENP-F function in isolated cells. One hundred percent of these animals develop progressive dilated cardiomyopathy with heart block and scarring, and there is a 20% mortality rate. Importantly, although it has long been postulated that the MT cytoskeleton plays a role in the development of heart disease, this study is the first to reveal a direct genetic link between disruption of this network and cardiomyopathy. Finally, this study has broad implications for development and disease because CENP-F loss of function affects a diverse array of cell-type-specific activities in other organs.

  19. Computational Biology Tools for Identifying Specific Ligand Binding Residues for Novel Agrochemical and Drug Design.

    Science.gov (United States)

    Neshich, Izabella Agostinho Pena; Nishimura, Leticia; de Moraes, Fabio Rogerio; Salim, Jose Augusto; Villalta-Romero, Fabian; Borro, Luiz; Yano, Inacio Henrique; Mazoni, Ivan; Tasic, Ljubica; Jardine, Jose Gilberto; Neshich, Goran

    2015-01-01

    The term "agrochemicals" is used in its generic form to represent a spectrum of pesticides, such as insecticides, fungicides or bactericides. They contain active components designed for optimized pest management and control, therefore allowing for economically sound and labor efficient agricultural production. A "drug" on the other side is a term that is used for compounds designed for controlling human diseases. Although drugs are subjected to much more severe testing and regulation procedures before reaching the market, they might contain exactly the same active ingredient as certain agrochemicals, what is the case described in present work, showing how a small chemical compound might be used to control pathogenicity of Gram negative bacteria Xylella fastidiosa which devastates citrus plantations, as well as for control of, for example, meningitis in humans. It is also clear that so far the production of new agrochemicals is not benefiting as much from the in silico new chemical compound identification/discovery as pharmaceutical production. Rational drug design crucially depends on detailed knowledge of structural information about the receptor (target protein) and the ligand (drug/agrochemical). The interaction between the two molecules is the subject of analysis that aims to understand relationship between structure and function, mainly deciphering some fundamental elements of the nanoenvironment where the interaction occurs. In this work we will emphasize the role of understanding nanoenvironmental factors that guide recognition and interaction of target protein and its function modifier, an agrochemical or a drug. The repertoire of nanoenvironment descriptors is used for two selected and specific cases we have approached in order to offer a technological solution for some very important problems that needs special attention in agriculture: elimination of pathogenicity of a bacterium which is attacking citrus plants and formulation of a new fungicide. Finally

  20. Glycyrrhizic acid prevents astrocyte death by neuromyelitis optica-specific IgG via inhibition of C1q binding.

    Science.gov (United States)

    Kim, Ji-Sun; Cheon, Soyoung; Kim, Seung Woo; Kim, Boram; Kim, Heejaung; Park, Ki Duk; Kim, Sung-Min

    2016-09-16

    Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system and is mediated by complement-dependent cytotoxicity (CDC) of NMO-specific immunoglobulin G (IgG) antibodies (NMO-IgG). Glycyrrhizic acid (GA) has numerous pharmacological effects including inhibition of the complement pathway. We aimed to study the influence of GA on NMO-IgG-induced CDC. NMO-IgG samples from 7 patients with NMO, together with human complement, induced CDC in an aquaporin 4 M23-overexpressing glial cell line, an in vitro NMO model. GA attenuated NMO-IgG-induced CDC in a dose-dependent manner. The mechanism of the GA-related CDC inhibition was sequentially dissected and found to involve inhibition of C1q binding to NMO-IgG. Consequently, GA attenuates NMO-IgG-induced CDC and may be a promising novel therapeutic agent against NMO. PMID:27462020

  1. Binding of a candidate splice regulator to a calcitonin-specific splice enhancer regulates calcitonin/CGRP pre-mRNA splicing.

    Science.gov (United States)

    Coleman, Timothy P; Tran, Quincy; Roesser, James R

    2003-01-27

    The calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA is alternatively processed in a tissue-specific manner leading to the production of calcitonin mRNA in thyroid C cells and CGRP mRNA in neurons. A candidate calcitonin/CGRP splice regulator (CSR) isolated from rat brain was shown to inhibit calcitonin-specific splicing in vitro. CSR specifically binds to two regions in the calcitonin-specific exon 4 RNA previously demonstrated to function as a bipartate exonic splice enhancer (ESE). The two regions, A and B element, are necessary for inclusion of exon 4 into calcitonin mRNA. A novel RNA footprinting method based on the UV cross-linking assay was used to define the site of interaction between CSR and B element RNA. Base changes at the CSR binding site prevented CSR binding to B element RNA and CSR was unable to inhibit in vitro splicing of pre-mRNAs containing the mutated CSR binding site. When expressed in cells that normally produce predominantly CGRP mRNA, a calcitonin/CGRP gene containing the mutated CSR binding site expressed predominantly calcitonin mRNA. These observations demonstrate that CSR binding to the calcitonin-specific ESE regulates calcitonin/CGRP pre-mRNA splicing.

  2. 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.

  3. Specific reduction of calcium-binding protein (28-kilodalton calbindin-D) gene expression in aging and neurodegenerative diseases

    International Nuclear Information System (INIS)

    The present studies establish that there are specific, significant decreases in the neuronal calcium-binding protein (28-kDa calbindin-D) gene expression in aging and in neurodegenerative diseases. The specificity of the changes observed in calbindin mRNA levels was tested by reprobing blots with calmodulin, cyclophilin, and B-actin cDNAs. Gross brain regions of the aging rat exhibited specific, significant decreases in calbindin·mRNA and protein levels in the cerebellum, corpus striatum, and brain-stem region but not in the cerebral cortex or hippocampus. Discrete areas of the aging human brain exhibited significant decreases in calbindin protein and mRNA in the cerebellum, corpus striatum, and nucleus basalis but not in the neocortex, hippocampus, amygdala, locus ceruleus, or nucleus raphe dorsalis. Comparison of diseased human brain tissue with age- and sex-matched controls yielded significant decreases calbindin protein and mRNA in the substantia nigra (Parkinson disease), in the corpus striatum (Huntington disease), in the nucleus basalis (Alzheimer disease), and in the hippocampus and nucleus raphe dorsalis (Parkinson, Huntington, and Alzheimer diseases) but not in the cerebellum, neocortex, amygdala, or locus ceruleus. These findings suggest that decreased calbindin gene expression may lead to a failure of calcium buffering or intraneuronal calcium homeostasis, which contributes to calcium-mediated cytotoxic events during aging and in the pathogenesis of neurodegenerative diseases

  4. In Vivo Selection of Phage Sequences and Characterization of Peptide-specific Binding to Breast Cancer Cells

    Institute of Scientific and Technical Information of China (English)

    Rui Wang; Ruifang Niu; Lin Zhang; Hongkai Zhang; Xiyin Wei; Yi Yang; Shiwu Zhang; Jing Wu; Min Wu; Youjia Cao

    2008-01-01

    OBJECTIVE To screen specific polypeptide target binding to breast cancer xenografts in vivo from a phage-displayed peptide library in order to provide peptide sequences for breast cancer tumor-targeting diagnosis and therapy.METHODS A mouse model for carrying breast cancer xenografts was established using Tientsin Albinao Ⅱ mice (TAII). A 12-peptide library was biopanned through 4 rounds.Phages were recovered and titrated from tumor xenografts and control tissue (liver). The distribution of phages was detected by immunohistochemical staining.RESULTS Phage homing to breast cancer was enriched through 4 rounds of biopanning, being 14-fold of that recovered from liver tissue. A peptide sequence, ASANPFPTKALL was characterized by randomly picked-up clones which appeared most frequently.Immunohistochemical staining revealed phage localization in cancer xenografts 40 min after injection of the enriched phages.When a specific phage was tested individually, the phage reclaimed from breast cancer xenografts was 14 times as those from control tissues.CONCLUSION Tumor-specific homing peptides may provide an effective tool for breast cancer target therapy. The in vivo phage display selection technique employed in this study was feasible and applicable to screening peptides that home to.breast cells.

  5. Evolutionary and structural perspectives of plant cyclic nucleotide-gated cation channels

    KAUST Repository

    Zelman, Alice K.

    2012-05-29

    Ligand-gated cation channels are a frequent component of signaling cascades in eukaryotes. Eukaryotes contain numerous diverse gene families encoding ion channels, some of which are shared and some of which are unique to particular kingdoms. Among the many different types are cyclic nucleotide-gated channels (CNGCs). CNGCs are cation channels with varying degrees of ion conduction selectivity. They are implicated in numerous signaling pathways and permit diffusion of divalent and monovalent cations, including Ca2+ and K+. CNGCs are present in both plant and animal cells, typically in the plasma membrane; recent studies have also documented their presence in prokaryotes. All eukaryote CNGC polypeptides have a cyclic nucleotide-binding domain and a calmodulin binding domain as well as a six transmembrane/one pore tertiary structure. This review summarizes existing knowledge about the functional domains present in these cation-conducting channels, and considers the evidence indicating that plant and animal CNGCs evolved separately. Additionally, an amino acid motif that is only found in the phosphate binding cassette and hinge regions of plant CNGCs, and is present in all experimentally confirmed CNGCs but no other channels was identified. This CNGC-specific amino acid motif provides an additional diagnostic tool to identify plant CNGCs, and can increase confidence in the annotation of open reading frames in newly sequenced genomes as putative CNGCs. Conversely, the absence of the motif in some plant sequences currently identified as probable CNGCs may suggest that they are misannotated or protein fragments. 2012 Zelman, Dawe, Gehring and Berkowitz.

  6. Isolation and Characterization of a Monobody with a Fibronectin Domain III Scaffold That Specifically Binds EphA2.

    Science.gov (United States)

    Park, Seung-Hwan; Park, Sukho; Kim, Dong-Yeon; Pyo, Ayoung; Kimura, Richard H; Sathirachinda, Ataya; Choy, Hyon E; Min, Jung-Joon; Gambhir, Sanjiv Sam; Hong, Yeongjin

    2015-01-01

    Monobodies are binding scaffold proteins originating from a human fibronectin domain III (Fn3) scaffold that can be easily engineered with specificity and affinity. Human EphA2 (hEphA2) is an early detection marker protein for various tumors including lung, breast, and colon cancer. In this study, we isolated two hEphA2-specific monobodies (E1 and E10) by screening a yeast surface display library. They showed the same amino acid sequence except in the DE loop and had high affinity (~2 nM Kd) against hEphA2. E1 bound only hEphA2 and mEphA2, although it bound hEphA2 with an affinity 2-fold higher than that of mEphA2. However, E10 also bound the mEphA6 and mEphA8 homologs as well as hEphA2 and mEphA2. Thus, E1 but not E10 was highly specific for hEphA2. E1 specifically bound human cells and xenograft tumor tissues expressing hEphA on the cell surface. In vivo optical imaging showed strong targeting of Cy5.5-labeled E1 to mouse tumor tissue induced by PC3 cells, a human prostate cancer cell line that expresses a high level of hEphA2. In conclusion, the highly specific monobody E1 is useful as a hEphA2 probe candidate for in vivo diagnosis and therapy. PMID:26177208

  7. Isolation and Characterization of a Monobody with a Fibronectin Domain III Scaffold That Specifically Binds EphA2.

    Directory of Open Access Journals (Sweden)

    Seung-Hwan Park

    Full Text Available Monobodies are binding scaffold proteins originating from a human fibronectin domain III (Fn3 scaffold that can be easily engineered with specificity and affinity. Human EphA2 (hEphA2 is an early detection marker protein for various tumors including lung, breast, and colon cancer. In this study, we isolated two hEphA2-specific monobodies (E1 and E10 by screening a yeast surface display library. They showed the same amino acid sequence except in the DE loop and had high affinity (~2 nM Kd against hEphA2. E1 bound only hEphA2 and mEphA2, although it bound hEphA2 with an affinity 2-fold higher than that of mEphA2. However, E10 also bound the mEphA6 and mEphA8 homologs as well as hEphA2 and mEphA2. Thus, E1 but not E10 was highly specific for hEphA2. E1 specifically bound human cells and xenograft tumor tissues expressing hEphA on the cell surface. In vivo optical imaging showed strong targeting of Cy5.5-labeled E1 to mouse tumor tissue induced by PC3 cells, a human prostate cancer cell line that expresses a high level of hEphA2. In conclusion, the highly specific monobody E1 is useful as a hEphA2 probe candidate for in vivo diagnosis and therapy.

  8. Structural basis for the ligand-binding specificity of fatty acid-binding proteins (pFABP4 and pFABP5) in gentoo penguin.

    Science.gov (United States)

    Lee, Chang Woo; Kim, Jung Eun; Do, Hackwon; Kim, Ryeo-Ok; Lee, Sung Gu; Park, Hyun Ho; Chang, Jeong Ho; Yim, Joung Han; Park, Hyun; Kim, Il-Chan; Lee, Jun Hyuck

    2015-09-11

    Fatty acid-binding proteins (FABPs) are involved in transporting hydrophobic fatty acids between various aqueous compartments of the cell by directly binding ligands inside their β-barrel cavities. Here, we report the crystal structures of ligand-unbound pFABP4, linoleate-bound pFABP4, and palmitate-bound pFABP5, obtained from gentoo penguin (Pygoscelis papua), at a resolution of 2.1 Å, 2.2 Å, and 2.3 Å, respectively. The pFABP4 and pFABP5 proteins have a canonical β-barrel structure with two short α-helices that form a cap region and fatty acid ligand binding sites in the hydrophobic cavity within the β-barrel structure. Linoleate-bound pFABP4 and palmitate-bound pFABP5 possess different ligand-binding modes and a unique ligand-binding pocket due to several sequence dissimilarities (A76/L78, T30/M32, underlining indicates pFABP4 residues) between the two proteins. Structural comparison revealed significantly different conformational changes in the β3-β4 loop region (residues 57-62) as well as the flipped Phe60 residue of pFABP5 than that in pFABP4 (the corresponding residue is Phe58). A ligand-binding study using fluorophore displacement assays shows that pFABP4 has a relatively strong affinity for linoleate as compared to pFABP5. In contrast, pFABP5 exhibits higher affinity for palmitate than that for pFABP4. In conclusion, our high-resolution structures and ligand-binding studies provide useful insights into the ligand-binding preferences of pFABPs based on key protein-ligand interactions.

  9. Interference of peptides and specific antibodies with the function of the Actinobacillus pleuropneumoniae transferrin-binding protein.

    OpenAIRE

    Strutzberg, K; Franz, B.; Gerlach, G F

    1997-01-01

    Multiple-antigenic peptides (MAPs) containing transferrin-binding domains of the Actinobacillus pleuropneumoniae serotype 7-derived transferrin-binding protein (TfbA) (K. Strutzberg, L. von Olleschik, B. Franz, C. Pyne, M. A. Schmidt, and G.-F. Gerlach, Infect. Immun. 63:3846-3850, 1995) were constructed. It was found that the MAPs inhibited transferrin binding of the recombinant TfbA protein, whereas antibodies directed against transferrin-binding domains failed to do so.

  10. Isolation and characterisation of a 17-kDa staphylococcal heparin-binding protein with broad specificity

    OpenAIRE

    Fallgren, Corina; Utt, Meeme; Ljungh, Åsa

    2001-01-01

    A previous study reported the ability of staphylococci to bind heparin and heparin-dependent host growth factors. The present study isolated and identified heparin- and basic fibroblast growth factor (bFGF)-binding surface components of S. epidermidis strain RP12 and S. haemolyticus strain SM 131. The staphylococcal heparin-binding component(s) were purified by affinity chromatography on heparin-Sepharose and a major heparin-binding protein, here designated HBP, was identified by immunoblot i...

  11. Influence of interlayer cations on organic intercalation of montmorillonite.

    Science.gov (United States)

    Wu, Limei; Liao, Libing; Lv, Guocheng

    2015-09-15

    The influence of the types of interlayer cations on organic intercalation of montmorillonite (Mt) was studied in this paper. The distribution of Na(+), K(+), Mg(2+), Ca(2+) and Fe(3+) in montmorillonite interlayer, their interaction with structure layers and the effect of interlayer cations on the basal spacing of Mt, the amount of binding water for different interlayer cations and the binding force between them were investigated systematically. 1-Hexadecy1-3-methylimidazolium chloride monohydrate (C16mimCl) was intercalated into montmorillonites with different interlayer cations. The influence of interlayer cations on organic intercalation was investigated. Molecular dynamics (MD) modeling was used to speculate the interlayer microstructures of the organically intercalated Mt with different interlayer cations. These simulations help to predict the microstructure of organo-Mt and guide their relevant engineering applications. PMID:26001131

  12. Structure and cell-specific expression of a cloned human retinol binding protein gene: the 5'-flanking region contains hepatoma specific transcriptional signals.

    Science.gov (United States)

    D'Onofrio, C; Colantuoni, V; Cortese, R

    1985-08-01

    Human plasma retinol binding protein (RBP) is coded by a single gene and is specifically synthesized in the liver. We have characterized a lambda clone, from a human DNA library, carrying the gene coding for plasma RBP. Southern blot analysis and DNA sequencing show that the gene is composed of six exons and five introns. Primer elongation and S1 mapping experiments allowed the definition of the initiation of transcription and the identification of the putative promoter. The 5'-flanking region of the RBP gene was fused upstream to the coding sequence of the bacterial enzyme chloramphenicol acetyl transferase (CAT): the chimeric gene was introduced, by calcium phosphate precipitation, into the human hepatoma cell line Hep G2 and into HeLa cells. Efficient expression of CAT was obtained only in Hep G2. Primer elongation analysis of the RNA extracted from transfected Hep G2 showed that initiation of transcription of the transfected chimeric gene occurs at a position identical to that of the natural gene. Transcriptional analysis of Bal31 deletions from the 3' end of the RBP 5'-flanking DNA allowed the identification of the RBP gene promoter.

  13. The size, shape and specificity of the sugar-binding site of the jacalin-related lectins is profoundly affected by the proteolytic cleavage of the subunits.

    Science.gov (United States)

    Houlès Astoul, Corinne; Peumans, Willy J; van Damme, Els J M; Barre, Annick; Bourne, Yves; Rougé, Pierre

    2002-11-01

    Mannose-specific lectins with high sequence similarity to jacalin and the Maclura pomifera agglutinin have been isolated from species belonging to the families Moraceae, Convolvulaceae, Brassicaceae, Asteraceae, Poaceae and Musaceae. Although these novel mannose-specific lectins are undoubtedly related to the galactose-specific Moraceae lectins there are several important differences. Apart from the obvious differences in specificity, the mannose- and galactose-specific jacalin-related lectins differ in what concerns their biosynthesis and processing, intracellular location and degree of oligomerization of the protomers. Taking into consideration that the mannose-specific lectins are widely distributed in higher plants, whereas their galactose-specific counterparts are confined to a subgroup of the Moraceae sp. one can reasonably assume that the galactose-specific Moraceae lectins are a small-side group of the main family. The major change that took place in the structure of the binding site of the diverging Moraceae lectins concerns a proteolytic cleavage close to the N-terminus of the protomer. To corroborate the impact of this change, the specificity of jacalin was re-investigated using surface plasmon resonance analysis. This approach revealed that in addition to galactose and N -acetylgalactosamine, the carbohydrate-binding specificity of jacalin extends to mannose, glucose, N -acetylmuramic acid and N -acetylneuraminic acid. Owing to this broad carbohydrate-binding specificity, jacalin is capable of recognizing complex glycans from plant pathogens or predators.

  14. Stream specificity and asymmetries in feature binding and content-addressable access in visual encoding and memory.

    Science.gov (United States)

    Huynh, Duong L; Tripathy, Srimant P; Bedell, Harold E; Ögmen, Haluk

    2015-01-01

    Human memory is content addressable-i.e., contents of the memory can be accessed using partial information about the bound features of a stored item. In this study, we used a cross-feature cuing technique to examine how the human visual system encodes, binds, and retains information about multiple stimulus features within a set of moving objects. We sought to characterize the roles of three different features (position, color, and direction of motion, the latter two of which are processed preferentially within the ventral and dorsal visual streams, respectively) in the construction and maintenance of object representations. We investigated the extent to which these features are bound together across the following processing stages: during stimulus encoding, sensory (iconic) memory, and visual short-term memory. Whereas all features examined here can serve as cues for addressing content, their effectiveness shows asymmetries and varies according to cue-report pairings and the stage of information processing and storage. Position-based indexing theories predict that position should be more effective as a cue compared to other features. While we found a privileged role for position as a cue at the stimulus-encoding stage, position was not the privileged cue at the sensory and visual short-term memory stages. Instead, the pattern that emerged from our findings is one that mirrors the parallel processing streams in the visual system. This stream-specific binding and cuing effectiveness manifests itself in all three stages of information processing examined here. Finally, we find that the Leaky Flask model proposed in our previous study is applicable to all three features. PMID:26382005

  15. Stream specificity and asymmetries in feature binding and content-addressable access in visual encoding and memory.

    Science.gov (United States)

    Huynh, Duong L; Tripathy, Srimant P; Bedell, Harold E; Ögmen, Haluk

    2015-01-01

    Human memory is content addressable-i.e., contents of the memory can be accessed using partial information about the bound features of a stored item. In this study, we used a cross-feature cuing technique to examine how the human visual system encodes, binds, and retains information about multiple stimulus features within a set of moving objects. We sought to characterize the roles of three different features (position, color, and direction of motion, the latter two of which are processed preferentially within the ventral and dorsal visual streams, respectively) in the construction and maintenance of object representations. We investigated the extent to which these features are bound together across the following processing stages: during stimulus encoding, sensory (iconic) memory, and visual short-term memory. Whereas all features examined here can serve as cues for addressing content, their effectiveness shows asymmetries and varies according to cue-report pairings and the stage of information processing and storage. Position-based indexing theories predict that position should be more effective as a cue compared to other features. While we found a privileged role for position as a cue at the stimulus-encoding stage, position was not the privileged cue at the sensory and visual short-term memory stages. Instead, the pattern that emerged from our findings is one that mirrors the parallel processing streams in the visual system. This stream-specific binding and cuing effectiveness manifests itself in all three stages of information processing examined here. Finally, we find that the Leaky Flask model proposed in our previous study is applicable to all three features.

