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Sample records for oxygen-binding protein myoglobin

  1. Expression and Purification of Sperm Whale Myoglobin

    Science.gov (United States)

    Miller, Stephen; Indivero, Virginia; Burkhard, Caroline

    2010-01-01

    We present a multiweek laboratory exercise that exposes students to the fundamental techniques of bacterial expression and protein purification through the preparation of sperm whale myoglobin. Myoglobin, a robust oxygen-binding protein, contains a single heme that gives the protein a reddish color, making it an ideal subject for the teaching…

  2. Positively selected sites in cetacean myoglobins contribute to protein stability

    DEFF Research Database (Denmark)

    Dasmeh, Pouria; Serohijos, Adrian W R; Kepp, Kasper P

    2013-01-01

    Since divergence ∼50 Ma ago from their terrestrial ancestors, cetaceans underwent a series of adaptations such as a ∼10-20 fold increase in myoglobin (Mb) concentration in skeletal muscle, critical for increasing oxygen storage capacity and prolonging dive time. Whereas the O2-binding affinity...... between Mb folding stability and protein abundance, suggesting that a selection pressure for stability acts proportionally to higher expression. We also identify a major divergence event leading to the common ancestor of whales, during which major stabilization occurred. Most of the positively selected...

  3. Myoglobin Expression in Chelonia mydas Brain, Heart and Liver Tissues

    Directory of Open Access Journals (Sweden)

    RINI PUSPITANINGRUM

    2010-09-01

    Full Text Available An understanding of the underpinning physiology and biochemistry of animals is essential to properly understand the impact of anthropogenic changes and natural catastrophes upon the conservation of endangered species. An observation on the tissue location of the key respiratory protein, myoglobin, now opens up new opportunities for understanding how hypoxia tolerance impacts on diving lifestyle in turtles. The respiratory protein, myoglobin has functions other than oxygen binding which are involved in hypoxia tolerance, including metabolism of reactive oxygen species and of the vascular function by metabolism of nitric oxide. Our work aims to determine whether myoglobin expression in the green turtle exists in multiple non muscle tissues and to confirm the hypothesis that reptiles also have a distributed myoglobin expression which is linked to the hypoxiatolerant trait. This initial work in turtle hatch Chelonia mydas confirms the presence of myoglobin transcriptin brain, heart and liver tissues. Furthermore, it will serve as a tool for completing the sequence and generating an in situ hybridization probe for verifying of cell location in expressing tissues.

  4. Myoglobin Expression in Chelonia mydas Brain, Heart and Liver Tissues

    Directory of Open Access Journals (Sweden)

    RINI PUSPITANINGRUM

    2010-09-01

    Full Text Available An understanding of the underpinning physiology and biochemistry of animals is essential to properly understand the impact of anthropogenic changes and natural catastrophes upon the conservation of endangered species. An observation on the tissue location of the key respiratory protein, myoglobin, now opens up new opportunities for understanding how hypoxia tolerance impacts on diving lifestyle in turtles. The respiratory protein, myoglobin has functions other than oxygen binding which are involved in hypoxia tolerance, including metabolism of reactive oxygen species and of the vascular function by metabolism of nitric oxide. Our work aims to determine whether myoglobin expression in the green turtle exists in multiple non muscle tissues and to confirm the hypothesis that reptiles also have a distributed myoglobin expression which is linked to the hypoxia-tolerant trait. This initial work in turtle hatch Chelonia mydas confirms the presence of myoglobin transcriptin brain, heart and liver tissues. Furthermore, it will serve as a tool for completing the sequence and generating an in situ hybridization probe for verifying of cell location in expressing tissues.