  16. Binding Specificities of the Telomere Phage ϕKO2 Prophage Repressor CB and Lytic Repressor Cro.

    Science.gov (United States)

    Hammerl, Jens Andre; Jäckel, Claudia; Lanka, Erich; Roschanski, Nicole; Hertwig, Stefan

    2016-01-01

    Temperate bacteriophages possess a genetic switch which regulates the lytic and lysogenic cycle. The genomes of the temperate telomere phages N15, PY54, and ϕKO2 harbor a primary immunity region (immB) comprising genes for the prophage repressor (cI or cB), the lytic repressor (cro) and a putative antiterminator (q). The roles of these products are thought to be similar to those of the lambda proteins CI (CI prophage repressor), Cro (Cro repressor), and Q (antiterminator Q), respectively. Moreover, the gene order and the location of several operator sites in the prototype telomere phage N15 and in ϕKO2 are reminiscent of lambda-like phages. We determined binding sites of the ϕKO2 prophage repressor CB and lytic repressor Cro on the ϕKO2 genome in detail by electrophoretic mobility shift assay (EMSA) studies. Unexpectedly, ϕKO2 CB and Cro revealed different binding specificities. CB was bound to three OR operators in the intergenic region between cB and cro, two OL operators between cB and the replication gene repA and even to operators of N15. Cro bound exclusively to the 16 bp operator site OR3 upstream of the ϕKO2 prophage repressor gene. The ϕKO2 genes cB and cro are regulated by several strong promoters overlapping with the OR operators. The data suggest that Cro represses cB transcription but not its own synthesis, as already reported for PY54 Cro. Thus, not only PY54, but also phage ϕKO2 possesses a genetic switch that diverges significantly from the switch of lambda-like phages. PMID:27527206

  17. Neuroendocrine differentiation factor, IA-1, is a transcriptional repressor and contains a specific DNA-binding domain: identification of consensus IA-1 binding sequence

    OpenAIRE

    Breslin, Mary B; Zhu, Min; Notkins, Abner L.; Lan, Michael S.

    2002-01-01

    A novel cDNA, insulinoma-associated antigen-1 (IA-1), containing five zinc-finger DNA-binding motifs, was isolated from a human insulinoma subtraction library. IA-1 expression is restricted to fetal but not adult pancreatic and brain tissues as well as tumors of neuroendocrine origin. Using various GAL4 DNA binding domain (DBD)/IA-1 fusion protein constructs, we demonstrated that IA-1 functions as a transcriptional repressor and that the region between amino acids 168 and 263 contains the maj...

  18. Specific binding of the activator Mga to promoter sequences of the emm and scpA genes in the group A streptococcus.

    OpenAIRE

    McIver, K S; Heath, A S; Green, B.D.; J. R. Scott

    1995-01-01

    Transcription of the surface-associated virulence factors of the group A streptococcus (GAS) Streptococcus pyogenes, M protein (emm) and the C5a peptidase (scpA), is activated by a protein called Mga (formerly Mry or VirR). To determine whether Mga binds directly to the promoters of the genes it regulates, a protein resulting from the fusion of Mga to the C-terminal end of maltose-binding protein was purified from Escherichia coli. Specific binding to the promoter regions of the scpA and emm ...

  19. Structural insights into parasite eIF4E binding specificity for m7G and m2,2,7G mRNA caps.

    Science.gov (United States)

    Liu, Weizhi; Zhao, Rui; McFarland, Craig; Kieft, Jeffrey; Niedzwiecka, Anna; Jankowska-Anyszka, Marzena; Stepinski, Janusz; Darzynkiewicz, Edward; Jones, David N M; Davis, Richard E

    2009-11-01

    The eukaryotic translation initiation factor eIF4E recognizes the mRNA cap, a key step in translation initiation. Here we have characterized eIF4E from the human parasite Schistosoma mansoni. Schistosome mRNAs have either the typical monomethylguanosine (m(7)G) or a trimethylguanosine (m(2,2,7)G) cap derived from spliced leader trans-splicing. Quantitative fluorescence titration analyses demonstrated that schistosome eIF4E has similar binding specificity for both caps. We present the first crystal structure of an eIF4E with similar binding specificity for m(7)G and m(2,2,7)G caps. The eIF4E.m(7)GpppG structure demonstrates that the schistosome protein binds monomethyl cap in a manner similar to that of single specificity eIF4Es and exhibits a structure similar to other known eIF4Es. The structure suggests an alternate orientation of a conserved, key Glu-90 in the cap-binding pocket that may contribute to dual binding specificity and a position for mRNA bound to eIF4E consistent with biochemical data. Comparison of NMR chemical shift perturbations in schistosome eIF4E on binding m(7)GpppG and m(2,2,7)GpppG identified key differences between the two complexes. Isothermal titration calorimetry demonstrated significant thermodynamics differences for the binding process with the two caps (m(7)G versus m(2,2,7)G). Overall the NMR and isothermal titration calorimetry data suggest the importance of intrinsic conformational flexibility in the schistosome eIF4E that enables binding to m(2,2,7)G cap. PMID:19710013

  20. Expression and inducibility in Staphylococcus aureus of the mecA gene, which encodes a methicillin-resistant S. aureus-specific penicillin-binding protein.

    OpenAIRE

    Ubukata, K; Nonoguchi, R; Matsuhashi, M; Konno, M

    1989-01-01

    A beta-lactam-sensitive strain of Staphylococcus aureus could be converted to methicillin resistance by the introduction of a plasmid carrying the 4.3-kilobase HindIII chromosomal DNA fragment which encoded the mecA gene from a methicillin-resistant S. aureus. Transformant cells produced methicillin-resistant S. aureus-specific penicillin-binding protein constitutively, and additional insertion of an inducible penicillinase plasmid caused production of the pencillin-binding protein to become ...

  1. B- and C-RAF display essential differences in their binding to Ras: the isotype-specific N terminus of B-RAF facilitates Ras binding.

    Science.gov (United States)

    Fischer, Andreas; Hekman, Mirko; Kuhlmann, Jürgen; Rubio, Ignacio; Wiese, Stefan; Rapp, Ulf R

    2007-09-01

    Recruitment of RAF kinases to the plasma membrane was initially proposed to be mediated by Ras proteins via interaction with the RAF Ras binding domain (RBD). Data reporting that RAF kinases possess high affinities for particular membrane lipids support a new model in which Ras-RAF interactions may be spatially restricted to the plane of the membrane. Although the coupling features of Ras binding to the isolated RAF RBD were investigated in great detail, little is known about the interactions of the processed Ras with the functional and full-length RAF kinases. Here we present a quantitative analysis of the binding properties of farnesylated and nonfarnesylated H-Ras to both full-length B- and C-RAF in the presence and absence of lipid environment. Although isolated RBD fragments associate with high affinity to both farnesylated and nonfarnesylated H-Ras, the full-length RAF kinases revealed fundamental differences with respect to Ras binding. In contrast to C-RAF that requires farnesylated H-Ras, cytosolic B-RAF associates effectively and with significantly higher affinity with both farnesylated and nonfarnesylated H-Ras. To investigate the potential farnesyl binding site(s) we prepared several N-terminal fragments of C-RAF and found that in the presence of cysteine-rich domain only the farnesylated form of H-Ras binds with high association rates. The extreme N terminus of B-RAF turned out to be responsible for the facilitation of lipid independent Ras binding to B-RAF, since truncation of this region resulted in a protein that changed its kinase properties and resembles C-RAF. In vivo studies using PC12 and COS7 cells support in vitro results. Co-localization measurements using labeled Ras and RAF documented essential differences between B- and C-RAF with respect to association with Ras. Taken together, these data suggest that the activation of B-RAF, in contrast to C-RAF, may take place both at the plasma membrane and in the cytosolic environment.

  2. Crystallography of a Lewis-binding norovirus, elucidation of strain-specificity to the polymorphic human histo-blood group antigens.

    Directory of Open Access Journals (Sweden)

    Yutao Chen

    2011-07-01

    Full Text Available Noroviruses, an important cause of acute gastroenteritis in humans, recognize the histo-blood group antigens (HBGAs as host susceptible factors in a strain-specific manner. The crystal structures of the HBGA-binding interfaces of two A/B/H-binding noroviruses, the prototype Norwalk virus (GI.1 and a predominant GII.4 strain (VA387, have been elucidated. In this study we determined the crystal structures of the P domain protein of the first Lewis-binding norovirus (VA207, GII.9 that has a distinct binding property from those of Norwalk virus and VA387. Co-crystallization of the VA207 P dimer with Le(y or sialyl Le(x tetrasaccharides showed that VA207 interacts with these antigens through a common site found on the VA387 P protein which is highly conserved among most GII noroviruses. However, the HBGA-binding site of VA207 targeted at the Lewis antigens through the α-1, 3 fucose (the Lewis epitope as major and the β-N-acetyl glucosamine of the precursor as minor interacting sites. This completely differs from the binding mode of VA387 and Norwalk virus that target at the secretor epitopes. Binding pocket of VA207 is formed by seven amino acids, of which five residues build up the core structure that is essential for the basic binding function, while the other two are involved in strain-specificity. Our results elucidate for the first time the genetic and structural basis of strain-specificity by a direct comparison of two genetically related noroviruses in their interaction with different HBGAs. The results provide insight into the complex interaction between the diverse noroviruses and the polymorphic HBGAs and highlight the role of human HBGA as a critical factor in norovirus evolution.

  3. The lectin domains of polypeptide GalNAc-transferases exhibit carbohydrate-binding specificity for GalNAc: lectin binding to GalNAc-glycopeptide substrates is required for high density GalNAc-O-glycosylation

    DEFF Research Database (Denmark)

    Wandall, Hans H; Irazoqui, Fernando; Tarp, Mads Agervig;

    2007-01-01

    to the enzyme. We have previously shown that the lectin domain of GalNAc-T4 modulates its substrate specificity to enable unique GalNAc-glycopeptide specificities and that this effect is selectively inhibitable by GalNAc; however, direct evidence of carbohydrate binding of GalNAc-transferase lectins has......-T2). Both lectins exhibited specificity for binding of free GalNAc. Kinetic and time-course analysis of GalNAc-T2 demonstrated that the lectin domain did not affect transfer to initial glycosylation sites, but selectively modulated velocity of transfer to subsequent sites and affected the number......Initiation of mucin-type O-glycosylation is controlled by a large family of UDP GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). Most GalNAc-transferases contain a ricin-like lectin domain in the C-terminal end, which may confer GalNAc-glycopeptide substrate specificity...

  4. Direct binding of specific AUF1 isoforms to tandem zinc finger domains of tristetraprolin (TTP) family proteins.

    Science.gov (United States)

    Kedar, Vishram P; Zucconi, Beth E; Wilson, Gerald M; Blackshear, Perry J

    2012-02-17

    Tristetraprolin (TTP) is the prototype of a family of CCCH tandem zinc finger proteins that can bind to AU-rich elements in mRNAs and promote their decay. TTP binds to mRNA through its central tandem zinc finger domain; it then promotes mRNA deadenylation, considered to be the rate-limiting step in eukaryotic mRNA decay. We found that TTP and its related family members could bind to certain isoforms of another AU-rich element-binding protein, HNRNPD/AUF1, as well as a related protein, laAUF1. The interaction domain within AUF1p45 appeared to be a C-terminal "GY" region, and the interaction domain within TTP was the tandem zinc finger domain. Surprisingly, binding of AUF1p45 to TTP occurred even with TTP mutants that lacked RNA binding activity. In cell extracts, binding of AUF1p45 to TTP potentiated TTP binding to ARE-containing RNA probes, as determined by RNA gel shift assays; AUF1p45 did not bind to the RNA probes under these conditions. Using purified, recombinant proteins and a synthetic RNA target in FRET assays, we demonstrated that AUF1p45, but not AUF1p37, increased TTP binding affinity for RNA ∼5-fold. These data suggest that certain isoforms of AUF1 can serve as "co-activators" of TTP family protein binding to RNA. The results raise interesting questions about the ability of AUF1 isoforms to regulate the mRNA binding and decay-promoting activities of TTP and its family members as well as the ability of AUF1 proteins to serve as possible physical links between TTP and other mRNA decay proteins and structures.

  5. 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.

    2007-01-01

    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. Eac

  6. N,N',N"-tris(dihydroxyphosphorylmethyl)-1,4,7-triazacyclononane (Deofix) - a high-affinity, high-specificity chelator for first transition series metal cations with significant deodorant, antimicrobial, and antioxidant activity.

    Science.gov (United States)

    Laden, Karl; Zaklad, Haim; Simhon, Elliot D; Klein, Joseph Y; Cyjon, Rosa L; Winchell, Harry S

    2003-01-01

    Deofix, N,N',N"-tris(dihydroxyphosphorylmethyl)-1,4,7-triazacyclononane, is a high-affinity, high-specificity chelator for first transition series cations such as iron, zinc, manganese, and copper. A 1% solution in 50% ethanol was found to be significantly better at reducing underarm malodor than a solution of 0.3% Triclosan in 50% ethanol. Compared to a 50% alcohol control, Deofix was found to produce a significant reduction in malodor for at least 48 hours. Deofix appears to work by reducing the concentration of first transition series metal ions below the levels needed for microbial cell reproduction and by inhibiting oxidative processes by interfering with catalytic formation of free radicals. Deofix has very low levels of toxicity when measured via a number of screening techniques.

  7. Structural analysis reveals the substrate-binding mechanism for the expanded substrate specificity of mutant meso-diaminopimelate dehydrogenase.

    Science.gov (United States)

    Liu, Weidong; Guo, Rey-Ting; Chen, Xi; Li, Zhe; Gao, Xiuzhen; Huang, Chun-Hsiang; Wu, Qiaqing; Feng, Jinhui; Zhu, Dunming

    2015-04-13

    A meso-diaminopimelate dehydrogenase (DAPDH) from Clostridium tetani E88 (CtDAPDH) was found to have low activity toward the D-amino acids other than its native substrate. Site-directed mutagenesis similar to that carried out on the residues mutated by Vedha-Peters et al. resulted in a mutant enzyme with highly improved catalytic ability for the synthesis of D-amino acids. The crystal structures of the CtDAPDH mutant in apo form and in complex with meso-diaminopimelate (meso-DAP), D-leucine (D-leu), and 4-methyl-2-oxopentanoic acid (MOPA) were solved. meso-DAP was found in an area outside the catalytic cavity; this suggested a possible two-step substrate-binding mechanism for meso-DAP. D-leu and MOPA each bound both to Leu154 and to Gly155 in the open form of CtDAPDH, and structural analysis revealed the molecular basis for the expanded substrate specificity of the mutant meso-diaminopimelate dehydrogenases. PMID:25754803

  8. Strain-specific V3 and CD4 binding site autologous HIV-1 neutralizing antibodies select neutralization-resistant viruses

    Science.gov (United States)

    Moody, M. Anthony; Gao, Feng; Gurley, Thaddeus C.; Amos, Joshua D.; Kumar, Amit; Hora, Bhavna; Marshall, Dawn J.; Whitesides, John F.; Xia, Shi-Mao; Parks, Robert; Lloyd, Krissey E.; Hwang, Kwan-Ki; Lu, Xiaozhi; Bonsignori, Mattia; Finzi, Andrés; Vandergrift, Nathan A.; Alam, S. Munir; Ferrari, Guido; Shen, Xiaoying; Tomaras, Georgia D.; Kamanga, Gift; Cohen, Myron S.; Sam, Noel E.; Kapiga, Saidi; Gray, Elin S.; Tumba, Nancy L.; Morris, Lynn; Zolla-Pazner, Susan; Gorny, Miroslaw K.; Mascola, John R.; Hahn, Beatrice; Shaw, George M.; Sodroski, Joseph G.; Liao, Hua-Xin; Montefiori, David C.; Hraber, Peter T.; Korber, Bette T.; Haynes, Barton F.

    2015-01-01

    Summary The third variable (V3) loop and the CD4 binding site (CD4bs) of the HIV-1 envelope are frequently targeted by neutralizing antibodies (nAbs) in infected individuals. In chronic infection, HIV-1 escape mutants repopulate the plasma, and V3 and CD4bs nAbs emerge that can neutralize heterologous tier 1 easy-to-neutralize, but not tier 2 difficult-to-neutralize HIV-1 isolates. However, neutralization sensitivity of autologous plasma viruses to this type of nAb response has not been studied. We describe the development and evolution in vivo of antibodies distinguished by their target specificity for V3and CD4bs epitopes on autologous tier 2 viruses but not on heterologous tier 2 viruses. A surprisingly high fraction of autologous circulating viruses was sensitive to these antibodies. These findings demonstrate a role for V3 and CD4bs antibodies in constraining the native envelope trimer in vivo to a neutralization-resistant phenotype, explaining why HIV-1 transmission generally occurs by tier 2 neutralization-resistant viruses. PMID:26355218

  9. Structural studies of ROK fructokinase YdhR from Bacillus subtilis : insights into substrate binding and fructose specificity.

    Energy Technology Data Exchange (ETDEWEB)

    Nocek, B.; Stein, A.; Jedrzejczak, R.; Cuff, M.; Li, H.; Volkart, L.; Joachimiak, A.; Biosciences Division

    2011-02-18

    The main pathway of bacterial sugar phosphorylation utilizes specific phosphoenolpyruvate phosphotransferase system (PTS) enzymes. In addition to the classic PTS system, a PTS-independent secondary system has been described in which nucleotide-dependent sugar kinases are used for monosaccharide phosphorylation. Fructokinase (FK), which phosphorylates d-fructose with ATP as a cofactor, has been shown to be a member of this secondary system. Bioinformatic analysis has shown that FK is a member of the 'ROK' (bacterial Repressors, uncharacterized Open reading frames, and sugar Kinases) sequence family. In this study, we report the crystal structures of ROK FK from Bacillus subtilis (YdhR) (a) apo and in the presence of (b) ADP and (c) ADP/d-fructose. All structures show that YdhR is a homodimer with a monomer composed of two similar {alpha}/{beta} domains forming a large cleft between domains that bind ADP and d-fructose. Enzymatic activity assays support YdhR function as an ATP-dependent fructose kinase.

  10. Microplate-based assay for identifying small molecules that bind a specific intersubunit interface within the assembled HIV-1 capsid.

    Science.gov (United States)

    Halambage, Upul D; Wong, Jason P; Melancon, Bruce J; Lindsley, Craig W; Aiken, Christopher

    2015-09-01

    Despite the availability of >30 effective drugs for managing HIV-1 infection, no current therapy is curative, and long-term management is challenging owing to the emergence and spread of drug-resistant mutants. Identification of drugs against novel HIV-1 targets would expand the current treatment options and help to control resistance. The highly conserved HIV-1 capsid protein represents an attractive target because of its multiple roles in replication of the virus. However, the low antiviral potencies of the reported HIV-1 capsid-targeting inhibitors render them unattractive for therapeutic development. To facilitate the identification of more-potent HIV-1 capsid inhibitors, we developed a scintillation proximity assay to screen for small molecules that target a biologically active and specific intersubunit interface in the HIV-1 capsid. The assay, which is based on competitive displacement of a known capsid-binding small-molecule inhibitor, exhibited a signal-to-noise ratio of >9 and a Z factor of >0.8. In a pilot screen of a chemical library containing 2,400 druglike compounds, we obtained a hit rate of 1.8%. This assay has properties that are suitable for screening large compound libraries to identify novel HIV-1 capsid ligands with antiviral activity. PMID:26077250

  11. Combinatorial binding leads to diverse regulatory responses: Lmd is a tissue-specific modulator of Mef2 activity.

    Directory of Open Access Journals (Sweden)

    Paulo M F Cunha

    2010-07-01

    Full Text Available Understanding how complex patterns of temporal and spatial expression are regulated is central to deciphering genetic programs that drive development. Gene expression is initiated through the action of transcription factors and their cofactors converging on enhancer elements leading to a defined activity. Specific constellations of combinatorial occupancy are therefore often conceptualized as rigid binding codes that give rise to a common output of spatio-temporal expression. Here, we assessed this assumption using the regulatory input of two essential transcription factors within the Drosophila myogenic network. Mutations in either Myocyte enhancing factor 2 (Mef2 or the zinc-finger transcription factor lame duck (lmd lead to very similar defects in myoblast fusion, yet the underlying molecular mechanism for this shared phenotype is not understood. Using a combination of ChIP-on-chip analysis and expression profiling of loss-of-function mutants, we obtained a global view of the regulatory input of both factors during development. The majority of Lmd-bound enhancers are co-bound by Mef2, representing a subset of Mef2's transcriptional input during these stages of development. Systematic analyses of the regulatory contribution of both factors demonstrate diverse regulatory roles, despite their co-occupancy of shared enhancer elements. These results indicate that Lmd is a tissue-specific modulator of Mef2 activity, acting as both a transcriptional activator and repressor, which has important implications for myogenesis. More generally, this study demonstrates considerable flexibility in the regulatory output of two factors, leading to additive, cooperative, and repressive modes of co-regulation.

  12. 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.

  13. Crystal and solution studies of the "Plus-C" odorant-binding protein 48 from Anopheles gambiae: control of binding specificity through three-dimensional domain swapping.