  5. Met-myoglobin formation, accumulation, degradation, and myoglobin oxygenation monitoring based on multiwavelength attenuance measurement in porcine meat

    Science.gov (United States)

    Nguyen, Thien; Phan, Kien Nguyen; Lee, Jee-Bum; Kim, Jae Gwan

    2016-05-01

    We propose a simple, rapid, and nondestructive method to investigate formation, accumulation, and degradation of met-myoglobin (met-Mb) and myoglobin oxygenation from the interior of porcine meat. For the experiment, color photos and attenuance spectra of porcine meat (well-bled muscle, fat, and mixed) were collected daily to perform colorimetric analysis and to obtain the differences of attenuance between 578 and 567 nm (A578-A567) and between 615 and 630 nm (A630-A615), respectively. Oxy-, deoxy-, and met-myoglobin concentration changes over storage time were also calculated using Beer-Lamberts' law with reflectance intensities at 557, 582, and 630 nm. The change of A578-A567 was well matched with the change of myoglobin oxygenation, and the change of A630-A615 corresponded well with the formation and degradation of met-Mb. In addition, attenuation differences, A578-A567 and A630-A615, were able to show the formation of met-Mb earlier than colorimetric analysis. Therefore, the attenuance differences between wavelengths can be indicators for estimating myoglobin oxygenation and met-Mb formation, accumulation, and degradation, which enable us to design a simple device to monitor myoglobin activities in porcine meat.

  6. Myoglobin oxygen affinity in aquatic and terrestrial birds and mammals.

    Science.gov (United States)

    Wright, Traver J; Davis, Randall W

    2015-07-01

    Myoglobin (Mb) is an oxygen binding protein found in vertebrate skeletal muscle, where it facilitates intracellular transport and storage of oxygen. This protein has evolved to suit unique physiological needs in the muscle of diving vertebrates that express Mb at much greater concentrations than their terrestrial counterparts. In this study, we characterized Mb oxygen affinity (P50) from 25 species of aquatic and terrestrial birds and mammals. Among diving species, we tested for correlations between Mb P50 and routine dive duration. Across all species examined, Mb P50 ranged from 2.40 to 4.85 mmHg. The mean P50 of Mb from terrestrial ungulates was 3.72±0.15 mmHg (range 3.70-3.74 mmHg). The P50 of cetaceans was similar to terrestrial ungulates ranging from 3.54 to 3.82 mmHg, with the exception of the melon-headed whale, which had a significantly higher P50 of 4.85 mmHg. Among pinnipeds, the P50 ranged from 3.23 to 3.81 mmHg and showed a trend for higher oxygen affinity in species with longer dive durations. Among diving birds, the P50 ranged from 2.40 to 3.36 mmHg and also showed a trend of higher affinities in species with longer dive durations. In pinnipeds and birds, low Mb P50 was associated with species whose muscles are metabolically active under hypoxic conditions associated with aerobic dives. Given the broad range of potential globin oxygen affinities, Mb P50 from diverse vertebrate species appears constrained within a relatively narrow range. High Mb oxygen affinity within this range may be adaptive for some vertebrates that make prolonged dives. © 2015. Published by The Company of Biologists Ltd.

  7. Crosstalk between nitrite, myoglobin and reactive oxygen species to regulate vasodilation under hypoxia.

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    Matthias Totzeck

    Full Text Available The systemic response to decreasing oxygen levels is hypoxic vasodilation. While this mechanism has been known for more than a century, the underlying cellular events have remained incompletely understood. Nitrite signaling is critically involved in vessel relaxation under hypoxia. This can be attributed to the presence of myoglobin in the vessel wall together with other potential nitrite reductases, which generate nitric oxide, one of the most potent vasodilatory signaling molecules. Questions remain relating to the precise concentration of nitrite and the exact dose-response relations between nitrite and myoglobin under hypoxia. It is furthermore unclear whether regulatory mechanisms exist which balance this interaction. Nitrite tissue levels were similar across all species investigated. We then investigated the exact fractional myoglobin desaturation in an ex vivo approach when gassing with 1% oxygen. Within a short time frame myoglobin desaturated to 58±12%. Given that myoglobin significantly contributes to nitrite reduction under hypoxia, dose-response experiments using physiological to pharmacological nitrite concentrations were conducted. Along all concentrations, abrogation of myoglobin in mice impaired vasodilation. As reactive oxygen species may counteract the vasodilatory response, we used superoxide dismutase and its mimic tempol as well as catalase and ebselen to reduce the levels of reactive oxygen species during hypoxic vasodilation. Incubation of tempol in conjunction with catalase alone and catalase/ebselen increased the vasodilatory response to nitrite. Our study shows that modest hypoxia leads to a significant nitrite-dependent vessel relaxation. This requires the presence of vascular myoglobin for both physiological and pharmacological nitrite levels. Reactive oxygen species, in turn, modulate this vasodilation response.