    Science.gov (United States)

    Tsitsanou, Katerina E; Drakou, Christina E; Thireou, Trias; Vitlin Gruber, Anna; Kythreoti, Georgia; Azem, Abdussalam; Fessas, Dimitrios; Eliopoulos, Elias; Iatrou, Kostas; Zographos, Spyros E

    2013-11-15

    Much physiological and behavioral evidence has been provided suggesting that insect odorant-binding proteins (OBPs) are indispensable for odorant recognition and thus are appealing targets for structure-based discovery and design of novel host-seeking disruptors. Despite the fact that more than 60 putative OBP-encoding genes have been identified in the malaria vector Anopheles gambiae, the crystal structures of only six of them are known. It is therefore clear that OBP structure determination constitutes the bottleneck for structure-based approaches to mosquito repellent/attractant discovery. Here, we describe the three-dimensional structure of an A. gambiae "Plus-C" group OBP (AgamOBP48), which exhibits the second highest expression levels in female antennae. This structure represents the first example of a three-dimensional domain-swapped dimer in dipteran species. A combined binding site is formed at the dimer interface by equal contribution of each monomer. Structural comparisons with the monomeric AgamOBP47 revealed that the major structural difference between the two Plus-C proteins localizes in their N- and C-terminal regions, and their concerted conformational change may account for monomer-swapped dimer conversion and furthermore the formation of novel binding pockets. Using a combination of gel filtration chromatography, differential scanning calorimetry, and analytical ultracentrifugation, we demonstrate the AgamOBP48 dimerization in solution. Eventually, molecular modeling calculations were used to predict the binding mode of the most potent synthetic ligand of AgamOBP48 known so far, discovered by ligand- and structure-based virtual screening. The structure-aided identification of multiple OBP binders represents a powerful tool to be employed in the effort to control transmission of the vector-borne diseases. PMID:24097978

  14. De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks

    KAUST Repository

    Mahfouz, Magdy M.

    2011-01-24

    Site-specific and rare cutting nucleases are valuable tools for genome engineering. The generation of double-strand DNA breaks (DSBs) promotes homologous recombination in eukaryotes and can facilitate gene targeting, additions, deletions, and inactivation. Zinc finger nucleases have been used to generate DSBs and subsequently, for genome editing but with low efficiency and reproducibility. The transcription activator-like family of type III effectors (TALEs) contains a central domain of tandem repeats that could be engineered to bind specific DNA targets. Here, we report the generation of a Hax3-based hybrid TALE nuclease with a user-selected DNA binding specificity. We show that the engineered TALE nuclease can bind to its target sequence in vitro and that the homodimeric TALE nuclease can cleave double-stranded DNA in vitro if the DNA binding sites have the proper spacing and orientation. Transient expression assays in tobacco leaves suggest that the hybrid nuclease creates DSB in its target sequence, which is subsequently repaired by nonhomologous end-joining repair. Taken together, our data show the feasibility of engineering TALE-based hybrid nucleases capable of generating site-specific DSBs and the great potential for site-specific genome modification in plants and eukaryotes in general.

  15. Effect of phosphorothioate modifications on the ability of GTn oligodeoxynucleotides to specifically recognize single-stranded DNA-binding proteins and to affect human cancer cellular growth.

    Science.gov (United States)

    Morassutti, C; Scaggiante, B; Dapas, B; Xodo, L; Tell, G; Quadrifoglio, F

    1999-12-01

    We have previously identified phosphodiester oligonucleotides exclusively made of G and T bases, named GTn, that significantly inhibit human cancer cell growth and recognize specific nuclear single-stranded DNA binding proteins. We wished to examine the ability of the modified GTn oligonucleotides with different degrees of phosphorothioate modifications to bind specifically to the same nuclear proteins recognized by the GTn phosphodiester analogues and their cytotoxic effect on the human T-lymphoblastic CCRF-CEM cell line. We showed that the full phosphorothioate GTn oligonucleotide was neither able to specifically recognize those nuclear proteins, nor cytotoxic. In contrast, the 3'-phosphorothioate-protected GTn oligonucleotides can maintain the specific protein-binding activity. The end-modified phosphorothioate oligonucleotides were also able to elicit the dose-dependent cell growth inhibition effect, but a loss in the cytotoxic ability was observed increasing the extent of sulphur modification of the sequences. Our results indicate that phosphorothioate oligonucleotides directed at specific single-stranded DNA-binding proteins should contain a number of phosphorothioate end-linkages which should be related to the length of the sequence, in order to maintain the same biological activities exerted by their phosphodiester analogues.

  16. BayesPI - a new model to study protein-DNA interactions: a case study of condition-specific protein binding parameters for Yeast transcription factors

    Directory of Open Access Journals (Sweden)

    Morigen

    2009-10-01

    Full Text Available Abstract Background We have incorporated Bayesian model regularization with biophysical modeling of protein-DNA interactions, and of genome-wide nucleosome positioning to study protein-DNA interactions, using a high-throughput dataset. The newly developed method (BayesPI includes the estimation of a transcription factor (TF binding energy matrices, the computation of binding affinity of a TF target site and the corresponding chemical potential. Results The method was successfully tested on synthetic ChIP-chip datasets, real yeast ChIP-chip experiments. Subsequently, it was used to estimate condition-specific and species-specific protein-DNA interaction for several yeast TFs. Conclusion The results revealed that the modification of the protein binding parameters and the variation of the individual nucleotide affinity in either recognition or flanking sequences occurred under different stresses and in different species. The findings suggest that such modifications may be adaptive and play roles in the formation of the environment-specific binding patterns of yeast TFs and in the divergence of TF binding sites across the related yeast species.

  17. A novel site contributing to growth-arrest-specific gene 6 binding to its receptors as revealed by a human monoclonal antibody

    Science.gov (United States)

    2004-01-01

    Gas6 (growth-arrest-specific gene 6) is a vitamin K-dependent protein known to activate the Axl family of receptor tyrosine kinases. It is an important regulator of thrombosis and many other biological functions. The C-terminus of Gas6 binds to receptors and consists of two laminin-like globular domains LG1 and LG2. It has been reported that a Ca2+-binding site at the junction of LG1 and LG2 domains and a hydrophobic patch at the LG2 domain are important for receptor binding [Sasaki, Knyazev, Cheburkin, Gohring, Tisi, Ullrich, Timpl and Hohenester (2002) J. Biol. Chem. 277, 44164–44170]. In the present study, we developed a neutralizing human monoclonal antibody, named CNTO300, for Gas6. The antibody was generated by immunization of human IgG-expressing transgenic mice with recombinant human Gas6 protein and the anti-Gas6 IgG sequences were rescued from an unstable hybridoma clone. Binding of Gas6 to its receptors was partially inhibited by the CNTO300 antibody in a dose-dependent manner. To characterize further the interaction between Gas6 and this antibody, the binding kinetics of CNTO300 for recombinant Gas6 were compared with independently expressed LG1 and LG2. The CNTO300 antibody showed comparable binding affinity, yet different dependence on Ca2+, to Gas6 and LG1. No binding to LG2 was detected. In the presence of EDTA, binding of the antibody to Gas6 was disrupted, but no significant effect of EDTA on LG1 binding was evident. Further epitope mapping identified a Gas6 peptide sequence recognized by the CNTO300 antibody. This peptide sequence was found to be located at the LG1 domain distant from the Ca2+-binding site and the hydrophobic patch. Co-interaction of Gas6 with its receptor and CNTO300 antibody was detected by BIAcore analysis, suggesting a second receptor-binding site on the LG1 domain. This hypothesis was further supported by direct binding of Gas6 receptors to an independently expressed LG1 domain. Our results revealed, for the first time, a

  18. Impact of cadmium, cobalt and nickel on sequence-specific DNA binding of p63 and p73 in vitro and in cells

    International Nuclear Information System (INIS)

    Highlights: • DNA binding of p53 family core domains is inhibited by cadmium, cobalt and nickel. • Binding to DNA protects p53 family core domains from metal induced inhibition. • Cadmium, cobalt and nickel induced inhibition was reverted by EDTA in vitro. - Abstract: Site-specific DNA recognition and binding activity belong to common attributes of all three members of tumor suppressor p53 family proteins: p53, p63 and p73. It was previously shown that heavy metals can affect p53 conformation, sequence-specific binding and suppress p53 response to DNA damage. Here we report for the first time that cadmium, nickel and cobalt, which have already been shown to disturb various DNA repair mechanisms, can also influence p63 and p73 sequence-specific DNA binding activity and transactivation of p53 family target genes. Based on results of electrophoretic mobility shift assay and luciferase reporter assay, we conclude that cadmium inhibits sequence-specific binding of all three core domains to p53 consensus sequences and abolishes transactivation of several promoters (e.g. BAX and MDM2) by 50 μM concentrations. In the presence of specific DNA, all p53 family core domains were partially protected against loss of DNA binding activity due to cadmium treatment. Effective cadmium concentration to abolish DNA–protein interactions was about two times higher for p63 and p73 proteins than for p53. Furthermore, we detected partial reversibility of cadmium inhibition for all p53 family members by EDTA. DTT was able to reverse cadmium inhibition only for p53 and p73. Nickel and cobalt abolished DNA–p53 interaction at sub-millimolar concentrations while inhibition of p63 and p73 DNA binding was observed at millimolar concentrations. In summary, cadmium strongly inhibits p53, p63 and p73 DNA binding in vitro and in cells in comparison to nickel and cobalt. The role of cadmium inhibition of p53 tumor suppressor family in carcinogenesis is discussed

  19. Impact of cadmium, cobalt and nickel on sequence-specific DNA binding of p63 and p73 in vitro and in cells

    Energy Technology Data Exchange (ETDEWEB)

    Adámik, Matej [Institute of Biophysics, Academy of Science of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno (Czech Republic); Bažantová, Pavla [Institute of Biophysics, Academy of Science of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno (Czech Republic); Department of Biology and Ecology, Faculty of Science, University of Ostrava, Chittussiho 10, 701 03 Ostrava (Czech Republic); Navrátilová, Lucie; Polášková, Alena [Institute of Biophysics, Academy of Science of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno (Czech Republic); Pečinka, Petr [Institute of Biophysics, Academy of Science of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno (Czech Republic); Department of Biology and Ecology, Faculty of Science, University of Ostrava, Chittussiho 10, 701 03 Ostrava (Czech Republic); Holaňová, Lucie [Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Palackého 1/3, 61242 Brno (Czech Republic); Tichý, Vlastimil [Institute of Biophysics, Academy of Science of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno (Czech Republic); Brázdová, Marie, E-mail: maruska@ibp.cz [Institute of Biophysics, Academy of Science of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno (Czech Republic); Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Palackého 1/3, 61242 Brno (Czech Republic)

    2015-01-02

    Highlights: • DNA binding of p53 family core domains is inhibited by cadmium, cobalt and nickel. • Binding to DNA protects p53 family core domains from metal induced inhibition. • Cadmium, cobalt and nickel induced inhibition was reverted by EDTA in vitro. - Abstract: Site-specific DNA recognition and binding activity belong to common attributes of all three members of tumor suppressor p53 family proteins: p53, p63 and p73. It was previously shown that heavy metals can affect p53 conformation, sequence-specific binding and suppress p53 response to DNA damage. Here we report for the first time that cadmium, nickel and cobalt, which have already been shown to disturb various DNA repair mechanisms, can also influence p63 and p73 sequence-specific DNA binding activity and transactivation of p53 family target genes. Based on results of electrophoretic mobility shift assay and luciferase reporter assay, we conclude that cadmium inhibits sequence-specific binding of all three core domains to p53 consensus sequences and abolishes transactivation of several promoters (e.g. BAX and MDM2) by 50 μM concentrations. In the presence of specific DNA, all p53 family core domains were partially protected against loss of DNA binding activity due to cadmium treatment. Effective cadmium concentration to abolish DNA–protein interactions was about two times higher for p63 and p73 proteins than for p53. Furthermore, we detected partial reversibility of cadmium inhibition for all p53 family members by EDTA. DTT was able to reverse cadmium inhibition only for p53 and p73. Nickel and cobalt abolished DNA–p53 interaction at sub-millimolar concentrations while inhibition of p63 and p73 DNA binding was observed at millimolar concentrations. In summary, cadmium strongly inhibits p53, p63 and p73 DNA binding in vitro and in cells in comparison to nickel and cobalt. The role of cadmium inhibition of p53 tumor suppressor family in carcinogenesis is discussed.

  20. Investigation of the Binding Interaction of Fatty Acids with Human G Protein-Coupled Receptor 40 Using a Site-Specific Fluorescence Probe by Flow Cytometry.

    Science.gov (United States)

    Ren, Xiao-Min; Cao, Lin-Ying; Zhang, Jing; Qin, Wei-Ping; Yang, Yu; Wan, Bin; Guo, Liang-Hong

    2016-04-01

    Human G protein-coupled receptor 40 (hGPR40), with medium- and long-chain free fatty acids (FFAs) as its natural ligands, plays an important role in the enhancement of glucose-dependent insulin secretion. To date, information about the direct binding of FFAs to hGPR40 is very limited, and how carbon-chain length affects the activities of FFAs on hGPR40 is not yet understood. In this study, a fluorescein-fasiglifam analogue (F-TAK-875A) conjugate was designed and synthesized as a site-specific fluorescence probe to study the interaction of FFAs with hGPR40. hGPR40 was expressed in human embryonic kidney 293 cells and labeled with F-TAK-875A. By using flow cytometry, competitive binding of FFA and F-TAK-875A to hGPR40-expressed cells was measured. Binding affinities of 18 saturated FFAs, with carbon-chain lengths ranging from C6 to C23, were analyzed. The results showed that the binding potencies of FFAs to hGPR40 were dependent on carbon length. There was a positive correlation between length and binding potency for seven FFAs (C9-C15), with myristic acid (C15) showing the highest potency, 0.2% relative to TAK-875. For FFAs with a length of fewer than C9 or more than C15, they had very weak or no binding. Molecular docking results showed that the binding pocket of TAK-875 in hGPR40 could enclose FFAs with lengths of C15 or fewer. However, for FFAs with lengths longer than C15, part of the alkyl chain extended out of the binding pocket. This study provided insights into the structural dependence of FFAs binding to and activation of hGPR40.

  1. Material Specific Rational Design of A1B2C3O7 High-Tc Superconductors without Copper [A, B, C = Cations

    Science.gov (United States)

    Isikaku-Ironkwe, O'paul; Schaffer, Michael J.

    Soon after the discovery of YBa2Cu3O7 with Tc = 93K, a similar structured system with Ag replacing Cu was discovered with a Tc = 50K. Also, the discovery of Ba0 . 6 K0 . 4 BiO3 with Tc = 30K indicated that Cu was not indispensable for high temperature superconductivity (HTSC). Latter, the discoveries of the Pnictide and Chalcogenide high-Tc superconductors confirmed those earlier experimental indications. Using our recently developed Material Specific Characterization Dataset (MSCD) model for analysis and design of superconductors, we have computed many designs that satisfy the MSCD characteristics of YBa2Cu3O7 as a design model. Our design recognizes the valence state characteristics that make YBa2Cu3O6 a semiconductor, while YBa2Cu3O7is a superconductor. Here we present ten material specific rational design examples of potential A1B2C3O7 HTSCs without Cu, using the YBa2Cu3O7 design model. This MSCD design model opens the possibility for search and discovery of high-Tc oxide superconductor systems without copper.

  2. Identification of the development stage—specific factors in mouse fetal liver binding to the human β—globin gene promoter

    Institute of Scientific and Technical Information of China (English)

    CHENYADI; YULONGHU; 等

    1994-01-01

    In order to elucidate the molecular mechanisms of globin gene expression during embryonic development,the nuclear extracts from mouse hematopoietic tissue at different stages of development have been prepared.By using DNase I footprinting and gel mobility shift assays,the binding of protein factors in these extracts to the human β-globin promoter was analyzed.The differences in the binding patterns of protein factors during development were observed.An erythroid-specific and stage-specific nuclear protein in the nuclear extrace from d 18 mouse fetal liver was identified,which can bind to the sequence(from-66bp to-90bp) of human β-globin promoter.We therefore speculate that the function of this cis-acting element may be similar to stage selector element(SSE) in chicken βA-promoter.

  3. Multiple POU-binding motifs, recognized by tissue-specific nuclear factors, are important for Dll1 gene expression in neural stem cells

    International Nuclear Information System (INIS)

    We cloned the 5'-flanking region of the mouse homolog of the Delta gene (Dll1) and demonstrated that the sequence between nucleotide position -514 and -484 in the 5'-flanking region of Dll1 played a critical role in the regulation of its tissue-specific expression in neural stem cells (NSCs). Further, we showed that multiple POU-binding motifs, located within this short sequence of 30 bp, were essential for transcriptional activation of Dll1 and also that multiple tissue-specific nuclear factors recognized these POU-binding motifs in various combinations through differentiation of NSCs. Thus, POU-binding factors may play an important role in Dll1 expression in developing NSCs

  4. Transcriptional activation requires protection of the TATA-binding protein Tbp1 by the ubiquitin-specific protease Ubp3.

    Science.gov (United States)

    Chew, Boon Shang; Siew, Wee Leng; Xiao, Benjamin; Lehming, Norbert

    2010-11-01

    Tbp1, the TATA-binding protein, is essential for transcriptional activation, and Gal4 and Gcn4 are unable to fully activate transcription in a Saccharomyces cerevisiae TBP1E86D mutant strain. In the present study we have shown that the Tbp1E186D mutant protein is proteolytically instable, and we have isolated intragenic and extragenic suppressors of the transcription defects of the TBP1E186D mutant strain. The TBP1R6S mutation stabilizes the Tbp1E186D mutant protein and suppresses the defects of the TBP1E186D mutant strain. Furthermore, we found that the overexpression of the de-ubiquitinating enzyme Ubp3 (ubiquitin-specific protease 3) also stabilizes the Tbp1E186D mutant protein and suppresses of the defects of the TBP1E186D mutant strain. Importantly, the deletion of UBP3 and its cofactor BRE5 lead to increased degradation of wild-type Tbp1 protein and to defects in transcriptional activation by Gal4 and Gcn4. Purified GST (glutathione transferase)-Ubp3 reversed Tbp1 ubiquitination, and the deletion of UBP3 lead to the accumulation of poly-ubiquitinated species of Tbp1 in a proteaseome-deficient genetic background, demonstrating that Ubp3 reverses ubiquitination of Tbp1 in vitro and in vivo. Chromatin immunoprecipitation showed that Ubp3 was recruited to the GAL1 and HIS3 promoters upon the induction of the respective gene, indicating that protection of promoter-bound Tbp1 by Ubp3 is required for transcriptional activation.

  5. Drosophila Syncrip binds the gurken mRNA localisation signal and regulates localised transcripts during axis specification

    Directory of Open Access Journals (Sweden)

    Suzanne M. McDermott

    2012-04-01

    In the Drosophila oocyte, mRNA transport and localised translation play a fundamental role in axis determination and germline formation of the future embryo. gurken mRNA encodes a secreted TGF-α signal that specifies dorsal structures, and is localised to the dorso-anterior corner of the oocyte via a cis-acting 64 nucleotide gurken localisation signal. Using GRNA chromatography, we characterised the biochemical composition of the ribonucleoprotein complexes that form around the gurken mRNA localisation signal in the oocyte. We identified a number of the factors already known to be involved in gurken localisation and translational regulation, such as Squid and Imp, in addition to a number of factors with known links to mRNA localisation, such as Me31B and Exu. We also identified previously uncharacterised Drosophila proteins, including the fly homologue of mammalian SYNCRIP/hnRNPQ, a component of RNA transport granules in the dendrites of mammalian hippocampal neurons. We show that Drosophila Syncrip binds specifically to gurken and oskar, but not bicoid transcripts. The loss-of-function and overexpression phenotypes of syncrip in Drosophila egg chambers show that the protein is required for correct grk and osk mRNA localisation and translational regulation. We conclude that Drosophila Syncrip is a new factor required for localisation and translational regulation of oskar and gurken mRNA in the oocyte. We propose that Syncrip/SYNCRIP is part of a conserved complex associated with localised transcripts and required for their correct translational regulation in flies and mammals.

  6. Serum amyloid A binds specific extracellular matrix glycoproteins and induces the adhesion of resting CD4+ T cells.

    Science.gov (United States)

    Preciado-Patt, L; Hershkoviz, R; Fridkin, M; Lider, O

    1996-02-01

    Serum amyloid A (SAA), a prototypic acute phase protein reactant, exists naturally in the serum of healthy individuals. However, the levels of SAA in serum and its presence in sites of inflammation increase during certain chronic diseases associated with a local elevation of cytokine concentrations. Although the chemical structure of SAA is defined, its putative immunologic role(s) is still obscure. Nevertheless, it has been shown that 1) SAA acts as a chemoattractant and regulator of the migration of monocytes, polymorphonuclear cells, and T lymphocytes through endothelial cell monolayers; and 2) SAA and its proteolytically degraded N-terminal amyloid A fragment contain an extracellular matrix (ECM)-related cell adhesion epitopes. Herein, we examined whether SAA can associate with specific ECM moieties, and whether immobilized SAA-ECM complexes affect T lymphocyte adhesion. Radiolabeled human rSAA ([125I]rSAA) interacted avidly (Kd = 10(-9) M) and transiently with intact ECM, laminin, and vitronectin, but not with fibronectin or collagen type II. The binding of [125I]rSAA to ECM and laminin was inhibited by unlabeled rSAA and by the AA fragment, but not by the C-terminal portion of SAA (amino acid residues 2-82 and 77-104, respectively). Upon interactions with SAA or amyloid A, immobilized ECM, laminin, and vitronectin induced the adhesion of resting human CD4+ T cells in an apparently beta 1-integrin-mediated manner. Thus, the ECM appears to serve as a temporary anchorage site for SAA and amyloid A, and these ECM-complexed molecules seem to be involved in regulating the recruitment and accumulation of immunocytes in extravascular inflammatory compartments. PMID:8557997

  7. Mouse PRDM9 DNA-binding specificity determines sites of histone H3 lysine 4 trimethylation for initiation of meiotic recombination.