  8. Aerobic dive limits of seals with mutant myoglobin using combined thermochemical and physiological data

    DEFF Research Database (Denmark)

    Dasmeh, Pouria; Davis, Randall W.; Kepp, Kasper Planeta

    2013-01-01

    This paper presents an integrated model of convective O2-transport, aerobic dive limits (ADL), and thermochemical data for oxygen binding to mutant myoglobin (Mb), used to quantify the impact of mutations in Mb on the dive limits of Weddell seals (Leptonychotes weddellii). We find that wild-type ...... that such conditions are mostly selected upon in seals. The model is capable of roughly quantifying the physiological impact of single-protein mutations and thus bridges an important gap between animal physiology and molecular (protein) evolution.......This paper presents an integrated model of convective O2-transport, aerobic dive limits (ADL), and thermochemical data for oxygen binding to mutant myoglobin (Mb), used to quantify the impact of mutations in Mb on the dive limits of Weddell seals (Leptonychotes weddellii). We find that wild-type Mb...... traits are only superior under specific behavioral and physiological conditions that critically prolong the ADL, action radius, and fitness of the seals. As an extreme example, the mutations in the conserved His-64 reduce ADL up to 14±2 min for routine aerobic dives, whereas many other mutations...

  9. Myoglobins: the link between discoloration and lipid oxidation in muscle and meat

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    Jens K. S. Møller

    2006-12-01

    Full Text Available Aerobic metabolism changes rapidly to glycolysis post-mortem resulting in a pH-decrease during the transformation of muscle in to meat affecting ligand binding and redox potential of the heme iron in myoglobin, the meat pigment. The "inorganic chemistry" of meat involves (i redox-cycling between iron(II, iron(III, and iron(IV/protein radicals; (ii ligand exchange processes; and (iii spin-equilibra with a change in coordination number for the heme iron. In addition to the function of myoglobin for oxygen storage, new physiological roles of myoglobin are currently being discovered, which notably find close parallels in the processes in fresh meat and nitrite-cured meat products. Myoglobin may be characterized as a bioreactor for small molecules like O2, NO, CO, CO2, H2O, and HNO with importance in bio-regulation and in protection against oxidative stress in vivo otherwise affecting lipids in membranes. Many of these processes may be recognised as colour changes in fresh meat and cured meat products under different atmospheric conditions, and could also be instructive for teaching purposes.

  10. The Nernst equation applied to oxidation-reduction reactions in myoglobin and hemoglobin. Evaluation of the parameters.

    Science.gov (United States)

    Saroff, Harry A

    Analyses of the binding of oxygen to monomers such as myoglobin employ the Mass Action equation. The Mass Action equation, as such, is not directly applicable for the analysis of the binding of oxygen to oligomers such as hemoglobin. When the binding of oxygen to hemoglobin is analyzed, models incorporating extensions of mass action are employed. Oxidation-reduction reactions of the heme group in myoglobin and hemoglobin involve the binding and dissociation of electrons. This reaction is described with the Nernst equation. The Nernst equation is applicable only to a monomeric species even if the number of electrons involved is greater than unity. To analyze the oxidation-reduction reaction in a molecule such as hemoglobin a model is required which incorporates extensions of the Nernst equation. This communication develops models employing the Nernst equation for oxidation-reduction reactions analogous to those employed for hemoglobin in the analysis of the oxygenation (binding of oxygen) reaction.

  11. The myoglobin of Emperor penguin (Aptenodytes forsteri): amino acid sequence and functional adaptation to extreme conditions.

    Science.gov (United States)

    Tamburrini, M; Romano, M; Giardina, B; di Prisco, G

    1999-02-01

    In the framework of a study on molecular adaptations of the oxygen-transport and storage systems to extreme conditions in Antarctic marine organisms, we have investigated the structure/function relationship in Emperor penguin (Aptenodytes forsteri) myoglobin, in search of correlation with the bird life style. In contrast with previous reports, the revised amino acid sequence contains one additional residue and 15 differences. The oxygen-binding parameters seem well adapted to the diving behaviour of the penguin and to the environmental conditions of the Antarctic habitat. Addition of lactate has no major effect on myoglobin oxygenation over a large temperature range. Therefore, metabolic acidosis does not impair myoglobin function under conditions of prolonged physical effort, such as diving.

  12. Artificial membrane-binding proteins stimulate oxygenation of stem cells during engineering of large cartilage tissue

    Science.gov (United States)

    Armstrong, James P. K.; Shakur, Rameen; Horne, Joseph P.; Dickinson, Sally C.; Armstrong, Craig T.; Lau, Katherine; Kadiwala, Juned; Lowe, Robert; Seddon, Annela; Mann, Stephen; Anderson, J. L. Ross; Perriman, Adam W.; Hollander, Anthony P.

    2015-06-01

    Restricted oxygen diffusion can result in central cell necrosis in engineered tissue, a problem that is exacerbated when engineering large tissue constructs for clinical application. Here we show that pre-treating human mesenchymal stem cells (hMSCs) with synthetic membrane-active myoglobin-polymer-surfactant complexes can provide a reservoir of oxygen capable of alleviating necrosis at the centre of hyaline cartilage. This is achieved through the development of a new cell functionalization methodology based on polymer-surfactant conjugation, which allows the delivery of functional proteins to the hMSC membrane. This new approach circumvents the need for cell surface engineering using protein chimerization or genetic transfection, and we demonstrate that the surface-modified hMSCs retain their ability to proliferate and to undergo multilineage differentiation. The functionalization technology is facile, versatile and non-disruptive, and in addition to tissue oxygenation, it should have far-reaching application in a host of tissue engineering and cell-based therapies.

  13. Myoglobin structure and function: A multiweek biochemistry laboratory project.

    Science.gov (United States)

    Silverstein, Todd P; Kirk, Sarah R; Meyer, Scott C; Holman, Karen L McFarlane

    2015-01-01

    We have developed a multiweek laboratory project in which students isolate myoglobin and characterize its structure, function, and redox state. The important laboratory techniques covered in this project include size-exclusion chromatography, electrophoresis, spectrophotometric titration, and FTIR spectroscopy. Regarding protein structure, students work with computer modeling and visualization of myoglobin and its homologues, after which they spectroscopically characterize its thermal denaturation. Students also study protein function (ligand binding equilibrium) and are instructed on topics in data analysis (calibration curves, nonlinear vs. linear regression). This upper division biochemistry laboratory project is a challenging and rewarding one that not only exposes students to a wide variety of important biochemical laboratory techniques but also ties those techniques together to work with a single readily available and easily characterized protein, myoglobin. © 2015 International Union of Biochemistry and Molecular Biology.

  14. Oxygen binding properties of non-mammalian nerve globins

    DEFF Research Database (Denmark)

    Hundahl, Christian; Fago, Angela; Dewilde, Sylvia

    2006-01-01

    Oxygen-binding globins occur in the nervous systems of both invertebrates and vertebrates. While the function of invertebrate nerve haemoglobins as oxygen stores that extend neural excitability under hypoxia has been convincingly demonstrated, the physiological role of vertebrate neuroglobins...... is less well understood. Here we provide a detailed analysis of the oxygenation characteristics of nerve haemoglobins from an annelid (Aphrodite aculeata), a nemertean (Cerebratulus lacteus) and a bivalve (Spisula solidissima) and of neuroglobin from zebrafish (Danio rerio). The functional differences...... have been related to haem coordination: the haem is pentacoordinate (as in human haemoglobin and myoglobin) in A. aculeata and C. lacteus nerve haemoglobins and hexacoordinate in S. solidissima nerve haemoglobin and D. rerio neuroglobin. Whereas pentacoordinate nerve globins lacked Bohr effects at all...