    Directory of Open Access Journals (Sweden)

    Corinne Grey

    2011-10-01

    Full Text Available Meiotic recombination generates reciprocal exchanges between homologous chromosomes (also called crossovers, COs that are essential for proper chromosome segregation during meiosis and are a major source of genome diversity by generating new allele combinations. COs have two striking properties: they occur at specific sites, called hotspots, and these sites evolve rapidly. In mammals, the Prdm9 gene, which encodes a meiosis-specific histone H3 methyltransferase, has recently been identified as a determinant of CO hotspots. Here, using transgenic mice, we show that the sole modification of PRDM9 zinc fingers leads to changes in hotspot activity, histone H3 lysine 4 trimethylation (H3K4me3 levels, and chromosome-wide distribution of COs. We further demonstrate by an in vitro assay that the PRDM9 variant associated with hotspot activity binds specifically to DNA sequences located at the center of the three hotspots tested. Remarkably, we show that mutations in cis located at hotspot centers and associated with a decrease of hotspot activity affect PRDM9 binding. Taken together, these results provide the direct demonstration that Prdm9 is a master regulator of hotspot localization through the DNA binding specificity of its zinc finger array and that binding of PRDM9 at hotspots promotes local H3K4me3 enrichment.

  8. 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

    Aberrant methylation of seven potential binding sites of the CTCF factor in the differentially methylated region upstream of the H19 gene (H19-DMR) has been suggested as critical for the regulation of IGF2 and H19 imprinted genes. In this study, we analyzed the allele-specific methylation pattern...

  9. The fission yeast RNA binding protein Mmi1 regulates meiotic genes by controlling intron specific splicing and polyadenylation coupled RNA turnover.

    Directory of Open Access Journals (Sweden)

    Huei-Mei Chen

    Full Text Available The polyA tails of mRNAs are monitored by the exosome as a quality control mechanism. We find that fission yeast, Schizosaccharomyces pombe, adopts this RNA quality control mechanism to regulate a group of 30 or more meiotic genes at the level of both splicing and RNA turnover. In vegetative cells the RNA binding protein Mmi1 binds to the primary transcripts of these genes. We find the novel motif U(U/C/GAAAC highly over-represented in targets of Mmi1. Mmi1 can specifically regulate the splicing of particular introns in a transcript: it inhibits the splicing of introns that are in the vicinity of putative Mmi1 binding sites, while allowing the splicing of other introns that are far from such sites. In addition, binding of Mmi1, particularly near the 3' end, alters 3' processing to promote extremely long polyA tails of up to a kilobase. The hyperadenylated transcripts are then targeted for degradation by the nuclear exonuclease Rrp6. The nuclear polyA binding protein Pab2 assists this hyperadenylation-mediated RNA decay. Rrp6 also targets other hyperadenylated transcripts, which become hyperadenylated in an unknown, but Mmi1-independent way. Thus, hyperadenylation may be a general signal for RNA degradation. In addition, binding of Mmi1 can affect the efficiency of 3' cleavage. Inactivation of Mmi1 in meiosis allows meiotic expression, through splicing and RNA stabilization, of at least 29 target genes, which are apparently constitutively transcribed.

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

    International Nuclear Information System (INIS)

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1988-11-01

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

  12. Mapping the tail fiber as the receptor binding protein responsible for differential host specificity of Pseudomonas aeruginosa bacteriophages PaP1 and JG004.

    Directory of Open Access Journals (Sweden)

    Shuai Le

    Full Text Available The first step in bacteriophage infection is recognition and binding to the host receptor, which is mediated by the phage receptor binding protein (RBP. Different RBPs can lead to differential host specificity. In many bacteriophages, such as Escherichia coli and Lactococcal phages, RBPs have been identified as the tail fiber or protruding baseplate proteins. However, the tail fiber-dependent host specificity in Pseudomonas aeruginosa phages has not been well studied. This study aimed to identify and investigate the binding specificity of the RBP of P. aeruginosa phages PaP1 and JG004. These two phages share high DNA sequence homology but exhibit different host specificities. A spontaneous mutant phage was isolated and exhibited broader host range compared with the parental phage JG004. Sequencing of its putative tail fiber and baseplate region indicated a single point mutation in ORF84 (a putative tail fiber gene, which resulted in the replacement of a positively charged lysine (K by an uncharged asparagine (N. We further demonstrated that the replacement of the tail fiber gene (ORF69 of PaP1 with the corresponding gene from phage JG004 resulted in a recombinant phage that displayed altered host specificity. Our study revealed the tail fiber-dependent host specificity in P. aeruginosa phages and provided an effective tool for its alteration. These contributions may have potential value in phage therapy.

  13. Human cyclophilin 33 (hCyP33) in T-cell binds specifically to poly(A)~+RNA (mRNA)

    Institute of Scientific and Technical Information of China (English)

    张万起; 袁直; 宓怀风; 元云峰; 何炳林; 王亦农

    2002-01-01

    Human cyclophilin 33 (hCyP33), found in 1996, consists of an RNA-binding domain in N-terminus, a cyclophilin domain in C-terminus and a connected part between the two domains. RNA-binding proteins concern functions, such as splicing, modification and transport, after transcription in eukaryotic cells. Cyclophilins (CyPs) possess enzymatic activity, namely peptidyl-proryl cis-trans isomerase (PPlase). They are involved in folding, transport and interaction of proteins. Cyclosporin A (CsA), an immunosuppressant used by organ transplantation, binds to CyPs and suppresses their enzymatic activity. However, up to now it is unknown that which cellular and physiological roles hCyP33, which possesses the above-mentioned both functions, plays. In this paper the binding specificity of hCyP33 to different cellular RNA is investigated by means of ion-exchange chromatography and affinity adsorption. The results show that it binds specifically to poly(A) tailed mRNA, namely poly(A)+RNA.

  14. Specific binding sites in the alcR and alcA promoters of the ethanol regulon for the CREA repressor mediating carbon catabolite repression in Aspergillus nidulans.

    Science.gov (United States)

    Kulmburg, P; Mathieu, M; Dowzer, C; Kelly, J; Felenbok, B

    1993-03-01

    The CREA repressor responsible for carbon catabolite repression in Aspergillus nidulans represses the transcription of the ethanol regulon. The N-terminal part of the CREA protein encompassing the two zinc fingers (C2H2 class family) and an alanine-rich region was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase. Our results show that CREA is a DNA-binding protein able to bind to the promoters of both the specific trans-acting gene, alcR, and of the structural gene, alcA, encoding the alcohol dehydrogenase I. DNase I protection footprinting experiments revealed several specific binding sites in the alcR and in the alcA promoters having the consensus sequence 5'-G/CPyGGGG-3'. The disruption of one of these CREA-binding sites in the alcR promoter overlapping the induction target for the trans-activator ALCR results in a partially derepressed alc phenotype and derepressed alcR transcription, showing that this binding site is functional in vivo. Our data suggest that CREA represses the ethanol regulon by a double lock mechanism repressing both the trans-acting gene, alcR, and the structural gene, alcA.

  15. Binding of the biogenic polyamines to deoxyribonucleic acids of varying base composition: base specificity and the associated energetics of the interaction.

    Directory of Open Access Journals (Sweden)

    Ayesha Kabir

    Full Text Available BACKGROUND: The thermodynamics of the base pair specificity of the binding of the polyamines spermine, spermidine, putrescine, and cadaverine with three genomic DNAs Clostridium perfringens, 27% GC, Escherichia coli, 50% GC and Micrococcus lysodeikticus, 72% GC have been studied using titration calorimetry and the data supplemented with melting studies, ethidium displacement and circular dichroism spectroscopy results. METHODOLOGY/PRINCIPAL FINDINGS: Isothermal titration calorimetry, differential scanning calorimetry, optical melting studies, ethidium displacement, circular dichroism spectroscopy are the various techniques employed to characterize the interaction of four polyamines, spermine, spermidine, putersine and cadaverine with the DNAs. Polyamines bound stronger with AT rich DNA compared to the GC rich DNA and the binding varied depending on the charge on the polyamine as spermine>spermidine >putrescine>cadaverine. Thermodynamics of the interaction revealed that the binding was entropy driven with small enthalpy contribution. The binding was influenced by salt concentration suggesting the contribution from electrostatic forces to the Gibbs energy of binding to be the dominant contributor. Each system studied exhibited enthalpy-entropy compensation. The negative heat capacity changes suggested a role for hydrophobic interactions which may arise due to the non polar interactions between DNA and polyamines. CONCLUSION/SIGNIFICANCE: From a thermodynamic analysis, the AT base specificity of polyamines to DNAs has been elucidated for the first time and supplemented by structural studies.

  16. Cationic liposomes enhance targeted delivery and expression of exogenous DNA mediated by N-terminal modified poly(L-lysine)-antibody conjugate in mouse lung endothelial cells.

    Science.gov (United States)

    Trubetskoy, V S; Torchilin, V P; Kennel, S; Huang, L

    1992-07-15

    A new and improved system for targeted gene delivery and expression is described. Transfection efficiency of N-terminal modified poly(L-lysine) (NPLL) conjugated with anti-thrombomodulin antibody 34A can be improved by adding to the system a lipophilic component, cationic liposomes. DNA, antibody conjugate and cationic liposomes form a ternary electrostatic complex which preserves the ability to bind specifically to the target cells. At the same time the addition of liposomes enhance the specific transfection efficiency of antibody-polylysine/DNA binary complex by 10 to 20-fold in mouse lung endothelial cells in culture.

  17. CpG methylation directly inhibits binding of the human papillomavirus type 16 E2 protein to specific DNA sequences.

    OpenAIRE

    Thain, A.; Jenkins, O; Clarke, A R; Gaston, K

    1996-01-01

    CpG methylation of the human papillomavirus upstream regulatory region has previously been shown to reduce virus promoter activity. Here, we demonstrate that methylation of the CpG dinucleotides contained within the binding site of the human papillomavirus type 16 E2 protein has a direct effect on the interaction of this protein with DNA. Methylation of both CpG dinucleotides within the E2 site abolishes the binding of E2.

  18. Coinvasion of dentinal tubules by Porphyromonas gingivalis and Streptococcus gordonii depends upon binding specificity of streptococcal antigen I/II adhesin.

    Science.gov (United States)

    Love, R M; McMillan, M D; Park, Y; Jenkinson, H F

    2000-03-01

    Cell wall-anchored polypeptides of the antigen I/II family are produced by many species of oral streptococci. These proteins mediate adhesion of streptococci to salivary glycoproteins and to other oral microorganisms and promote binding of cells to collagen type I and invasion of dentinal tubules. Since infections of the root canal system have a mixed anaerobic bacterial etiology, we investigated the hypothesis that coadhesion of anaerobic bacteria with streptococci may facilitate invasive endodontic disease. Porphyromonas gingivalis ATCC 33277 cells were able to invade dentinal tubules when cocultured with Streptococcus gordonii DL1 (Challis) but not when cocultured with Streptococcus mutans NG8. An isogenic noninvasive mutant of S. gordonii, with production of SspA and SspB (antigen I/II family) polypeptides abrogated, was deficient in binding to collagen and had a 40% reduced ability to support adhesion of P. gingivalis. Heterologous expression of the S. mutans SpaP (antigen I/II) protein in this mutant restored collagen binding and tubule invasion but not adhesion to P. gingivalis or the ability to promote P. gingivalis coinvasion of dentin. An isogenic afimbrial mutant of P. gingivalis had 50% reduced binding to S. gordonii cells but was unaffected in the ability to coinvade dentinal tubules with S. gordonii wild-type cells. Expression of the S. gordonii SspA or SspB polypeptide on the surface of Lactococcus lactis cells endowed these bacteria with the abilities to bind P. gingivalis, penetrate dentinal tubules, and promote P. gingivalis coinvasion of dentin. The results demonstrate that collagen-binding and P. gingivalis-binding properties of antigen I/II polypeptides are discrete functions. Specificity of antigen I/II polypeptide recognition accounts for the ability of P. gingivalis to coinvade dentinal tubules with S. gordonii but not with S. mutans. This provides evidence that the specificity of interbacterial coadhesion may influence directly the etiology

  19. Biochemical studies of olfaction: binding specificity of odorants to a cilia preparation from rainbow trout olfactory rosettes

    Energy Technology Data Exchange (ETDEWEB)

    Rhein, L.D.; Cagan, R.H.

    1983-08-01

    Cilia isolated from the olfactory epithelium (olfactory rosettes) of rainbow trout (Salmo gairdneri) bind amino acids, which are odor stimuli to this species. We demonstrate that L-threonine, L-serine, and L-alanine bind to a common site, TSA, in the cilia preparation. All possible mixtures of two of the amino acids as competitors, with the third as the /sup 3/H-labeled ligand, were studied. The effect of two combined (unlabeled) competitors was always substantially less than additive compared with their actions singly. Along with additional inhibition studies using mixtures of inhibitors, the data show that the three odorants must interact with at least one common binding site, TSA. Binding of L-(/sup 3/H)lysine to site L was unaffected by addition of L-threonine, L-serine, or L-alanine, establishing its independence from site TSA. L-Arginine inhibited binding of L-(/sup 3/H)lysine, showing that both of these basic amino acids interact with site L. The data establish the presence, in trout olfactory cilia, of at least two separate and noninteracting populations of odorant binding sites, TSA and L.

  20. The non-selective voltage-activated cation channel in the human red blood cell membrane: reconciliation between two conflicting reports and further characterisation

    DEFF Research Database (Denmark)

    Kaestner, Lars; Christophersen, Palle; Bernhardt, Ingolf;

    2000-01-01

    Erythrocyte; Patch-clamp; Non-specific; cation channel; Voltage dependence; Acetylcholin receptor......Erythrocyte; Patch-clamp; Non-specific; cation channel; Voltage dependence; Acetylcholin receptor...

  1. High Resolution Structures of Periplasmic Glucose-binding Protein of Pseudomonas putida CSV86 Reveal Structural Basis of Its Substrate Specificity.

    Science.gov (United States)

    Pandey, Suman; Modak, Arnab; Phale, Prashant S; Bhaumik, Prasenjit

    2016-04-01

    Periplasmic substrate-binding proteins (SBPs) bind to the specific ligand with high affinity and mediate their transport into the cytoplasm via the cognate inner membrane ATP-binding cassette proteins. Because of low sequence identities, understanding the structural basis of substrate recognition by SBPs has remained very challenging. There are several structures available for the ligand-bound sugar SBPs, but very few unliganded structures are reported. No structural data are available for sugar SBPs fromPseudomonassp. to date. This study reports the first high resolution crystal structures of periplasmic glucose-binding protein fromPseudomonas putidaCSV86 (ppGBP) in unliganded form (2.5 Å) and complexed with glucose (1.25 Å) and galactose (1.8 Å). Asymmetric domain closure of ppGBP was observed upon substrate binding. The ppGBP was found to have an affinity of ∼ 0.3 μmfor glucose. The structural analysis showed that the sugars are bound to the protein mainly by hydrogen bonds, and the loss of two strong hydrogen bonds between ppGBP and galactose compared with glucose may be responsible for lowering its affinity toward galactose. The higher stability of ppGBP-glucose complex was also indicated by an 8 °C increase in the melting temperature compared with unliganded form and ppGBP-galactose complex. ppGBP binds to monosaccharide, but the structural features revealed it to have an oligosaccharide-binding protein fold, indicating that during evolution the sugar binding pocket may have undergone structural modulation to accommodate monosaccharide only.

  2. Detection of C3d-Binding Donor-Specific Anti-HLA Antibodies at Diagnosis of Humoral Rejection Predicts Renal Graft Loss

    Science.gov (United States)

    Sicard, Antoine; Ducreux, Stéphanie; Rabeyrin, Maud; Couzi, Lionel; McGregor, Brigitte; Badet, Lionel; Scoazec, Jean Yves; Bachelet, Thomas; Lepreux, Sébastien; Visentin, Jonathan; Merville, Pierre; Fremeaux-Bacchi, Véronique; Morelon, Emmanuel; Taupin, Jean-Luc; Dubois, Valérie

    2015-01-01

    Antibody-mediated rejection (AMR) is a major cause of kidney graft loss, yet assessment of individual risk at diagnosis is impeded by the lack of a reliable prognosis assay. Here, we tested whether the capacity of anti-HLA antibodies to bind complement components allows accurate risk stratification at the time of AMR diagnosis. Among 938 kidney transplant recipients for whom a graft biopsy was performed between 2004 and 2012 at the Lyon University Hospitals, 69 fulfilled the diagnosis criteria for AMR and were enrolled. Sera banked at the time of the biopsy were screened for the presence of donor-specific anti-HLA antibodies (DSAs) and their ability to bind C1q and C3d using flow bead assays. In contrast with C4d graft deposition, the presence of C3d-binding DSA was associated with a higher risk of graft loss (P<0.001). Despite similar trend, the difference did not reach significance with a C1q-binding assay (P=0.06). The prognostic value of a C3d-binding assay was further confirmed in an independent cohort of 39 patients with AMR (P=0.04). Patients with C3d-binding antibodies had worse eGFR and higher DSA mean fluorescence intensity. In a multivariate analysis, only eGFR<30 ml/min per 1.73 m2 (hazard ratio [HR], 3.56; 95% confidence interval [CI], 1.46 to 8.70; P=0.005) and the presence of circulating C3d-binding DSA (HR, 2.80; 95% CI, 1.12 to 6.95; P=0.03) were independent predictors for allograft loss at AMR diagnosis. We conclude that assessment of the C3d-binding capacity of DSA at the time of AMR diagnosis allows for identification of patients at risk for allograft loss. PMID:25125383

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

    Science.gov (United States)

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

    2013-11-01

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

  4. Nonbonded interactions in membrane active cyclic biopolymers. IV - Cation dependence

    Science.gov (United States)

    Radhakrishnan, R.; Srinivasan, S.; Prasad, C. V.; Brinda, S. R.; Macelroy, R. D.; Sundaram, K.

    1980-01-01

    Interactions of valinomycin and form of its analogs in several conformations with the central ions Li(+), Na(+), K(+), Rb(+) and Cs(+) are investigated as part of a study of the specific preference of valinomycin for potassium and the mechanisms of carrier-mediated ion transport across membranes. Ion binding energies and conformational potential energies are calculated taking into account polarization energy formulas and repulsive energy between the central ion and the ligand atoms for conformations representing various stages in ion capture and release for each of the two ring chiralities of valinomycin and its analogs. Results allow the prediction of the chirality and conformation most likely to be observed for a given analog, and may be used to synthesize analogs with a desired rigidity or flexibility. The binding energies with the alkali metal cations are found to decrease with increasing ion size, and to be smaller than the corresponding ion hydration energies. It is pointed out that the observed potassium preference may be explainable in terms of differences between binding and hydration energies. Binding energies are also noted to depend on ligand conformation.

  5. Regulation of mga Transcription in the Group A Streptococcus: Specific Binding of Mga within Its Own Promoter and Evidence for a Negative Regulator

    OpenAIRE

    McIver, Kevin S.; Thurman, Alec S.; Scott, June R.

    1999-01-01

    Transcription of mga, encoding the multiple virulence gene regulator of the group A streptococcus, is positively autoregulated. This regulation requires a DNA region (Pmga) that contains both a promoter proximal to mga (P2) and a promoter located further upstream (P1). To determine if Mga has a direct role in this process, its ability to bind to specific sequences within Pmga was tested. A purified fusion of Mga to the C-terminal end of maltose-binding protein (MBP-Mga), encoded by malE-mga, ...

  6. Dialysis purification of integrase-DNA complexes provides high-resolution atomic force microscopy images: dimeric recombinant HIV-1 integrase binding and specific looping on DNA.

    Directory of Open Access Journals (Sweden)

    Tatsuaki Tsuruyama

    Full Text Available It remains difficult to obtain high-resolution atomic force microscopy images of HIV-1 integrase bound to DNA in a dimeric or tetrameric fashion. We therefore constructed specific target DNAs to assess HIV-1 integrase binding and purified the complex by dialysis prior to analysis. Our resulting atomic force microscopy analyses indicated precise size of binding human immunodeficiency virus type 1 (HIV-1 recombinant integrase in a tetrameric manner, inducing formation of a loop-like or figure-eight-like secondary structure in the target DNA. Our findings regarding the target DNA secondary structure provide new insights into the intermediate states of retroviral integration.

  7. Dialysis purification of integrase-DNA complexes provides high-resolution atomic force microscopy images: dimeric recombinant HIV-1 integrase binding and specific looping on DNA.

    Science.gov (United States)

    Tsuruyama, Tatsuaki; Nakai, Tonau; Ohmori, Rei; Ozeki, Munetaka; Tamaki, Keiji; Yoshikawa, Kenichi

    2013-01-01

    It remains difficult to obtain high-resolution atomic force microscopy images of HIV-1 integrase bound to DNA in a dimeric or tetrameric fashion. We therefore constructed specific target DNAs to assess HIV-1 integrase binding and purified the complex by dialysis prior to analysis. Our resulting atomic force microscopy analyses indicated precise size of binding human immunodeficiency virus type 1 (HIV-1) recombinant integrase in a tetrameric manner, inducing formation of a loop-like or figure-eight-like secondary structure in the target DNA. Our findings regarding the target DNA secondary structure provide new insights into the intermediate states of retroviral integration.

  8. Specific binding of collagen Q to the neuromuscular junction is exploited to cure congenital myasthenia and to explore bases of myasthenia gravis

    OpenAIRE

    Ohno, Kinji; Ito, Mikako; Kawakami, Yu; Krejci, Eric; Engel, Andrew G.

    2012-01-01

    Acetylcholinesterase (AChE) at the neuromuscular junction (NMJ) is anchored to the synaptic basal lamina via a triple helical collagen Q (ColQ) in the form of asymmetric AChE (AChE/ColQ). The C-terminal domain of ColQ binds to MuSK, the muscle-specific receptor tyrosine kinase, that mediates a signal for acetylcholine receptor (AChR) clustering at the NMJ. ColQ also binds to heparan sulfate proteoglycans including perlecan.

  9. The Ewing sarcoma protein (EWS) binds directly to the proximal elements of the macrophage-specific promoter of the CSF-1 receptor (csf1r) gene.

    Science.gov (United States)

    Hume, David A; Sasmono, Tedjo; Himes, S Roy; Sharma, Sudarshana M; Bronisz, Agnieszka; Constantin, Myrna; Ostrowski, Michael C; Ross, Ian L

    2008-05-15

    Many macrophage-specific promoters lack classical transcriptional start site elements such as TATA boxes and Sp1 sites. One example is the CSF-1 receptor (CSF-1R, CD115, c-fms), which is used as a model of the transcriptional regulation of macrophage genes. To understand the molecular basis of start site recognition in this gene, we identified cellular proteins binding specifically to the transcriptional start site (TSS) region. The mouse and human csf1r TSS were identified using cap analysis gene expression (CAGE) data. Conserved elements flanking the TSS cluster were analyzed using EMSAs to identify discrete DNA-binding factors in primary bone marrow macrophages as candidate transcriptional regulators. Two complexes were identified that bind in a highly sequence-specific manner to the mouse and human TSS proximal region and also to high-affinity sites recognized by myeloid zinc finger protein 1 (Mzf1). The murine proteins were purified by DNA affinity isolation from the RAW264.7 macrophage cell line and identified by mass spectrometry as EWS and FUS/TLS, closely related DNA and RNA-binding proteins. Chromatin immunoprecipitation experiments in bone marrow macrophages confirmed that EWS, but not FUS/TLS, was present in vivo on the CSF-1R proximal promoter in unstimulated primary macrophages. Transfection assays suggest that EWS does not act as a conventional transcriptional activator or repressor. We hypothesize that EWS contributes to start site recognition in TATA-less mammalian promoters.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-11-04

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

  11. Specifications

    International Nuclear Information System (INIS)

    As part of the Danish RERTR Program, three fuel elements with LEU U3O8-Al fuel and three fuel elements with LEU U3Si2-Al fuel were manufactured by NUKEM for irradiation testing in the DR-3 reactor at the Risoe National Laboratory in Denmark. The specifications for the elements with U3O8-Al fuel are presented here as an illustration only. Specifications for the elements with U3Si2-Al fuel were very similar. In this example, materials, material numbers, documents numbers, and drawing numbers specific to a single fabricator have been deleted. (author)

  12. Measurement of {sup 11}C-raclopride binding in micropig brain with high and low specific activities

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Su Jin; Lee, Jae Sung; Eo, Jae Seon [Seoul National University College of Medicine, Seoul (Korea, Republic of)] (and others)

    2005-07-01

    In vitro, a saturation hyperbola or Scatchard plot is applied for the determination of receptor density (Bmax) and affinity (Kd). Simillary, Bmax and Kd could be obtained in vivo by performing two or more PET experiments with high and low specific activities (SA). To measure these parameters, the binding potential (BP) of 11C-raclopride for striatal D2 receptor in micropig brain at high and low SA was measured in this study. A normal male PWG micropig (weight: 38 kg, age: 24 months) was used in this study. The animals were anesthetized with ketamine (2 mL/10 kg, i.m.) and xylazine (1mL/10kg, i.m.), and placed in a supine position. Dynamic PET data was acquired for 60 min after injection of 11C-raclopride (2.5 mCi) through the catheter placed in a femoral vein. High SA and low SA (6.8 uCi/nmol) PET and T1 SPGR MRI scans were performed. MR image was co-registered to the static PET images and ROIs were drawn on striatum and cerebellum to obtain the time activity curve. The BP in striatum was computed by both the Lammertsma and Logan reference tissue methods using cerebellum tissue input function. BP parametric images were also generated using the Logan method. The value of striatum BP was 1.46/0.32 (high SA / low SA) and 1.34/0.31 in Lammertsma and Logan methods, respectively. The D2 occupancy by the cold raclopride was approximately 78% in micropig striatum. The Logan BP parametric image visualized well the change of receptor occupancy between the two scans. In this study, BP values at high and low SA and their percent change estimated by the two different methods were correlated well. This preliminary result suggests that the experimental procedures established in this study would be useful for the quantification of the density of D2 receptor and affinity in the micropig brain.

  13. A Specific Cholesterol Binding Site Is Established by the 2.8 Å Structure of the Human [beta][subscript 2]-Adrenergic Receptor

    Energy Technology Data Exchange (ETDEWEB)

    Hanson, Michael A.; Cherezov, Vadim; Griffith, Mark T.; Roth, Christopher B.; Jaakola, Veli-Pekka; Chien, Ellen Y.T.; Velasquez, Jeffrey; Kuhn, Peter; Stevens, Raymond C. (Scripps)

    2008-07-08

    The role of cholesterol in eukaryotic membrane protein function has been attributed primarily to an influence on membrane fluidity and curvature. We present the 2.8 {angstrom} resolution crystal structure of a thermally stabilized human {beta}{sub 2}-adrenergic receptor bound to cholesterol and the partial inverse agonist timolol. The receptors pack as monomers in an antiparallel association with two distinct cholesterol molecules bound per receptor, but not in the packing interface, thereby indicating a structurally relevant cholesterol-binding site between helices I, II, III, and IV. Thermal stability analysis using isothermal denaturation confirms that a cholesterol analog significantly enhances the stability of the receptor. A consensus motif is defined that predicts cholesterol binding for 44% of human class A receptors, suggesting that specific sterol binding is important to the structure and stability of other G protein-coupled receptors, and that this site may provide a target for therapeutic discovery.

  14. Endonuclease-based Method for Detecting the Sequence Specific DNA Binding Protein on Double-stranded DNA Microarray

    Institute of Scientific and Technical Information of China (English)

    Yun Fei BAI; Qin Yu GE; Tong Xiang LI; Jin Ke WANG; Quan Jun LIU; Zu Hong LU

    2005-01-01

    The double-stranded DNA (dsDNA) probe contains two different protein binding sites.One is for DNA- binding proteins to be detected and the other is for a DNA restriction enzyme.The two sites were arranged together with no base interval. The working principle of the capturing dsDNA probe is described as follows: the capturing probe can be cut with the DNA restriction enzyme (such as EcoR I) to cause a sticky terminal, if the probe is not bound with a target protein, and the sticky terminal can be extended and labeled with Cy3-dUTP by DNA polymerase. When the probe is bound with a target protein, the probe is not capable to be cut by the restriction enzyme because of space obstruction. The amount of the target DNA binding proteins can be measured according to the variations of fluorescent signals of the corresponding probes.

  15. Structural Basis for Specific Binding of Human MPP8 Chromodomain to Histone H3 Methylated at Lysine 9

    Energy Technology Data Exchange (ETDEWEB)

    Li, Jing; Li, Zhihong; Ruan, Jianbin; Xu, Chao; Tong, Yufeng; Pan, Patricia W.; Tempel, Wolfram; Crombet, Lissete; Min, Jinrong; Zang, Jianye (Toronto); (Chinese Aca. Sci.)

    2012-02-27

    M-phase phosphoprotein 8 (MPP8) was initially identified to be a component of the RanBPM-containing large protein complex, and has recently been shown to bind to methylated H3K9 both in vivo and in vitro. MPP8 binding to methylated H3K9 is suggested to recruit the H3K9 methyltransferases GLP and ESET, and DNA methyltransferase 3A to the promoter of the E-cadherin gene, mediating the E-cadherin gene silencing and promote tumor cell motility and invasion. MPP8 contains a chromodomain in its N-terminus, which is used to bind the methylated H3K9. Here, we reported the crystal structures of human MPP8 chromodomain alone and in complex with the trimethylated histone H3K9 peptide (residue 1-15). The complex structure unveils that the human MPP8 chromodomain binds methylated H3K9 through a conserved recognition mechanism, which was also observed in Drosophila HP1, a chromodomain containing protein that binds to methylated H3K9 as well. The structure also reveals that the human MPP8 chromodomain forms homodimer, which is mediated via an unexpected domain swapping interaction through two {beta} strands from the two protomer subunits. Our findings reveal the molecular mechanism of selective binding of human MPP8 chromodomain to methylated histone H3K9. The observation of human MPP8 chromodomain in both solution and crystal lattice may provide clues to study MPP8-mediated gene regulation furthermore.

  16. Structural basis for specific binding of human MPP8 chromodomain to histone H3 methylated at lysine 9.

    Directory of Open Access Journals (Sweden)

    Jing Li

    Full Text Available BACKGROUND: M-phase phosphoprotein 8 (MPP8 was initially identified to be a component of the RanBPM-containing large protein complex, and has recently been shown to bind to methylated H3K9 both in vivo and in vitro. MPP8 binding to methylated H3K9 is suggested to recruit the H3K9 methyltransferases GLP and ESET, and DNA methyltransferase 3A to the promoter of the E-cadherin gene, mediating the E-cadherin gene silencing and promote tumor cell motility and invasion. MPP8 contains a chromodomain in its N-terminus, which is used to bind the methylated H3K9. METHODOLOGY/PRINCIPAL FINDINGS: Here, we reported the crystal structures of human MPP8 chromodomain alone and in complex with the trimethylated histone H3K9 peptide (residue 1-15. The complex structure unveils that the human MPP8 chromodomain binds methylated H3K9 through a conserved recognition mechanism, which was also observed in Drosophila HP1, a chromodomain containing protein that binds to methylated H3K9 as well. The structure also reveals that the human MPP8 chromodomain forms homodimer, which is mediated via an unexpected domain swapping interaction through two β strands from the two protomer subunits. CONCLUSIONS/SIGNIFICANCE: Our findings reveal the molecular mechanism of selective binding of human MPP8 chromodomain to methylated histone H3K9. The observation of human MPP8 chromodomain in both solution and crystal lattice may provide clues to study MPP8-mediated gene regulation furthermore.

  17. Specific binding sites for the activator protein, ALCR, in the alcA promoter of the ethanol regulon of Aspergillus nidulans.

    Science.gov (United States)

    Kulmburg, P; Judewicz, N; Mathieu, M; Lenouvel, F; Sequeval, D; Felenbok, B

    1992-10-15

    ALCR is the specific activator of the Aspergillus nidulans ethanol-utilization pathway, mediating the induction of its own transcription and that of the structural genes alcA and aldA, encoding respectively, alcohol dehydrogenase I and aldehyde dehydrogenase. ALCR is a DNA binding protein in which 6 cysteines are coordinated in a zinc binuclear cluster. This domain was fused to glutathione-S-transferase (GST) and isolated as a GST-ALCR(7-58*) fusion protein from Escherichia coli. Mobility shift assays showed that the ALCR fusion protein binds at sites upstream of the alcA promoter. DNaseI protection footprinting experiments revealed three specific binding sites, two that are direct repeats and one that is an inverted repeat with the same half-site 5'-CCGCA-3'. The half-sites are separated by a variable number of nucleotides in both types of target. The interaction of the ALCR fusion protein with direct and inverted repeats were examined by using interference and protection footprinting assays. In both binding sites, modification of the guanines in the half-sites interfered with the formation of the DNA complex, but the adjacent ones did not. Our results suggest that the ALCR protein makes contact in the major groove of the DNA helix of the half-sites. The functionality of two out of three binding sites of the GST-ALCR protein was demonstrated after their deletion. Therefore, the region encompassing these binding sites is a cis-acting element involved in the full induction of the alcA gene.

  18. Functional evolution of IGF2:IGF2R domain 11 binding generates novel structural interactions and a specific IGF2 antagonist.

    Science.gov (United States)

    Frago, Susana; Nicholls, Ryan D; Strickland, Madeleine; Hughes, Jennifer; Williams, Christopher; Garner, Lee; Surakhy, Mirvat; Maclean, Rory; Rezgui, Dellel; Prince, Stuart N; Zaccheo, Oliver J; Ebner, Daniel; Sanegre, Sabina; Yu, Sheng; Buffa, Francesca M; Crump, Matthew P; Hassan, Andrew Bassim

    2016-05-17

    Among the 15 extracellular domains of the mannose 6-phosphate/insulin-like growth factor-2 receptor (M6P/IGF2R), domain 11 has evolved a binding site for IGF2 to negatively regulate ligand bioavailability and mammalian growth. Despite the highly evolved structural loops of the IGF2:domain 11 binding site, affinity-enhancing AB loop mutations suggest that binding is modifiable. Here we examine the extent to which IGF2:domain 11 affinity, and its specificity over IGF1, can be enhanced, and we examine the structural basis of the mechanistic and functional consequences. Domain 11 binding loop mutants were selected by yeast surface display combined with high-resolution structure-based predictions, and validated by surface plasmon resonance. We discovered previously unidentified mutations in the ligand-interacting surface binding loops (AB, CD, FG, and HI). Five combined mutations increased rigidity of the AB loop, as confirmed by NMR. When added to three independently identified CD and FG loop mutations that reduced the koff value by twofold, these mutations resulted in an overall selective 100-fold improvement in affinity. The structural basis of the evolved affinity was improved shape complementarity established by interloop (AB-CD) and intraloop (FG-FG) side chain interactions. The high affinity of the combinatorial domain 11 Fc fusion proteins functioned as ligand-soluble antagonists or traps that depleted pathological IGF2 isoforms from serum and abrogated IGF2-dependent signaling in vivo. An evolved and reengineered high-specificity M6P/IGF2R domain 11 binding site for IGF2 may improve therapeutic targeting of the frequent IGF2 gain of function observed in human cancer. PMID:27140600

  19. Massive parallel analysis of the binding specificity of histone-like protein HU to single- and double-stranded DNA with generic oligodeoxyribonucleotide microchips.

    Energy Technology Data Exchange (ETDEWEB)

    Krylov, A. S.; Zasedateleva, O. A.; Prokopenko, D. V.; Rouviere-Yaniv, J.; Mirzabekov, A. D.; Biochip Technology Center; Engelhardt Inst. of Molecular Biology; Inst. de Biologie Physico-Chimique

    2001-06-15

    A generic hexadeoxyribonucleotide microchip has been applied to test the DNA-binding properties of HU histone-like bacterial protein, which is known to have a low sequence specificity. All 4096 hexamers flanked within 8mers by degenerate bases at both the 3'- and 5'-ends were immobilized within the 100 x 100 x 20 mm polyacrylamide gel pads of the microchip. Single-stranded immobilized oligonucleotides were converted in some experiments to the double-stranded form by hybridization with a specified mixture of 8mers. The DNA interaction with HU was characterized by three type of measurements: (i) binding of FITC-labeled HU to microchip oligonucleotides; (ii) melting curves of complexes of labeled HU with single-stranded microchip oligonucleotides; (iii) the effect of HU binding on melting curves of microchip double-stranded DNA labeled with another fluorescent dye, Texas Red. Large numbers of measurements of these parameters were carried out in parallel for all or many generic microchip elements in real time with a multi-wavelength fluorescence microscope. Statistical analysis of these data suggests some preference for HU binding to G/C-rich single-stranded oligonucleotides. HU complexes with double-stranded microchip 8mers can be divided into two groups in which HU binding either increased the melting temperature (T{sub m}) of duplexes or decreased it. The stabilized duplexes showed some preference for presence of the sequence motifs AAG, AGA and AAGA. In the second type of complex, enriched with A/T base pairs, the destabilization effect was higher for longer stretches of A/T duplexes. Binding of HU to labeled duplexes in the second type of complex caused some decrease in fluorescence. This decrease also correlates with the higher A/T content and lower T{sub m}. The results demonstrate that generic microchips could be an efficient approach in analysis of sequence specificity of proteins.

  20. Spermidine/spermine N-1-acetyltransferase specifically binds to the integrin alpha 9 subunit cytoplasmic domain and enhances cell migration

    OpenAIRE

    Chen, C.; Young, B A; Coleman, C S; Pegg, A E; Sheppard, D

    2004-01-01

    T he integrin alpha9beta1 is expressed on migrating cells, such as leukocytes, and binds to multiple ligands that are present at sites of tissue injury and inflammation. alpha9beta1, like the structurally related integrin alpha4beta1, mediates accelerated cell migration, an effect that depends on the beta cytoplasmic domain. alpha4beta1 enhances migration through reversible binding to the adapter protein, paxillin, but alpha9beta1-dependent migration is paxillin independent. Using yeast two-h...

  1. In vitro effects of buyang huanwu decoction and its ingredients on inhibiting the specific binding of 3H-platelet activating factor to its receptor in rabbits

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    BACKGROUND: Pharmacologic action of traditional Chinese medicine compound is the comprehensive effect of various ingredients, and the interactions of various ingredients are closely correlated with the final effect. In order to reveal the compatibility mechanism of buyang huanwu decoction (BHD)'s prescription in treating and preventing ischemic cerebrovascular disease, we need to explore the effect and relation of ingredients in prescription except for considering the effect of each ingredient on the whole prescription.OBJECTIVE: To study the effect of BHD and its ingredients in the prescription on the specific binding of 3H-platelet activating factor (PAF) to its receptor (PAFR)in rabbits in vitro, and to analyze the action of each ingredient in the prescription.DESIGN: A decomposed recipe study based on orthogonal test.SETTING: Guangzhou University of Traditional Chinese Medicine.MATERIALS: Five healthy adult New Zealand rabbits of either gender were provided by the Experimental Animal Center of Guangzhou University of Traditional Chinese medicine. The prescription herbal pieces were purchased from Foshan Kangpu Pharmaceuticals Company and Jianmin Pharmaceuticals Company, and were appraised by Professor Yanchen Xu from College of Traditional Chinese Medicine, Guangzhou University of Traditional Chinese Medicine. 3H-PAF was supplied by Amersham Co.,Ltd.(Specific activity:6.475 TBq/mmol;batch number:200402); PAF standard by Biomol Co., Ltd.(batch number: P1318V).METHODS: This experiment was carried out in the Laboratory of Nuclear Medicine, Guangzhou University of Traditional Chinese Medicine between September and December 2004. ① The seven influencing factors were selected: such as Shenghuangqi , Dangguiwei, Chishao, Dilong, Taoren, Honghua, Chuanxiong. Each factor was divided into two levels, selected or not selected. The tests were arranged according to L8 (27) orthogonal test table. ②The specific binding of 3H-PAF to its receptors in rabbits was measured by

  2. Nucleotide sequence and structural determinants of specific binding of coat protein or coat protein peptides to the 3' untranslated region of alfalfa mosaic virus RNA 4.

    Science.gov (United States)

    Houser-Scott, F; Baer, M L; Liem, K F; Cai, J M; Gehrke, L

    1994-01-01

    The specific binding of alfalfa mosaic virus coat protein to viral RNA requires determinants in the 3' untranslated region (UTR). Coat protein and peptide binding sites in the 3' UTR of alfalfa mosaic virus RNA 4 have been analyzed by hydroxyl radical footprinting, deletion mapping, and site-directed mutagenesis experiments. The 3' UTR has several stable hairpins that are flanked by single-stranded (A/U)UGC sequences. Hydroxyl radical footprinting data show that five sites in the 3' UTR of alfalfa mosaic virus RNA 4 are protected by coat protein, and four of the five protected regions contain AUGC or UUGC. Electrophoretic mobility band shift results suggest four coat protein binding sites in the 3' UTR. A 3'-terminal 39-nucleotide RNA fragment containing four AUGC repeats bound coat protein and coat protein peptides with high affinity; however, coat protein bound poorly to antisense 3' UTR transcripts and poly(AUGC)10. Site-directed mutagenesis of AUGC865-868 resulted in a loss of coat protein binding and peptide binding by the RNA fragment. Alignment of alfalfa mosaic RNA sequences with those from several closely related ilarviruses demonstrates that AUGC865-868 is perfectly conserved; moreover, the RNAs are predicted to form similar 3'-terminal secondary structures. The data strongly suggest that alfalfa mosaic virus coat protein and ilavirus coat proteins recognize invariant AUGC sequences in the context of conserved structural elements. Images PMID:8139004

  3. Isolation of a gene encoding a developmentally regulated T cell-specific protein with a guanine nucleotide triphosphate-binding motif

    Energy Technology Data Exchange (ETDEWEB)

    Carlow, D.A.; Teh, H.S.; Marth, J. [Univ. of British Columbia, Vancouver (Canada)] [and others

    1995-02-15

    In this study, we describe a novel full length cDNA clone designated Tgtp that encodes a predicted 415-amino acid a T cell-specific guanine nucleotide triphosphate-binding protein (TGTP) bearing the characteristic motifs of a guanine nucleotide triphosphate (GTP) binding protein. Tgtp is expressed preferentially, if not exclusively, in T cells, and is up-regulated in both unfractionated and in purified CD4{sup +}8{sup +} thymocytes upon TCR cross-linking. In contrast, expression of Tgtp in peripheral T cells is maintained at relatively high levels and is not grossly affected by TCR cross-linking. Antiserum generated against synthetic peptides from the predicted TGTP amino acid sequence recognized a single protein with a molecular mass of {approx}50 kDa, corresponding well with the computed molecular mass of 47 kDa. The only known relative of Tgtp is MUSGTP, which is reportedly expressed in B cells and bears a GTP binding motif. Thus, the discovery of Tgtp resolves a subfamily of molecules with GTP binding motifs and apparent lymphoid lineage-restricted expression. Given the restricted expression pattern in T cells, the up-regulated expression observed in response to TCR signaling in immature thymocytes, and the presence of the motifs characteristic of GTP binding proteins, we suggest that TGTP may have an important function in T cell development and/or T cell activation. 51 refs., 6 figs.