  15. Dynamic factors affecting gaseous ligand binding in an artificial oxygen transport protein.

    Science.gov (United States)

    Zhang, Lei; Andersen, Eskil M E; Khajo, Abdelahad; Magliozzo, Richard S; Koder, Ronald L

    2013-01-22

    We report the functional analysis of an artificial hexacoordinate oxygen transport protein, HP7, which operates via a mechanism similar to that of human neuroglobin and cytoglobin: the destabilization of one of two heme-ligating histidine residues. In the case of HP7, this is the result of the coupling of histidine side chain ligation with the burial of three charged glutamate residues on the same helix. Here we compare gaseous ligand binding, including rates, affinities, and oxyferrous state lifetimes, of both heme binding sites in HP7. We find that despite the identical sequence of helices in both binding sites, there are differences in oxygen affinity and oxyferrous state lifetime that may be the result of differences in the freedom of motion imposed by the candelabra fold on the two sites of the protein. We further examine the effect of mutational removal of the buried glutamates on function. Heme iron in the ferrous state of this mutant is rapidly oxidized when exposed to oxygen. Compared to that of HP7, the distal histidine affinity is increased by a 22-fold decrease in the histidine ligand off rate. Electron paramagnetic resonance comparison of these ferric hemoproteins demonstrates that the mutation increases the level of disorder at the heme binding site. Nuclear magnetic resonance-detected deuterium exchange demonstrates that the mutation greatly increases the degree of penetration of water into the protein core. The inability of the mutant protein to bind oxygen may be due to an increased level of water penetration, the large decrease in binding rate caused by the increase in distal histidine affinity, or a combination of the two factors. Together, these data underline the importance of the control of protein dynamics in the design of functional artificial proteins.

  16. Hot atom labeling of myoglobin and hemoglobin and biophysical studies of oxygen and CO binding to carp hemoglobin

    International Nuclear Information System (INIS)

    Astatke, M.

    1992-01-01

    Human Hb, the monomeric Hb of Glycera dibranchiata and horse Mb were modified by replacement of the protoheme with 2,4-dibromodeuteroheme. Following neutron capture by 79 Br and 81 Br, the locations of radioactive Br were determined. Although human Hb had approximately four times the mass and volume of the other proteins, about 9% of the activated Br was inserted into each of the three globins. These results suggest that the insertion is short-range (within 15 angstrom) and that this method could be used to label target sites in various proteins and other biological structures. Carp Hb's containing proto-, meso-, deutero- and dibromoheme were prepared. Kinetic and thermodynamic parameters for oxygen and CO binding were determined at Ph 6 (+IHP) (T-state, low-affinity protein) and Ph 9 (R-state, high-affinity protein). Parameters for the binding of oxygen and CO were related to the properties of the four hemes to estimate the inductive and steric factors in the ligation process. The results suggest that the steric factors are more important for the T-state than for the R-state. The T-state carp Hbs were very readily oxidized. Two new procedures were developed for the rapid determination of oxygen equilibrium isotherms for the T-state carp Hbs. The kinetic and thermodynamic parameters for ligation of oxygen and CO with the isolated carp α-chains were determined. Carp α-chains are the only hemoglobin chains isolated to date that can be classified as T-state. The secondary thermodynamic parameter (δH degrees) was found to be essential for classifying hemoglobins as T- or R-state

  17. Effect of fatty acid interaction on myoglobin oxygen affinity and triglyceride metabolism.

    Science.gov (United States)

    Jue, Thomas; Simond, Gregory; Wright, Traver J; Shih, Lifan; Chung, Youngran; Sriram, Renuka; Kreutzer, Ulrike; Davis, Randall W

    2016-08-01

    Recent studies have suggested myoglobin (Mb) may have other cellular functions in addition to storing and transporting O 2 . Indeed, NMR experiments have shown that the saturated fatty acid (FA) palmitate (PA) can interact with myoglobin (Mb) in its ligated state (MbCO and MbCN) but does not interact with Mb in its deoxygenated state. The observation has led to the hypothesis that Mb can also serve as a fatty acid transporter. The present study further investigates fatty acid interaction with the physiological states of Mb using the more soluble but unsaturated fatty acid, oleic acid (OA). OA binds to MbCO but does not bind to deoxy Mb. OA binding to Mb, however, does not alter its O 2 affinity. Without any Mb, muscle has a significantly lower level of triglyceride (TG). In Mb knock-out (MbKO) mice, both heart and skeletal muscles have lower level of TG relative to the control mice. Training further decreases the relative TG in the MbKO skeletal muscle. Nevertheless, the absence of Mb and lower TG level in muscle does not impair the MbKO mouse performance as evidenced by voluntary wheel running measurements. The results support the hypothesis of a complex physiological role for Mb, especially with respect to fatty acid metabolism.