  4. Growth hormone binding to specific receptors stimulates growth and function of cloned insulin-producing rat insulinoma RIN-5AH cells

    DEFF Research Database (Denmark)

    Billestrup, Nils; Martin, J M

    1985-01-01

    of the insulinotropic effect showed that half-maximal and maximal stimulation were observed in cells cultured in the presence of 10 and 100 ng/ml, respectively. Insulin release to the medium during the 4-day culture period was not affected by hGH. These data suggest that GH, through binding to specific receptors......Binding of 125I-labeled human GH (hGH) to a cloned rat insulin-producing cell line RIN-5AH in monolayer culture was studied along with some physiological effects of the hormone on these cells. Binding was time and temperature dependent, and steady state binding was observed in 60 min at 37 C...... affinity binding sites were calculated. Culture of RIN-5AH in the presence of 1 microgram/ml hGH for 4 days resulted in an 80% increase in insulin content as well as an 18% increase in cell number and DNA and protein content compared to those in cells cultured in the absence of hGH. The dose dependence...

  5. Detection of a protein, AngCP, which binds specifically to the three upstream regions of glaA gene in A. niger T21

    Institute of Scientific and Technical Information of China (English)

    QIU; Runxiang(仇润祥); ZHU; Xingguo(朱兴国); LIU; Li(刘丽); TANG; Guomin(唐国敏)

    2002-01-01

    Electromobility shift assay (EMSA) was used to scan 600 bp of 5′cis regulatory sequence of Aspergillus niger (A. niger ) T21 glucoamylase gene (glaA) for binding by partially fractionated T21 protein extracted from starch-induced mycelia. In this process, one protein, AngCP, was detected to bind specifically to three regions covering -374 to -344, -484 to -414 and -580 to -540 relative to the glaA translational start codon. UV-crosslinking of DNA-protein complex showed that MW of AngCP was 10 ku. DNaseⅠfootprinting analysis demonstrated that AngCP specifically binds to two CCAAT containing sequences within the regions between -374 and -344 and -484 and -414 bp. And the region between -580 and -540 bp contains CCAAT similar box, CCTAT. The results indicated that AngCP is probably one of the members of CCAAT-binding protein families, which are generally involved in enhancement of gene expression in filamentous fungi. These findings suggested that AngCP should be a transcription activator for high-level expression of glaA gene.

  6. Phylogenetic divergence of CD47 interactions with human signal regulatory protein alpha reveals locus of species specificity. Implications for the binding site.

    Science.gov (United States)

    Subramanian, Shyamsundar; Boder, Eric T; Discher, Dennis E

    2007-01-19

    Cell-cell interactions between ubiquitously expressed integrin-associated protein (CD47) and its counterreceptor signal regulatory protein (SIRPalpha) on phagocytes regulate a wide range of adhesive signaling processes, including the inhibition of phagocytosis as documented in mice. We show that CD47-SIRPalpha binding interactions are different between mice and humans, and we exploit phylogenetic divergence to identify the species-specific binding locus on the immunoglobulin domain of human CD47. All of the studies are conducted in the physiological context of membrane protein display on Chinese hamster ovary (CHO) cells. Novel quantitative flow cytometry analyses with CD47-green fluorescent protein and soluble human SIRPalpha as a probe show that neither human CD47 nor SIRPalpha requires glycosylation for interaction. Human CD47-expressing CHO cells spread rapidly on SIRPalpha-coated glass surfaces, correlating well with the spreading of primary human T cells. In contrast, CHO cells expressing mouse CD47 spread minimally and show equally weak binding to soluble human SIRPalpha. Further phylogenetic analyses and multisite substitutions of the CD47 Ig domain show that human to cow mutation of a cluster of seven residues on adjacent strands near the middle of the domain decreases the association constant for human SIRPalpha to about one-third that of human CD47. Direct tests of cell-cell adhesion between human monocytes and CD47-displaying CHO cells affirm the species specificity as well as the importance of the newly identified binding locus in cell-cell interactions.

  7. The lectin domains of polypeptide GalNAc-transferases exhibit carbohydrate-binding specificity for GalNAc: lectin binding to GalNAc-glycopeptide substrates is required for high density GalNAc-O-glycosylation.

    Science.gov (United States)

    Wandall, Hans H; Irazoqui, Fernando; Tarp, Mads Agervig; Bennett, Eric P; Mandel, Ulla; Takeuchi, Hideyuki; Kato, Kentaro; Irimura, Tatsuro; Suryanarayanan, Ganesh; Hollingsworth, Michael A; Clausen, Henrik

    2007-04-01

    Initiation of mucin-type O-glycosylation is controlled by a large family of UDP GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). Most GalNAc-transferases contain a ricin-like lectin domain in the C-terminal end, which may confer GalNAc-glycopeptide substrate specificity to the enzyme. We have previously shown that the lectin domain of GalNAc-T4 modulates its substrate specificity to enable unique GalNAc-glycopeptide specificities and that this effect is selectively inhibitable by GalNAc; however, direct evidence of carbohydrate binding of GalNAc-transferase lectins has not been previously presented. Here, we report the direct carbohydrate binding of two GalNAc-transferase lectin domains, GalNAc-T4 and GalNAc-T2, representing isoforms reported to have distinct glycopeptide activity (GalNAc-T4) and isoforms without apparent distinct GalNAc-glycopeptide specificity (GalNAc-T2). Both lectins exhibited specificity for binding of free GalNAc. Kinetic and time-course analysis of GalNAc-T2 demonstrated that the lectin domain did not affect transfer to initial glycosylation sites, but selectively modulated velocity of transfer to subsequent sites and affected the number of acceptor sites utilized. The results suggest that GalNAc-transferase lectins serve to modulate the kinetic properties of the enzymes in the late stages of the initiation process of O-glycosylation to accomplish dense or complete O-glycan occupancy.

  8. Rifampicin-dependent antibodies bind a similar or identical epitope to glycoprotein IX-specific quinine-dependent antibodies

    NARCIS (Netherlands)

    Burgess, J K; Lopez, J A; Gaudry, L E; Chong, B H

    2000-01-01

    The drug-dependent antibody of a patient with rifampicin-induced thrombocytopenia was characterized using the antigen-capture enzyme-linked immunosorbent assay (MAIPA assay), flow cytometry, and immunoprecipitation. The antibody was found to bind glycoprotein (GP) Ib-IX but not GPIIb-IIIa because (1

  9. Design and synthesis of a stable oxidized phospholipid mimic with specific binding recognition for macrophage scavenger receptors

    DEFF Research Database (Denmark)

    Turner, William W; Hartvigsen, Karsten; Boullier, Agnes;

    2012-01-01

    Macrophage scavenger receptors appear to play a major role in the clearance of oxidized phospholipid (OxPL) products. Discrete peptide-phospholipid conjugates with the phosphatidylcholine headgroup have been shown to exhibit binding affinity for these receptors. We report the preparation of a wat...

  10. Acetylcholine-Binding Protein Engineered to Mimic the α4-α4 Binding Pocket in α4β2 Nicotinic Acetylcholine Receptors Reveals Interface Specific Interactions Important for Binding and Activity

    DEFF Research Database (Denmark)

    Shahsavar, Azadeh; Ahring, Philip K; Olsen, Jeppe A;

    2015-01-01

    Neuronal α4β2 nicotinic acetylcholine receptors are attractive drug targets for psychiatric and neurodegenerative disorders and smoking cessation aids. Recently, a third agonist binding site between two α4 subunits in the (α4)(3)(β2)(2) receptor subpopulation was discovered. In particular, three...

  11. Cell wall regeneration in Bangia atropurpurea (Rhodophyta) protoplasts observed using a mannan-specific carbohydrate-binding module.

    Science.gov (United States)

    Umemoto, Yoshiaki; Araki, Toshiyoshi

    2010-02-01

    The cell wall of the red alga Bangia atropurpurea is composed of three unique polysaccharides (beta-1,4-mannan, beta-1,3-xylan, and porphyran), similar to that in Porphyra. In this study, we visualized beta-mannan in the regenerating cell walls of B. atropurpurea protoplasts by using a fusion protein of a carbohydrate-binding module (CBM) and green fluorescent protein (GFP). A mannan-binding family 27 CBM (CBM27) of beta-1,4-mannanase (Man5C) from Vibrio sp. strain MA-138 was fused to GFP, and the resultant fusion protein (GFP-CBM27) was expressed in Escherichia coli. Native affinity gel electrophoresis revealed that GFP-CBM27 maintained its binding ability to soluble beta-mannans, while normal GFP could not bind to beta-mannans. Protoplasts were isolated from the fronds of B. atropurpurea by using three kinds of bacterial enzymes. The GFP-CBM27 was mixed with protoplasts from different growth stages, and the process of cell wall regeneration was observed by fluorescence microscopy. Some protoplasts began to excrete beta-mannan at certain areas of their cell surface after 12 h of culture. As the protoplast culture progressed, beta-mannans were spread on their entire cell surfaces. The percentages of protoplasts bound to GFP-CBM27 were 3%, 12%, 17%, 29%, and 25% after 12, 24, 36, 48, and 60 h of culture, respectively. Although GFP-CBM27 bound to cells at the initial growth stages, its binding to the mature fronds was not confirmed definitely. This is the first report on the visualization of beta-mannan in regenerating algal cell walls by using a fluorescence-labeled CBM. PMID:19466498

  12. A stimulus-specific role for CREB-binding protein (CBP) in T cell receptor-activated tumor necrosis factor gene expression

    Science.gov (United States)

    Falvo, James V.; Brinkman, Brigitta M. N.; Tsytsykova, Alla V.; Tsai, Eunice Y.; Yao, Tso-Pang; Kung, Andrew L.; Goldfeld, Anne E.

    2000-04-01

    The cAMP response element binding protein (CREB)-binding protein (CBP)/p300 family of coactivator proteins regulates gene transcription through the integration of multiple signal transduction pathways. Here, we show that induction of tumor necrosis factor (TNF-) gene expression in T cells stimulated by engagement of the T cell receptor (TCR) or by virus infection requires CBP/p300. Strikingly, in mice lacking one copy of the CBP gene, TNF- gene induction by TCR activation is inhibited, whereas virus induction of the TNF- gene is not affected. Consistent with these findings, the transcriptional activity of CBP is strongly potentiated by TCR activation but not by virus infection of T cells. Thus, CBP gene dosage and transcriptional activity are critical in TCR-dependent TNF-α gene expression, demonstrating a stimulus-specific requirement for CBP in the regulation of a specific gene.

  13. The N-terminal cellulose-binding domain of EGXA increases thermal stability of xylanase and changes its specific activities on different substrates

    Institute of Scientific and Technical Information of China (English)

    Ming Ding; Yigang Teng; Qiuyu Yin; Jie Zhao; Fukun Zhao

    2008-01-01

    A full-length EGXA enzyme from a mollusk, Ampullaria crossean, was cloned into pFastBac vector and then heterogeneously expressed in insect Tn5 cells. Its natural N-terminal signal peptide worked well in the insect Tn5 cells.The recombinant EGXA was a 63 kDa protein and had active endo-β-1,4-glucanase (EC 3.2.1.4) and endo-β-1,4-xylanase (EC 3.2.1.8). The specific activity of endo-β-1,4-xylanase was higher than in the EGX, which was purified from the stomach tissues of Ampullaria crossen. The N-terminal cellulosebinding domain of EGXA made it bind to cellulose and xylan more efficiently. This cellulose-binding domain also increased the thermal stability of this recombinant enzyme and decreased the recombinant EGXA's specific activities on p-nitrophenyi-β-D-cellobioside and sodium carboxymethyl cellulose.

  14. Neuroendocrine Cancer-Specific Up-Regulating Mechanism of Insulin-Like Growth Factor Binding Protein-2 in Small Cell Lung Cancer

    OpenAIRE

    Yazawa, Takuya; Sato, Hanako; Shimoyamada, Hiroaki; Okudela, Koji; Woo, Tetsukan; Tajiri, Michihiko; Ogura, Takashi; Ogawa, Nobuo; Suzuki, Takehisa; Mitsui, Hideaki; Ishii, Jun; Miyata, Chie; Sakaeda, Masashi; Goto, Kazuya; Kashiwagi, Korehito

    2009-01-01

    Small cell lung cancer (SCLC) exhibits insulin-like growth factor-dependent growth. SCLC is the most aggressive among known in vivo lung cancers, whereas in vitro growth of SCLC is paradoxically slow as compared with that of non-SCLC (NSCLC). In this study, we demonstrate that SCLC cells overexpress insulin-like growth factor binding protein (IGFBP)-2 via NeuroD, a neuroendocrine cell-specific transcription factor. Chromatin immunoprecipitation, electrophoretic mobility shift, and IGFBP-2 pro...

  15. Triplex targeting of human PDGF-B (c-sis, proto-oncogene) promoter specifically inhibits factors binding and PDGF-B transcription

    OpenAIRE

    Jing LIU; Xu, Ren-Huan; Jin, You-xin; Wang, De-Bao

    2001-01-01

    Human c-sis/PDGF-B proto-oncogene has been shown to be overexpressed in a large percentage of human tumor cells establishing a growth-promoting, autocrine growth circuit. Triplex forming oligonucleotides (TFOs) can recognize and bind sequences in duplex DNA, and have received considerable attention because of their potential for targeting specific genomic sites. The c-sis/PDGF-B promoter contains a unique homopurine/homopyrimidine sequence (SIS proximal element, ...

  16. Reversible supramolecular assembly at specific DNA sites: nickel-promoted bivalent DNA binding with designed peptide and bipyridyl-bis(benzamidine) components.

    Science.gov (United States)

    Sánchez, Mateo I; Mosquera, Jesús; Vázquez, M Eugenio; Mascareñas, José L

    2014-09-01

    At specific DNA sites, nickel(II) salts promote the assembly of designed components, namely a bis(histidine)-modified peptide that is derived from a bZIP transcription factor and a bis(benzamidine) unit that is equipped with a bipyridine. This programmed supramolecular system with emergent properties reproduces some key characteristics of naturally occurring DNA-binding proteins, such as bivalence, selectivity, responsiveness to external agents, and reversibility.

  17. Improved detection of deeply invasive candidiasis with DNA aptamers specific binding to (1→3)-β-D-glucans from Candida albicans.

    Science.gov (United States)

    Tang, X-L; Hua, Y; Guan, Q; Yuan, C-H

    2016-04-01

    Deeply invasive or disseminated candidiasis is a serious and often fatal complication that can occur frequently in immuno-compromised individuals. However, conventional diagnostic methods of Candida albicans display low sensitivity and lack of specificity; the development of rapid and accurate detection methods remains a high priority. Aptamers are single-strand DNA or RNA oligonucleotides that specifically bind to target molecules with high affinity. In this study, we sought to screen high-affinity DNA aptamers that specifically bound to (1→3)-β-D-glucans from cell wall of Candida albicans using a systematic evolution of ligands by exponential enrichment (SELEX) technique, and further evaluate the diagnostic potential for invasive or disseminated candidiasis with selected aptamers. (1→3)-β-D-glucans was purified from Candida albicans, and two single DNA aptamers (designated as AU1 and AD1) were selected. Analysis of dissociation constants and binding domains further revealed that these two selected single DNA aptamers (AU1 and AD1) showed high binding affinity (AD1: Kd = 79.76 nM, AD1: Kd = 103.7 nM) and did not bind to the same domain of (1→3)-β-D-glucans. Next, we further detected (1→3)-β-D-glucans in serum samples from different groups of patients with Candida albicans infection or simple bacterial infection by using a double-aptamer sandwich enzyme-linked oligonucleotide assay (ELONA). The results showed that the sensitivity and specificity of this aptamer-based sandwich ELONA were 92.31 % and 91.94 % respectively. Thus, our study suggests that AU1 and AD1 have potential application for the differentiate diagnosis of deeply invasive candidiasis and provide valuable clues for designing diagnostic agents for the identification of invasive fungal infection. PMID:26810058

  18. Interaction of actinide cations with synthetic polyelectrolytes

    International Nuclear Information System (INIS)

    The binding of Am+3, Th+4 and UO2+2 to polymaleic acid, polyethylenemaleic acid and polymethylvinylethermaleic acid has been measured by a solvent extraction technique at 250C and either 0.02 or 0.10 M ionic strength. The solutions were buffered over a pH range such that the percent of carboxylate groups ionized ranged from 25 to 74%. The binding was described by two constants, β1 and β2, which were evaluated after correction for complexation of the actinide cations by acetate and hydrolysis. For comparable degrees of ionization, all three polyelectrolytes showed similar binding strengths. In general, these results indicated that the binding of actinides to these synthetic polyelectrolytes is basically similar to that of natural polyelectrolytes such as humic and fulvic acids. (orig.)

  19. Alzheimer's therapeutics targeting amyloid beta 1-42 oligomers I: Abeta 42 oligomer binding to specific neuronal receptors is displaced by drug candidates that improve cognitive deficits.

    Directory of Open Access Journals (Sweden)

    Nicholas J Izzo

    Full Text Available Synaptic dysfunction and loss caused by age-dependent accumulation of synaptotoxic beta amyloid (Abeta 1-42 oligomers is proposed to underlie cognitive decline in Alzheimer's disease (AD. Alterations in membrane trafficking induced by Abeta oligomers mediates reduction in neuronal surface receptor expression that is the basis for inhibition of electrophysiological measures of synaptic plasticity and thus learning and memory. We have utilized phenotypic screens in mature, in vitro cultures of rat brain cells to identify small molecules which block or prevent the binding and effects of Abeta oligomers. Synthetic Abeta oligomers bind saturably to a single site on neuronal synapses and induce deficits in membrane trafficking in neuronal cultures with an EC50 that corresponds to its binding affinity. The therapeutic lead compounds we have found are pharmacological antagonists of Abeta oligomers, reducing the binding of Abeta oligomers to neurons in vitro, preventing spine loss in neurons and preventing and treating oligomer-induced deficits in membrane trafficking. These molecules are highly brain penetrant and prevent and restore cognitive deficits in mouse models of Alzheimer's disease. Counter-screening these compounds against a broad panel of potential CNS targets revealed they are highly potent and specific ligands of the sigma-2/PGRMC1 receptor. Brain concentrations of the compounds corresponding to greater than 80% receptor occupancy at the sigma-2/PGRMC1 receptor restore cognitive function in transgenic hAPP Swe/Ldn mice. These studies demonstrate that synthetic and human-derived Abeta oligomers act as pharmacologically-behaved ligands at neuronal receptors--i.e. they exhibit saturable binding to a target, they exert a functional effect related to their binding and their displacement by small molecule antagonists blocks their functional effect. The first-in-class small molecule receptor antagonists described here restore memory to normal in

  20. Alzheimer's therapeutics targeting amyloid beta 1-42 oligomers I: Abeta 42 oligomer binding to specific neuronal receptors is displaced by drug candidates that improve cognitive deficits.

    Science.gov (United States)

    Izzo, Nicholas J; Staniszewski, Agnes; To, Lillian; Fa, Mauro; Teich, Andrew F; Saeed, Faisal; Wostein, Harrison; Walko, Thomas; Vaswani, Anisha; Wardius, Meghan; Syed, Zanobia; Ravenscroft, Jessica; Mozzoni, Kelsie; Silky, Colleen; Rehak, Courtney; Yurko, Raymond; Finn, Patricia; Look, Gary; Rishton, Gilbert; Safferstein, Hank; Miller, Miles; Johanson, Conrad; Stopa, Edward; Windisch, Manfred; Hutter-Paier, Birgit; Shamloo, Mehrdad; Arancio, Ottavio; LeVine, Harry; Catalano, Susan M

    2014-01-01

    Synaptic dysfunction and loss caused by age-dependent accumulation of synaptotoxic beta amyloid (Abeta) 1-42 oligomers is proposed to underlie cognitive decline in Alzheimer's disease (AD). Alterations in membrane trafficking induced by Abeta oligomers mediates reduction in neuronal surface receptor expression that is the basis for inhibition of electrophysiological measures of synaptic plasticity and thus learning and memory. We have utilized phenotypic screens in mature, in vitro cultures of rat brain cells to identify small molecules which block or prevent the binding and effects of Abeta oligomers. Synthetic Abeta oligomers bind saturably to a single site on neuronal synapses and induce deficits in membrane trafficking in neuronal cultures with an EC50 that corresponds to its binding affinity. The therapeutic lead compounds we have found are pharmacological antagonists of Abeta oligomers, reducing the binding of Abeta oligomers to neurons in vitro, preventing spine loss in neurons and preventing and treating oligomer-induced deficits in membrane trafficking. These molecules are highly brain penetrant and prevent and restore cognitive deficits in mouse models of Alzheimer's disease. Counter-screening these compounds against a broad panel of potential CNS targets revealed they are highly potent and specific ligands of the sigma-2/PGRMC1 receptor. Brain concentrations of the compounds corresponding to greater than 80% receptor occupancy at the sigma-2/PGRMC1 receptor restore cognitive function in transgenic hAPP Swe/Ldn mice. These studies demonstrate that synthetic and human-derived Abeta oligomers act as pharmacologically-behaved ligands at neuronal receptors--i.e. they exhibit saturable binding to a target, they exert a functional effect related to their binding and their displacement by small molecule antagonists blocks their functional effect. The first-in-class small molecule receptor antagonists described here restore memory to normal in multiple AD models

  1. Alzheimer's therapeutics targeting amyloid beta 1-42 oligomers I: Abeta 42 oligomer binding to specific neuronal receptors is displaced by drug candidates that improve cognitive deficits.