  18. Molecular Evolution of the Oxygen-Binding Hemerythrin Domain.

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    Claudia Alvarez-Carreño

    Full Text Available The evolution of oxygenic photosynthesis during Precambrian times entailed the diversification of strategies minimizing reactive oxygen species-associated damage. Four families of oxygen-carrier proteins (hemoglobin, hemerythrin and the two non-homologous families of arthropodan and molluscan hemocyanins are known to have evolved independently the capacity to bind oxygen reversibly, providing cells with strategies to cope with the evolutionary pressure of oxygen accumulation. Oxygen-binding hemerythrin was first studied in marine invertebrates but further research has made it clear that it is present in the three domains of life, strongly suggesting that its origin predated the emergence of eukaryotes.Oxygen-binding hemerythrins are a monophyletic sub-group of the hemerythrin/HHE (histidine, histidine, glutamic acid cation-binding domain. Oxygen-binding hemerythrin homologs were unambiguously identified in 367/2236 bacterial, 21/150 archaeal and 4/135 eukaryotic genomes. Overall, oxygen-binding hemerythrin homologues were found in the same proportion as single-domain and as long protein sequences. The associated functions of protein domains in long hemerythrin sequences can be classified in three major groups: signal transduction, phosphorelay response regulation, and protein binding. This suggests that in many organisms the reversible oxygen-binding capacity was incorporated in signaling pathways. A maximum-likelihood tree of oxygen-binding hemerythrin homologues revealed a complex evolutionary history in which lateral gene transfer, duplications and gene losses appear to have played an important role.Hemerythrin is an ancient protein domain with a complex evolutionary history. The distinctive iron-binding coordination site of oxygen-binding hemerythrins evolved first in prokaryotes, very likely prior to the divergence of Firmicutes and Proteobacteria, and spread into many bacterial, archaeal and eukaryotic species. The later evolution of the

  19. Aggregation and conformational stability evaluation of myoglobin in the presence of ionic surfactant

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    Mohammad A. Alsenaidy

    2018-05-01

    Full Text Available Sodium lauroyl sarcosinate (SLS is frequently used for the solubilization of inclusion bodies in vitro due to its structural similarity to lipid plasma membrane. There are many factors that could influence protein aggregation propensity, including overall protein surface charge and hydrophobicity. Here, the aggregation pathway of myoglobin protein was studied under different conditions (pH 3.5 and 7.4 in the presence of varying concentrations of SLS to evaluate the underlying forces dictating protein aggregation. Data obtained from Rayleigh light scattering, ThT binding assay, and far-UV CD indicated that SLS have different effects on the protein depending on its concentration and environmental conditions. In the presence of low concentrations of SLS (0.05–0.1 mM, no aggregation was detected at both pH conditions tested. Whereas, as we reach higher SLS concentrations (0.5–10.0 mM, myoglobin started forming larger-sized aggregates at pH 3.5 and not pH 7.4. These results suggest that electrostatics interactions as well as hydrophobic forces play an important role in SLS-induced myoglobin aggregation. Keywords: Sodium lauroyl sarcosinate, Surfactant, Myoglobin, Protein aggregation, Amorphous aggregates, pH

  20. Structure-function paradigm in human myoglobin: how a single-residue substitution affects NO reactivity at low pO2.

    Science.gov (United States)

    Scorciapino, Mariano Andrea; Spiga, Enrico; Vezzoli, Alessandra; Mrakic-Sposta, Simona; Russo, Rosaria; Fink, Bruno; Casu, Mariano; Gussoni, Maristella; Ceccarelli, Matteo

    2013-05-22

    This work is focused on the two more expressed human myoglobin isoforms. In the literature, their different overexpression in high-altitude natives was proposed to be related to alternative/complementary functions in hypoxia. Interestingly, they differ only at residue-54, lysine or glutamate, which is external and far from the main binding site. In order to ascertain whether these two almost identical myoglobins might exert different functions and to contribute to a deeper understanding about myoglobin's oxygen-level dependent functioning, they have been compared with respect to dynamics, heme electronic structure, and NO reactivity at different O2 levels. Electron paramagnetic resonance (EPR) spectroscopy was employed to investigate the electronic structure of the nitrosyl-form, obtaining fundamental clues about a different bond interaction between the heme-iron and the proximal histidine and highlighting striking differences in NO reactivity, especially at a very low pO2. The experimental results well matched with the information provided by molecular dynamics simulations, which showed a significantly different dynamics for the two proteins only in the absence of O2. The single mutation differentiating the two myoglobins resulted in strongly affecting the plasticity of the CD-region (C-helix-loop-D-helix), whose fluctuations, being coupled to the solvent, were found to be correlated with the dynamics of the distal binding site. In the absence of O2, on the one hand a significantly different probability for the histidine-gate opening has been shown by MD simulations, and on the other a different yield of myoglobin-NO formation was experimentally observed through EPR.

  1. Ligand and proton exchange dynamics in recombinant human myoglobin mutants.

    Science.gov (United States)

    Lambright, D G; Balasubramanian, S; Boxer, S G

    1989-05-05

    Site-specific mutants of human myoglobin have been prepared in which lysine 45 is replaced by arginine (K45R) and aspartate 60 by glutamate (D60E), in order to examine the influence of these residues and their interaction on the dynamics of the protein. These proteins were studied by a variety of methods, including one and two-dimensional proton nuclear magnetic resonance spectroscopy, exchange kinetics for the distal and proximal histidine NH protons as a function of pH in the met cyano forms, flash photolysis of the CO forms, and ligand replacement kinetics. The electronic absorption and proton nuclear magnetic resonance spectra of the CO forms of these proteins are virtually identical, indicating that the structure of the heme pocket is unaltered by these mutations. There are, however, substantial changes in the dynamics of both CO binding and proton exchange for the mutant K45R, whereas the mutant D60E exhibits behavior indistinguishable from the reference human myoglobin. K45R has a faster CO bimolecular recombination rate and slower CO off-rate relative to the reference. The kinetics for CO binding are independent of pH (6.5 to 10) as well as ionic strength (0 to 1 M-NaCl). The exchange rate for the distal histidine NH is substantially lower for K45R than the reference, whereas the proximal histidine NH exchange rate is unaltered. The exchange behavior of the human proteins is similar to that reported for a comparison of the exchange rates for myoglobins having lysine at position 45 with sperm whale myoglobin, which has arginine at this position. This indicates that the differences in exchange rates reflects largely the Lys----Arg substitution. The lack of a simple correlation for the CO kinetics with this substitution means that these are sensitive to other factors as well. Specific kinetic models, whereby substitution of arginine for lysine at position 45 can affect ligand binding dynamics, are outlined. These experiments demonstrate that a relatively

  2. Factors correlating with significant differences between X-ray structures of myoglobin

    International Nuclear Information System (INIS)

    Rashin, Alexander A.; Domagalski, Marcin J.; Zimmermann, Michael T.; Minor, Wladek; Chruszcz, Maksymilian; Jernigan, Robert L.

    2014-01-01

    Conformational differences between myoglobin structures are studied. Most structural differences in whale myoglobin beyond the uncertainty threshold can be correlated with a few specific structural factors. There are always exceptions and a search for additional factors is needed. The results might have serious implications for biological insights from conformational differences. Validation of general ideas about the origins of conformational differences in proteins is critical in order to arrive at meaningful functional insights. Here, principal component analysis (PCA) and distance difference matrices are used to validate some such ideas about the conformational differences between 291 myoglobin structures from sperm whale, horse and pig. Almost all of the horse and pig structures form compact PCA clusters with only minor coordinate differences and outliers that are easily explained. The 222 whale structures form a few dense clusters with multiple outliers. A few whale outliers with a prominent distortion of the GH loop are very similar to the cluster of horse structures, which all have a similar GH-loop distortion apparently owing to intermolecular crystal lattice hydrogen bonds to the GH loop from residues near the distal histidine His64. The variations of the GH-loop coordinates in the whale structures are likely to be owing to the observed alternative intermolecular crystal lattice bond, with the change to the GH loop distorting bonds correlated with the binding of specific ‘unusual’ ligands. Such an alternative intermolecular bond is not observed in horse myoglobins, obliterating any correlation with the ligands. Intermolecular bonds do not usually cause significant coordinate differences and cannot be validated as their universal cause. Most of the native-like whale myoglobin structure outliers can be correlated with a few specific factors. However, these factors do not always lead to coordinate differences beyond the previously determined uncertainty