    Science.gov (United States)

    Izzo, Nicholas J; Staniszewski, Agnes; To, Lillian; Fa, Mauro; Teich, Andrew F; Saeed, Faisal; Wostein, Harrison; Walko, Thomas; Vaswani, Anisha; Wardius, Meghan; Syed, Zanobia; Ravenscroft, Jessica; Mozzoni, Kelsie; Silky, Colleen; Rehak, Courtney; Yurko, Raymond; Finn, Patricia; Look, Gary; Rishton, Gilbert; Safferstein, Hank; Miller, Miles; Johanson, Conrad; Stopa, Edward; Windisch, Manfred; Hutter-Paier, Birgit; Shamloo, Mehrdad; Arancio, Ottavio; LeVine, Harry; Catalano, Susan M

    2014-01-01

    Synaptic dysfunction and loss caused by age-dependent accumulation of synaptotoxic beta amyloid (Abeta) 1-42 oligomers is proposed to underlie cognitive decline in Alzheimer's disease (AD). Alterations in membrane trafficking induced by Abeta oligomers mediates reduction in neuronal surface receptor expression that is the basis for inhibition of electrophysiological measures of synaptic plasticity and thus learning and memory. We have utilized phenotypic screens in mature, in vitro cultures of rat brain cells to identify small molecules which block or prevent the binding and effects of Abeta oligomers. Synthetic Abeta oligomers bind saturably to a single site on neuronal synapses and induce deficits in membrane trafficking in neuronal cultures with an EC50 that corresponds to its binding affinity. The therapeutic lead compounds we have found are pharmacological antagonists of Abeta oligomers, reducing the binding of Abeta oligomers to neurons in vitro, preventing spine loss in neurons and preventing and treating oligomer-induced deficits in membrane trafficking. These molecules are highly brain penetrant and prevent and restore cognitive deficits in mouse models of Alzheimer's disease. Counter-screening these compounds against a broad panel of potential CNS targets revealed they are highly potent and specific ligands of the sigma-2/PGRMC1 receptor. Brain concentrations of the compounds corresponding to greater than 80% receptor occupancy at the sigma-2/PGRMC1 receptor restore cognitive function in transgenic hAPP Swe/Ldn mice. These studies demonstrate that synthetic and human-derived Abeta oligomers act as pharmacologically-behaved ligands at neuronal receptors--i.e. they exhibit saturable binding to a target, they exert a functional effect related to their binding and their displacement by small molecule antagonists blocks their functional effect. The first-in-class small molecule receptor antagonists described here restore memory to normal in multiple AD models

  2. Homology modelling of the core domain of the endogenous lectin comitin: structural basis for its mannose-binding specificity.

    Science.gov (United States)

    Barre, A; Van Damme, E J; Peumans, W J; Rougé, P

    1999-03-01

    The N-terminal core domain of comitin from the slime mold Dictyostelium discoideum has been modelled from the X-ray coordinates of the monocot mannose-binding lectin from snowdrop (Galanthus nivalis). Docking experiments performed on the three-dimensional model showed that two of the three mannose-binding sites of the comitin monomer are functional. They are located at both ends of the comitin dimer whereas the actin-interacting region occurs in the central hinge region where both monomers are non covalently associated. This distribution is fully consistent with the bifunctional character of comitin which is believed to link the Golgi vesicles exhibiting mannosylated membrane glycans to the actin cytoskeleton in the cell.

  3. Genus-specific protein binding to the large clusters of DNA repeats (short regularly spaced repeats) present in Sulfolobus genomes

    DEFF Research Database (Denmark)

    Peng, Xu; Brügger, Kim; Shen, Biao;

    2003-01-01

    Short regularly spaced repeats (SRSRs) occur in multiple large clusters in archaeal chromosomes and as smaller clusters in some archaeal conjugative plasmids and bacterial chromosomes. The sequence, size, and spacing of the repeats are generally constant within a cluster but vary between clusters....... For the crenarchaeon Sulfolobus solfataricus P2, the repeats in the genome fall mainly into two closely related sequence families that are arranged in seven clusters containing a total of 441 repeats which constitute ca. 1% of the genome. The Sulfolobus conjugative plasmid pNOB8 contains a small cluster of six repeats...... that are identical in sequence to one of the repeat variants in the S. solfataricus chromosome. Repeats from the pNOB8 cluster were amplified and tested for protein binding with cell extracts from S. solfataricus. A 17.5-kDa SRSR-binding protein was purified from the cell extracts and sequenced. The protein is N...

  4. Binding specificity and in vivo targets of the EH domain, a novel protein-protein interaction module

    DEFF Research Database (Denmark)

    Salcini, A E; Confalonieri, S; Doria, M;

    1997-01-01

    EH is a recently identified protein-protein interaction domain found in the signal transducers Eps15 and Eps15R and several other proteins of yeast nematode. We show that EH domains from Eps15 and Eps15R bind in vitro to peptides containing an asparagine-proline-phenylalanine (NPF) motif. Direct...... screening of expression libraries with EH domains yielded a number of putative EH interactors, all of which possessed NPF motifs that were shown to be responsible for the interaction. Among these interactors were the human homolog of NUMB, a developmentally reguated gene of Drosophila, and RAB, the cellular...... cofactor of the HIV REV protein. We demonstrated coimmunoprecipitation of Eps15 with NUMB and RAB. Finally, in vitro binding of NPF-containing peptides to cellular proteins and EST database screening established the existence of a family of EH-containing proteins in mammals. Based on the characteristics of...

  5. A Simple Method for Discovering Druggable, Specific Glycosaminoglycan-Protein Systems. Elucidation of Key Principles from Heparin/Heparan Sulfate-Binding Proteins.

    Science.gov (United States)

    Sarkar, Aurijit; Desai, Umesh R

    2015-01-01

    Glycosaminoglycans (GAGs) affect human physiology and pathology by modulating more than 500 proteins. GAG-protein interactions are generally assumed to be ionic and nonspecific, but specific interactions do exist. Here, we present a simple method to identify the GAG-binding site (GBS) on proteins that in turn helps predict high specific GAG-protein systems. Contrary to contemporary thinking, we found that the electrostatic potential at basic arginine and lysine residues neither identifies the GBS consistently, nor its specificity. GBSs are better identified by considering the potential at neutral hydrogen bond donors such as asparagine or glutamine sidechains. Our studies also reveal that an unusual constellation of ionic and non-ionic residues in the binding site leads to specificity. Nature engineers the local environment of Asn45 of antithrombin, Gln255 of 3-O-sulfotransferase 3, Gln163 and Asn167 of 3-O-sulfotransferase 1 and Asn27 of basic fibroblast growth factor in the respective GBSs to induce specificity. Such residues are distinct from other uncharged residues on the same protein structure in possessing a significantly higher electrostatic potential, resultant from the local topology. In contrast, uncharged residues on nonspecific GBSs such as thrombin and serum albumin possess a diffuse spread of electrostatic potential. Our findings also contradict the paradigm that GAG-binding sites are simply a collection of contiguous Arg/Lys residues. Our work demonstrates the basis for discovering specifically interacting and druggable GAG-protein systems based on the structure of protein alone, without requiring access to any structure-function relationship data.

  6. A Simple Method for Discovering Druggable, Specific Glycosaminoglycan-Protein Systems. Elucidation of Key Principles from Heparin/Heparan Sulfate-Binding Proteins.

    Directory of Open Access Journals (Sweden)

    Aurijit Sarkar

    Full Text Available Glycosaminoglycans (GAGs affect human physiology and pathology by modulating more than 500 proteins. GAG-protein interactions are generally assumed to be ionic and nonspecific, but specific interactions do exist. Here, we present a simple method to identify the GAG-binding site (GBS on proteins that in turn helps predict high specific GAG-protein systems. Contrary to contemporary thinking, we found that the electrostatic potential at basic arginine and lysine residues neither identifies the GBS consistently, nor its specificity. GBSs are better identified by considering the potential at neutral hydrogen bond donors such as asparagine or glutamine sidechains. Our studies also reveal that an unusual constellation of ionic and non-ionic residues in the binding site leads to specificity. Nature engineers the local environment of Asn45 of antithrombin, Gln255 of 3-O-sulfotransferase 3, Gln163 and Asn167 of 3-O-sulfotransferase 1 and Asn27 of basic fibroblast growth factor in the respective GBSs to induce specificity. Such residues are distinct from other uncharged residues on the same protein structure in possessing a significantly higher electrostatic potential, resultant from the local topology. In contrast, uncharged residues on nonspecific GBSs such as thrombin and serum albumin possess a diffuse spread of electrostatic potential. Our findings also contradict the paradigm that GAG-binding sites are simply a collection of contiguous Arg/Lys residues. Our work demonstrates the basis for discovering specifically interacting and druggable GAG-protein systems based on the structure of protein alone, without requiring access to any structure-function relationship data.

  7. Identification of Plasmodium falciparum RhopH3 protein peptides that specifically bind to erythrocytes and inhibit merozoite invasion

    OpenAIRE

    Pinzón, Carlos Giovanni; Curtidor, Hernando; Reyes, Claudia; Méndez, David; Patarroyo, Manuel Elkin

    2008-01-01

    The identification of sequences involved in binding to erythrocytes is an important step for understanding the molecular basis of merozoite–erythrocyte interactions that take place during invasion of the Plasmodium falciparum malaria parasite into host cells. Several molecules located in the apical organelles (micronemes, rhoptry, dense granules) of the invasive-stage parasite are essential for erythrocyte recognition, invasion, and establishment of the nascent parasitophorous vacuole. Partic...

  8. On the analysis and comparison of conformer-specific essential dynamics upon ligand binding to a protein

    Energy Technology Data Exchange (ETDEWEB)

    Grosso, Marcos; Kalstein, Adrian; Parisi, Gustavo; Fernandez-Alberti, Sebastian, E-mail: sfalberti@gmail.com [Universidad Nacional de Quilmes, Roque Saenz Peña 352, B1876BXD Bernal (Argentina); Roitberg, Adrian E. [Departments of Physics and Chemistry, University of Florida, Gainesville, Florida 32611 (United States)

    2015-06-28

    The native state of a protein consists of an equilibrium of conformational states on an energy landscape rather than existing as a single static state. The co-existence of conformers with different ligand-affinities in a dynamical equilibrium is the basis for the conformational selection model for ligand binding. In this context, the development of theoretical methods that allow us to analyze not only the structural changes but also changes in the fluctuation patterns between conformers will contribute to elucidate the differential properties acquired upon ligand binding. Molecular dynamics simulations can provide the required information to explore these features. Its use in combination with subsequent essential dynamics analysis allows separating large concerted conformational rearrangements from irrelevant fluctuations. We present a novel procedure to define the size and composition of essential dynamics subspaces associated with ligand-bound and ligand-free conformations. These definitions allow us to compare essential dynamics subspaces between different conformers. Our procedure attempts to emphasize the main similarities and differences between the different essential dynamics in an unbiased way. Essential dynamics subspaces associated to conformational transitions can also be analyzed. As a test case, we study the glutaminase interacting protein (GIP), composed of a single PDZ domain. Both GIP ligand-free state and glutaminase L peptide-bound states are analyzed. Our findings concerning the relative changes in the flexibility pattern upon binding are in good agreement with experimental Nuclear Magnetic Resonance data.

  9. On the analysis and comparison of conformer-specific essential dynamics upon ligand binding to a protein

    International Nuclear Information System (INIS)

    The native state of a protein consists of an equilibrium of conformational states on an energy landscape rather than existing as a single static state. The co-existence of conformers with different ligand-affinities in a dynamical equilibrium is the basis for the conformational selection model for ligand binding. In this context, the development of theoretical methods that allow us to analyze not only the structural changes but also changes in the fluctuation patterns between conformers will contribute to elucidate the differential properties acquired upon ligand binding. Molecular dynamics simulations can provide the required information to explore these features. Its use in combination with subsequent essential dynamics analysis allows separating large concerted conformational rearrangements from irrelevant fluctuations. We present a novel procedure to define the size and composition of essential dynamics subspaces associated with ligand-bound and ligand-free conformations. These definitions allow us to compare essential dynamics subspaces between different conformers. Our procedure attempts to emphasize the main similarities and differences between the different essential dynamics in an unbiased way. Essential dynamics subspaces associated to conformational transitions can also be analyzed. As a test case, we study the glutaminase interacting protein (GIP), composed of a single PDZ domain. Both GIP ligand-free state and glutaminase L peptide-bound states are analyzed. Our findings concerning the relative changes in the flexibility pattern upon binding are in good agreement with experimental Nuclear Magnetic Resonance data

  10. Specific binding of collagen Q to the neuromuscular junction is exploited to cure congenital myasthenia and to explore bases of myasthenia gravis.

    Science.gov (United States)

    Ohno, Kinji; Ito, Mikako; Kawakami, Yu; Krejci, Eric; Engel, Andrew G

    2013-03-25

    Acetylcholinesterase (AChE) at the neuromuscular junction (NMJ) is anchored to the synaptic basal lamina via a triple helical collagen Q (ColQ) in the form of asymmetric AChE (AChE/ColQ). The C-terminal domain of ColQ binds to MuSK, the muscle-specific receptor tyrosine kinase, that mediates a signal for acetylcholine receptor (AChR) clustering at the NMJ. ColQ also binds to heparan sulfate proteoglycans including perlecan. Congenital defects of ColQ cause endplate AChE deficiency. A single intravenous administration of adeno-associated virus serotype 8 (AAV8)-COLQ to Colq-/- mice rescued motor functions, synaptic transmission, and the ultrastructure of NMJ. We also injected AAV1-COLQ-IRES-EGFP to the left tibialis anterior and observed colocalization of AChE/ColQ at all the examined NMJs of the non-injected limbs. Additionally, injection of purified recombinant AChE/ColQ protein complex into gluteus maximus accumulated AChE in non-injected forelimbs. These observations suggest that the tissue-targeting signal of ColQ can be exploited to specifically deliver the transgene product to the target tissue. MuSK antibody-positive myasthenia gravis (MG) accounts for 5-15% of autoimmune MG. As AChR deficiency is typically mild and as cholinesterase inhibitors are generally ineffective or worsen myasthenic symptoms, we asked if the patient's MuSK-IgG interferes with binding of ColQ to MuSK. In vitro overlay of AChE/ColQ to muscle sections of Colq-/- mice revealed that MuSK-IgG blocks binding of ColQ to the NMJ. In vitro plate-binding of MuSK to ColQ disclosed that MuSK-IgG exerts a dose-dependent block of MuSK-ColQ interaction. In addition, passive transfer of MuSK-IgG to mice reduced the size and density of ColQ to ∼10% of controls and had a lesser effect on the sizes and densities of AChR and MuSK. Elucidation of molecular mechanisms of specific binding of ColQ to the NMJ enabled us to ameliorate devastating myasthenic symptoms of Colq-/- mice and to reveal bases of

  11. PET study using [11C]FTIMD with ultra-high specific activity to evaluate I2-imidazoline receptors binding in rat brains

    International Nuclear Information System (INIS)

    Introduction: We recently developed a selective 11C-labeled I2-imidazoline receptor (I2R) ligand, 2-(3-fluoro-4-[11C]tolyl)-4,5-dihydro-1H-imidazole ([11C]FTIMD). [11C]FTIMD showed specific binding to I2Rs in rat brains having a high density of I2R, as well as to I2Rs those in monkey brains, as illustrated by positron emission tomography (PET) and autoradiography. However, [11C]FTIMD also showed moderate non-specific binding in rat brains. In order to increase the specificity for I2R in rat brains, we synthesized [11C]FTIMD with ultra-high specific activity and evaluated its binding. Methods: [11C]FTIMD with ultra-high specific activity was prepared by a palladium-promoted cross-coupling reaction of the tributylstannyl precursor and [11C]methyl iodide, which was produced by iodination of [11C]methane using the single-pass method. Dynamic PET scans were conducted in rats, and the kinetic parameters were estimated. Results: [11C]FTIMD with ultra-high specific activity was successfully synthesized with an appropriate level of radioactivity and ultra-high specific activity (4470±1660 GBq/μmol at end of synthesis, n=11) for injection. In the PET study, distribution volume (VT) values in all the brain regions investigated whether I2R expression was greatly reduced in BU224-pretreatead rats compared with control rats (29–45% decrease). Differences in VT values between control and BU224-pretreated rats using [11C]FTIMD with ultra-high specific activity were greater than those using [11C]FTIMD with normal specific activity (17–34% decrease) in all brain regions investigated. Conclusion: Quantitative PET using [11C]FTIMD with ultra-high specific activity can contribute to the detection of small changes in I2R expression in the brain.

  12. Peptide-specific,allogeneic T cell response in vitro induced by a self-peptide binding to HLA-A2

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The role of the bound peptide in alloreactive T-cell recognition is controversial, ranging from pep-tide-independent to peptide-specific recognition of alloreactive T-cells. The aim of this study is to find the evidence that there exist peptide/MHC complex (pMHC)-specific CTLs among alloreactive T cells generated with long-term mixed lymphocytes culture (LTMLC). A single pMHC was manipulated by loading the TAP-defective, HLA-A2 expressing T2 cells with a viral peptide (LMP2A426-434) or a self-peptide (Tyr369-377). The PBLs samples from 4 HLA-A2 positive (HLA-A2+ve) and 4 HLA-A2 negative (HLA-A2-ve) donors were included in this study. The HLA-A2+ve PBL co-cultured with the LMP2A426-434 pulsed T2 (T2/LMP) stands for the nominal T-cell response to a viral antigen, and the HLA-A2-ve PBLs co-cultured with the Tyr369-377 pulsed T2 (T2/Tyr) for alloreactive T-cell response to an allogeneic antigen. The specificity of the expanded CTLs after the LTMLC was detected by their specific cytotoxicity and binding ability to specific pMHC-tetramer. An HLA-A2 restricted, HIV peptide (Gag77-85)was included for control. The cultural bulk of HLA-A2+ve PBLs with the T2/LMP showed an elevated specific cytotoxicity against the T2/LMP compared to that against the T2/HIV (26.52%±3.72% vs 7.01%±0.87%, P<0.001), and an increased frequency of binding to LMP-tetramer compared to that binding to HIV-tetramer (0.98%±0.33% vs 0.05%±0.01%, P=0.0014). The cultural bulk of HLA-A2-ve PBLs with the T2/Tyr showed a more active cytotoxicity against the T2/Tyr than that against T2/HIV (28.07%±2.58% vs 6.87%±0.01 %, P<0.001), and a higher frequency of binding to the Tyr-tetramer than that binding to the HIV-tetramer (0.88%±0.3% vs 0.06%±0.03%, P=0.0018). Our results indicate that the LTMLC is able to expand the viral antigen-specific CTLs as well as allogeneic antigen-specific CTLs. A relatively large proportion of alloreactive CTLs should be pMHC-specific, i.e., the specificity of the

  13. Peptide-specific, allogeneic T cell response in vitro induced by a self-peptide binding to HLA-A2

    Institute of Scientific and Technical Information of China (English)

    WENG XiuFang; LIANG ZhiHui; LU XiaoLing; ZHONG MaoHua; LU ShengJun; ZHANG CaiE; DENG Jing; WU XiongWen; GONG FeiLi

    2007-01-01

    The role of the bound peptide in alloreactive T-cell recognition is controversial, ranging from peptide-independent to peptide-specific recognition of alloreactive T-cells. The aim of this study is to find the evidence that there exist peptide/MHC complex (pMHC)-specific CTLs among alloreactive T cells generated with long-term mixed lymphocytes culture (LTMLC). A single pMHC was manipulated by loading the TAP-defective, HLA-A2 expressing T2 cells with a viral peptide (LMP2A426-434) or a self-peptide (Tyr369-377). The PBLs samples from 4 HLA-A2 positive (HLA-A2+ve) and 4 HLA-A2 negative (HLA-A2-ve) donors were included in this study. The HLA-A2+ve PBL co-cultured with the LMP2A426-434pulsed T2 (T2/LMP) stands for the nominal T-cell response to a viral antigen, and the HLA-A2-ve PBLs co-cultured with the Tyr369-377 pulsed T2 (T2/Tyr) for alloreactive T-cell response to an allogeneic antigen.The specificity of the expanded CTLs after the LTMLC was detected by their specific cytotoxicity and binding ability to specific pMHC-tetramer. An HLA-A2 restricted, HIV peptide (Gag77-85) was included for control. The cultural bulk of HLA-A2+ve PBLs with the T2/LMP showed an elevated specific cytotoxicity against the T2/LMP compared to that against the T2/HIV (26.52%±3.72% vs 7.01%±0.87%, P<0.001), and an increased frequency of binding to LMP-tetramer compared to that binding to HIV-tetramer (0.98%±0.33% vs 0.05%±0.01%, P=0.0014). The cultural bulk of HLA-A2-ve PBLs with the T2/Tyr showed a more active cytotoxicity against the T2/Tyr than that against T2/HIV (28.07%±2.58% vs 6.87%±1.01%,P<0.001), and a higher frequency of binding to the Tyr-tetramer than that binding to the HIV-tetramer (0.88%±0.3% vs 0.06%±0.03%, P=0.0018). Our results indicate that the LTMLC is able to expand the viral antigen-specific CTLs as well as allogeneic antigen-specific CTLs. A relatively large proportion of alloreactive CTLs should be pMHC-specific, i.e., the specificity of the

  14. Correction: Identification of specific calcitonin-like receptor residues important for calcitonin gene-related peptide high affinity binding

    OpenAIRE

    Banerjee, Sugato; Evanson, Janel; Harris, Erik; Lowe, Stephen L; Speth, Robert C.; Thomasson, Kathryn A; Porter, James E.

    2006-01-01

    This is a correction article. After publication of this work [1], we became aware of the fact that Robert C. Speth was not included as an author. Dr. Speth put a considerable amount of time and effort into developing and preparing the radiopeptide used to carry out the radioligand binding studies reported in this manuscript and therefore should have originally been included as an author. We apologize to Dr. Speth for any inconvenience that this oversight might have caused and thank him for hi...

  15. ARF-B2: A Protein Complex that Specifically Binds to Part of the Anaerobic Response Element of Maize Adh 11

    Science.gov (United States)

    Ferl, Robert J.

    1990-01-01

    Crude whole cell extracts from maize (Zea mays L.) suspension cells were examined for DNA binding proteins that specifically interact with a portion of the maize Adh 1 promoter that was previously shown to be in contact with a trans-acting factor in vivo. A 17 base pair, double-stranded oligonucleotide probe was constructed that centered around a strong in vivo dimethylsulfate footprint (B2) that coincides with part of the anaerobic response element (ARE). Gel retardation assays were used to characterize a major, specific DNA binding protein activity found in the crude extracts. The activity is present in both aerobic and hypoxically treated cultures and has been designated ARF-B2 (ARE binding factor). ARF-B2 appears to be a multicomponent complex, with a 54 kilodalton subunit termed ARF-B2α in primary contact with the target DNA. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:16667563

  16. Complexation Between Cationic Diblock Copolymers and Plasmid DNA

    Science.gov (United States)

    Jung, Seyoung; Reineke, Theresa; Lodge, Timothy

    Deoxyribonucleic acids (DNA), as polyanions, can spontaneously bind with polycations to form polyelectrolyte complexes. When the polycation is a diblock copolymer with one cationic block and one uncharged hydrophilic block, the polyelectrolyte complexes formed with plasmid DNA (pDNA) are often colloidally stable, and show great promise in the field of polymeric gene therapy. While the resulting properties (size, stability, and toxicity to biological systems) of the complexes have been studied for numerous cationic diblocks, the fundamentals of the pDNA-diblock binding process have not been extensively investigated. Herein, we report how the cationic block content of a diblock influences the pDNA-diblock interactions. pDNA with 7164 base pairs and poly(2-deoxy-2-methacrylamido glucopyranose)-block-poly(N-(2-aminoethyl) methacrylamide) (PMAG-b-PAEMA) are used as the model pDNA and cationic diblock, respectively. To vary the cationic block content, two PMAG-b-PAEMA copolymers with similar PMAG block lengths but distinct PAEMA block lengths and a PAEMA homopolymer are utilized. We show that the enthalpy change from pDNA-diblock interactions is dependent on the cationic diblock composition, and is closely associated with both the binding strength and the pDNA tertiary structure.

  17. Detection of Interaction of Binding Affinity of Aromatic Hydrocarbon Receptor to the Specific DNA by Exonuclease Protection Mediated PCR Assay

    Institute of Scientific and Technical Information of China (English)

    SUN Xi; XU Shunqing

    2005-01-01

    A novel exonuclease protection mediated PCR assay (EPM-PCR) to detect the interaction of protein and DNA at a dioxin-responsive enhancer (DRE) upstream of the CYP1A1 gene in rat hepatic cytosol was established. A double-stranded DNA fragment containing two binding sites was designed and incubated with the aryl hydrocarbon receptor (AhR) transformed by 2,3,7,8-tet rachlorodibenzo p dioxin (TCDD) to generate TCDD: AhR: DNA complex which could protect receptor-binding DNA against exonuclease Ⅲ (Exo Ⅲ) digestion. With ExoⅢ treatment, free DNAs were digested and receptor-bound DNAs remained that could be amplified by PCR. By agarose gel electrophoreses a clear band (285bp) was detected using TCDD-treated sample, while nothing with control samples. To detect transformed AhR-DRE complex, 2 fmol DNAs and 3 ug cytosol proteins were found to be sufficient in the experiment. Compared with gel retardation assay, this new method is more sensitive for monitoring the Ah receptor-enhancer interaction without radioactive pollution.

  18. The extracytoplasmic domain of the Mycobacterium tuberculosis Ser/Thr kinase PknB binds specific muropeptides and is required for PknB localization.

    Directory of Open Access Journals (Sweden)

    Mushtaq Mir

    2011-07-01

    Full Text Available The Mycobacterium tuberculosis Ser/Thr kinase PknB has been implicated in the regulation of cell growth and morphology in this organism. The extracytoplasmic domain of this membrane protein comprises four penicillin binding protein and Ser/Thr kinase associated (PASTA domains, which are predicted to bind stem peptides of peptidoglycan. Using a comprehensive library of synthetic muropeptides, we demonstrate that the extracytoplasmic domain of PknB binds muropeptides in a manner dependent on the presence of specific amino acids at the second and third positions of the stem peptide, and on the presence of the sugar moiety N-acetylmuramic acid linked to the peptide. We further show that PknB localizes strongly to the mid-cell and also to the cell poles, and that the extracytoplasmic domain is required for PknB localization. In contrast to strong growth stimulation by conditioned medium, we observe no growth stimulation of M. tuberculosis by a synthetic muropeptide with high affinity for the PknB PASTAs. We do find a moderate effect of a high affinity peptide on resuscitation of dormant cells. While the PASTA domains of PknB may play a role in stimulating growth by binding exogenous peptidoglycan fragments, our data indicate that a major function of these domains is for proper PknB localization, likely through binding of peptidoglycan fragments produced locally at the mid-cell and the cell poles. These data suggest a model in which PknB is targeted to the sites of peptidoglycan turnover to regulate cell growth and cell division.

  19. Role of cysteines in the stability and DNA-binding activity of the hypochlorite-specific transcription factor HypT.

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    Adrian Drazic

    Full Text Available Reactive oxygen species are important components of the immune response. Hypochlorite (HOCl is produced by neutrophils to kill invading microorganisms. The bactericidal activity of HOCl is due to proteome-wide unfolding and oxidation of proteins at cysteine and methionine residues. Escherichia coli cells are protected from HOCl-killing by the previously identified dodecameric transcription factor HypT (YjiE. Here, we aimed to unravel whether HOCl activates HypT directly or via a reaction product of HOCl with a cellular component. Bacterial viability assays and analysis of target gene regulation indicate that HypT is highly specific to activation by HOCl and that no reaction products of HOCl such as monochloramine, hydroxyl radicals, or methionine sulfoxide activate HypT in vivo. Surprisingly, purified HypT lost its DNA-binding activity upon incubation with HOCl or reaction products that oxidize HypT to form a disulfide-linked dimer, and regained DNA-binding activity upon reduction. Thus, we postulate that the cysteines in HypT contribute to control the DNA-binding activity of HypT in vitro. HypT contains five cysteine residues; a HypT mutant with all cysteines substituted by serine is aggregation-prone and forms tetramers in addition to the typical dodecamers. Using single and multiple cysteine-to-serine mutants, we identified Cys150 to be required for stability and Cys4 being important for oligomerization of HypT to dodecamers. Further, oxidation of Cys4 is responsible for the loss of DNA-binding of HypT upon oxidation in vitro. It appears that Cys4 oxidation upon conditions that are insufficient to stimulate the DNA-binding activity of HypT prevents unproductive interactions of HypT with DNA. Thus, Cys4 oxidation may be a check point in the activation process of HypT.

  20. Genome-wide and phase-specific DNA-binding rhythms of BMAL1 control circadian output functions in mouse liver.

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    Guillaume Rey

    2011-02-01

    Full Text Available The mammalian circadian clock uses interlocked negative feedback loops in which the heterodimeric basic helix-loop-helix transcription factor BMAL1/CLOCK is a master regulator. While there is prominent control of liver functions by the circadian clock, the detailed links between circadian regulators and downstream targets are poorly known. Using chromatin immunoprecipitation combined with deep sequencing we obtained a time-resolved and genome-wide map of BMAL1 binding in mouse liver, which allowed us to identify over 2,000 binding sites, with peak binding narrowly centered around Zeitgeber time 6. Annotation of BMAL1 targets confirms carbohydrate and lipid metabolism as the major output of the circadian clock in mouse liver. Moreover, transcription regulators are largely overrepresented, several of which also exhibit circadian activity. Genes of the core circadian oscillator stand out as strongly bound, often at promoter and distal sites. Genomic sequence analysis of the sites identified E-boxes and tandem E1-E2 consensus elements. Electromobility shift assays showed that E1-E2 sites are bound by a dimer of BMAL1/CLOCK heterodimers with a spacing-dependent cooperative interaction, a finding that was further validated in transactivation assays. BMAL1 target genes showed cyclic mRNA expression profiles with a phase distribution centered at Zeitgeber time 10. Importantly, sites with E1-E2 elements showed tighter phases both in binding and mRNA accumulation. Finally, analyzing the temporal profiles of BMAL1 binding, precursor mRNA and mature mRNA levels showed how transcriptional and post-transcriptional regulation contribute differentially to circadian expression phase. Together, our analysis of a dynamic protein-DNA interactome uncovered how genes of the core circadian oscillator crosstalk and drive phase-specific circadian output programs in a complex tissue.

  1. Efficient synthesis of metallated thioporphyrazines in task specific ionic liquids and their spectroscopic investigation of binding with selected transition metal ions

    Indian Academy of Sciences (India)

    POONAM; RITIKA NAGPAL; SMRITI ARORA; SHIVE M S CHAUHAN

    2016-09-01

    Tetramerization of substituted maleonitriles in task specific 2-hydroxylethyl based imidazolium ionic liquids at 120◦C gave corresponding electron rich peripheral substituted thioporphyrazines in moderate yield. The 2-hydroxylethyl imidazolium ionic liquids gave better yields of peripheral substituted thioporphyrazinesin comparison with non-hydroxyl functionalized ionic liquids. Further, these peripherally functionalized porphyrazines containing sulphur are used to investigate spectroscopically the binding studies with palladium(II) and mercury(II) ions. These metal ions are toxic in nature and deserve serious attention in the areaof design of effective separation and efficient micro-sensing techniques. The UV–Vis absorption spectroscopy and fluorescence signalling are mainly used to study peripheral binding of transition metal ions.

  2. Demonstration of specific binding of heparin to Plasmodium falciparum-infected vs. non-infected red blood cells by single-molecule force spectroscopy

    Science.gov (United States)

    Valle-Delgado, Juan José; Urbán, Patricia; Fernàndez-Busquets, Xavier

    2013-04-01

    Glycosaminoglycans (GAGs) play an important role in the sequestration of Plasmodium falciparum-infected red blood cells (pRBCs) in the microvascular endothelium of different tissues, as well as in the formation of small clusters (rosettes) between infected and non-infected red blood cells (RBCs). Both sequestration and rosetting have been recognized as characteristic events in severe malaria. Here we have used heparin and pRBCs infected by the 3D7 strain of P. falciparum as a model to study GAG-pRBC interactions. Fluorescence microscopy and fluorescence-assisted cell sorting assays have shown that exogenously added heparin has binding specificity for pRBCs (preferentially for those infected with late forms of the parasite) vs. RBCs. Heparin-pRBC adhesion has been probed by single-molecule force spectroscopy, obtaining an average binding force ranging between 28 and 46 pN depending on the loading rate. No significant binding of heparin to non-infected RBCs has been observed in control experiments. This work represents the first approach to quantitatively evaluate GAG-pRBC molecular interactions at the individual molecule level.Glycosaminoglycans (GAGs) play an important role in the sequestration of Plasmodium falciparum-infected red blood cells (pRBCs) in the microvascular endothelium of different tissues, as well as in the formation of small clusters (rosettes) between infected and non-infected red blood cells (RBCs). Both sequestration and rosetting have been recognized as characteristic events in severe malaria. Here we have used heparin and pRBCs infected by the 3D7 strain of P. falciparum as a model to study GAG-pRBC interactions. Fluorescence microscopy and fluorescence-assisted cell sorting assays have shown that exogenously added heparin has binding specificity for pRBCs (preferentially for those infected with late forms of the parasite) vs. RBCs. Heparin-pRBC adhesion has been probed by single-molecule force spectroscopy, obtaining an average binding force

  3. Specific binding of a mutated fragment of Clostridium perfringens enterotoxin to endothelial claudin-5 and its modulation of cerebral vascular permeability.

    Science.gov (United States)

    Liao, Zhuangbin; Yang, Zhenguo; Piontek, Anna; Eichner, Miriam; Krause, Gerd; Li, Longxuan; Piontek, Joerg; Zhang, Jingjing

    2016-07-01

    The vertebrate blood-brain barrier (BBB) creates an obstacle for central nervous system-related drug delivery. Claudin-5 (Cldn5), expressed in large quantities in BBB, plays a vital role in restricting BBB permeability. The C-terminal domain of Clostridium perfringens enterotoxin (cCPE) has been verified as binding to a subset of claudins (Cldns). The Cldn5-binding cCPE194-319 variant cCPEY306W/S313H was applied in this study to investigate its ability to modulate the permeability of zebrafish larval BBB. In vitro results showed that cCPEY306W/S313H is able to bind specifically to Cldn5 in murine brain vascular endothelial (bEnd.3) cells, and is transported along with Cldn5 from the cell membrane to the cytoplasm, which in turn results in a reduction in transendothelial electrical resistance (TEER). Conversely, this effect can be reversed by removal of cCPEY306W/S313H. In an in vivo experiment, this study estimates the capability of cCPEY306W/S313H to modulate Cldn5 using a rhodamine B-Dextran dye diffusion assay in zebrafish larval BBB. The results show that cCPEY306W/S313H co-localized with Cldn5 in zebrafish cerebral vascular cells and modulated BBB permeability, resulting in dye leakage. Taken together, this study suggests that cCPEY306W/S313H has the capability - both in vitro and in vivo - to modulate BBB permeability temporarily by specific binding to Cldn5. PMID:27095710

  4. Actinomyces naeslundii Displays Variant fimP and fimA Fimbrial Subunit Genes Corresponding to Different Types of Acidic Proline-Rich Protein and β-Linked Galactosamine Binding Specificity

    OpenAIRE

    Hallberg, K.; Holm, C.; Öhman, U.; Strömberg, N.

    1998-01-01

    Actinomyces naeslundii genospecies 1 and 2 bind to acidic proline-rich proteins (APRPs) and statherin via type 1 fimbriae and to β-linked galactosamine (GalNAcβ) structures via type 2 fimbriae. In addition, A. naeslundii displays two types of binding specificity for both APRPs-statherin and GalNAcβ, while Actinomyces odontolyticus binds to unknown structures. To study the molecular basis for these binding specificities, DNA fragments spanning the entire or central portions of fimP (type 1) an...

  5. Deciphering ligand specificity of a Clostridium thermocellum family 35 carbohydrate binding module (CtCBM35 for gluco- and galacto- substituted mannans and its calcium induced stability.

    Directory of Open Access Journals (Sweden)

    Arabinda Ghosh

    Full Text Available This study investigated the role of CBM35 from Clostridium thermocellum (CtCBM35 in polysaccharide recognition. CtCBM35 was cloned into pET28a (+ vector with an engineered His6 tag and expressed in Escherichia coli BL21 (DE3 cells. A homogenous 15 kDa protein was purified by immobilized metal ion chromatography (IMAC. Ligand binding analysis of CtCBM35 was carried out by affinity electrophoresis using various soluble ligands. CtCBM35 showed a manno-configured ligand specific binding displaying significant association with konjac glucomannan (Ka = 14.3×10(4 M(-1, carob galactomannan (Ka = 12.4×10(4 M(-1 and negligible association (Ka = 12 µM(-1 with insoluble mannan. Binding of CtCBM35 with polysaccharides which was calcium dependent exhibited two fold higher association in presence of 10 mM Ca(2+ ion with konjac glucomannan (Ka = 41×10(4 M(-1 and carob galactomannan (Ka = 30×10(4 M(-1. The polysaccharide binding was further investigated by fluorescence spectrophotometric studies. On binding with carob galactomannan and konjac glucomannan the conformation of CtCBM35 changed significantly with regular 21 nm peak shifts towards lower quantum yield. The degree of association (K a with konjac glucomannan and carob galactomannan, 14.3×10(4 M(-1 and 11.4×10(4 M(-1, respectively, corroborated the findings from affinity electrophoresis. The association of CtCBM35with konjac glucomannan led to higher free energy of binding (ΔG -25 kJ mole(-1 as compared to carob galactomannan (ΔG -22 kJ mole(-1. On binding CtCBM35 with konjac glucomannan and carob galactomannan the hydrodynamic radius (RH as analysed by dynamic light scattering (DLS study, increased to 8 nm and 6 nm, respectively, from 4.25 nm in absence of ligand. The presence of 10 mM Ca(2+ ions imparted stiffer orientation of CtCBM35 particles with increased RH of 4.52 nm. Due to such stiffer orientation CtCBM35 became more thermostable and its melting temperature was

  6. Strain-specific diversity of mucus-binding proteins in the adhesion and aggregation properties of Lactobacillus reuteri.

    Science.gov (United States)

    Mackenzie, Donald A; Jeffers, Faye; Parker, Mary L; Vibert-Vallet, Amandine; Bongaerts, Roy J; Roos, Stefan; Walter, Jens; Juge, Nathalie

    2010-11-01

    Mucus-binding proteins (MUBs) have been revealed as one of the effector molecules involved in mechanisms of the adherence of lactobacilli to the host; mub, or mub-like, genes are found in all of the six genomes of Lactobacillus reuteri that are available. We recently reported the crystal structure of a Mub repeat from L. reuteri ATCC 53608 (also designated strain 1063), revealing an unexpected recognition of immunoglobulins. In the current study, we explored the diversity of the ATCC 53608 mub gene, and MUB expression levels in a large collection of L. reuteri strains isolated from a range of vertebrate hosts. This analysis revealed that the MUB was only detectable on the cell surface of two highly related isolates when using antibodies that were raised against the protein. There was considerable variation in quantitative mucus adhesion in vitro among L. reuteri strains, and mucus binding showed excellent correlation with the presence of cell-surface ATCC 53608 MUB. ATCC 53608 MUB presence was further highly associated with the autoaggregation of L. reuteri strains in washed cell suspensions, suggesting a novel role of this surface protein in cell aggregation. We also characterized MUB expression in representative L. reuteri strains. This analysis revealed that one derivative of strain 1063 was a spontaneous mutant that expressed a C-terminally truncated version of MUB. This frameshift mutation was caused by the insertion of a duplicated 13 nt sequence at position 4867 nt in the mub gene, producing a truncated MUB also lacking the C-terminal LPxTG region, and thus unable to anchor to the cell wall. This mutant, designated 1063N (mub-4867(i)), displayed low mucus-binding and aggregation capacities, further providing evidence for the contribution of cell-wall-anchored MUB to such phenotypes. In conclusion, this study provided novel information on the functional attributes of MUB in L. reuteri, and further demonstrated that MUB and MUB-like proteins

  7. Sequence analyses of fimbriae subunit FimA proteins on Actinomyces naeslundii genospecies 1 and 2 and Actinomyces odontolyticus with variant carbohydrate binding specificities

    Directory of Open Access Journals (Sweden)

    Persson Karina

    2006-05-01

    Full Text Available Abstract Background Actinomyces naeslundii genospecies 1 and 2 express type-2 fimbriae (FimA subunit polymers with variant Galβ binding specificities and Actinomyces odontolyticus a sialic acid specificity to colonize different oral surfaces. However, the fimbrial nature of the sialic acid binding property and sequence information about FimA proteins from multiple strains are lacking. Results Here we have sequenced fimA genes from strains of A.naeslundii genospecies 1 (n = 4 and genospecies 2 (n = 4, both of which harboured variant Galβ-dependent hemagglutination (HA types, and from A.odontolyticus PK984 with a sialic acid-dependent HA pattern. Three unique subtypes of FimA proteins with 63.8–66.4% sequence identity were present in strains of A. naeslundii genospecies 1 and 2 and A. odontolyticus. The generally high FimA sequence identity (>97.2% within a genospecies revealed species specific sequences or segments that coincided with binding specificity. All three FimA protein variants contained a signal peptide, pilin motif, E box, proline-rich segment and an LPXTG sorting motif among other conserved segments for secretion, assembly and sorting of fimbrial proteins. The highly conserved pilin, E box and LPXTG motifs are present in fimbriae proteins from other Gram-positive bacteria. Moreover, only strains of genospecies 1 were agglutinated with type-2 fimbriae antisera derived from A. naeslundii genospecies 1 strain 12104, emphasizing that the overall folding of FimA may generate different functionalities. Western blot analyses with FimA antisera revealed monomers and oligomers of FimA in whole cell protein extracts and a purified recombinant FimA preparation, indicating a sortase-independent oligomerization of FimA. Conclusion The genus Actinomyces involves a diversity of unique FimA proteins with conserved pilin, E box and LPXTG motifs, depending on subspecies and associated binding specificity. In addition, a sortase independent

  8. 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

    The scorpion toxin BeKm-1 is unique among a variety of known short scorpion toxins affecting potassium channels in its selective action on ether-a-go-go-related gene (ERG)-type channels. BeKm-1 shares the common molecular scaffold with other short scorpion toxins. The toxin spatial structure...... 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...