  3. Factors correlating with significant differences between X-ray structures of myoglobin

    Energy Technology Data Exchange (ETDEWEB)

    Rashin, Alexander A., E-mail: alexander-rashin@hotmail.com [BioChemComp Inc., 543 Sagamore Avenue, Teaneck, NJ 07666 (United States); Iowa State University, 112 Office and Lab Bldg, Ames, IA 50011-3020 (United States); Domagalski, Marcin J. [University of Virginia, 1340 Jefferson Park Avenue, Jordan Hall, Room 4223, Charlottesville, VA 22908 (United States); Zimmermann, Michael T. [Iowa State University, 112 Office and Lab Bldg, Ames, IA 50011-3020 (United States); Minor, Wladek [University of Virginia, 1340 Jefferson Park Avenue, Jordan Hall, Room 4223, Charlottesville, VA 22908 (United States); Chruszcz, Maksymilian [University of Virginia, 1340 Jefferson Park Avenue, Jordan Hall, Room 4223, Charlottesville, VA 22908 (United States); University of South Carolina, 631 Sumter Street, Columbia, SC 29208 (United States); Jernigan, Robert L. [Iowa State University, 112 Office and Lab Bldg, Ames, IA 50011-3020 (United States); BioChemComp Inc., 543 Sagamore Avenue, Teaneck, NJ 07666 (United States)

    2014-02-01

    Conformational differences between myoglobin structures are studied. Most structural differences in whale myoglobin beyond the uncertainty threshold can be correlated with a few specific structural factors. There are always exceptions and a search for additional factors is needed. The results might have serious implications for biological insights from conformational differences. Validation of general ideas about the origins of conformational differences in proteins is critical in order to arrive at meaningful functional insights. Here, principal component analysis (PCA) and distance difference matrices are used to validate some such ideas about the conformational differences between 291 myoglobin structures from sperm whale, horse and pig. Almost all of the horse and pig structures form compact PCA clusters with only minor coordinate differences and outliers that are easily explained. The 222 whale structures form a few dense clusters with multiple outliers. A few whale outliers with a prominent distortion of the GH loop are very similar to the cluster of horse structures, which all have a similar GH-loop distortion apparently owing to intermolecular crystal lattice hydrogen bonds to the GH loop from residues near the distal histidine His64. The variations of the GH-loop coordinates in the whale structures are likely to be owing to the observed alternative intermolecular crystal lattice bond, with the change to the GH loop distorting bonds correlated with the binding of specific ‘unusual’ ligands. Such an alternative intermolecular bond is not observed in horse myoglobins, obliterating any correlation with the ligands. Intermolecular bonds do not usually cause significant coordinate differences and cannot be validated as their universal cause. Most of the native-like whale myoglobin structure outliers can be correlated with a few specific factors. However, these factors do not always lead to coordinate differences beyond the previously determined uncertainty

  4. How different oxidation states of crystalline myoglobin are influenced by X-rays.

    Science.gov (United States)

    Hersleth, Hans-Petter; Andersson, K Kristoffer

    2011-06-01

    X-ray induced radiation damage of protein crystals is well known to occur even at cryogenic temperatures. Redox active sites like metal sites seem especially vulnerable for these radiation-induced reductions. It is essential to know correctly the oxidation state of metal sites in protein crystal structures to be able to interpret the structure-function relation. Through previous structural studies, we have tried to characterise and understand the reactions between myoglobin and peroxides. These reaction intermediates are relevant because myoglobin is proposed to take part as scavenger of reactive oxygen species during oxidative stress, and because these intermediates are similar among the haem peroxidases and oxygenases. We have in our previous studies shown that these different myoglobin states are influenced by the X-rays used. In this study, we have in detail investigated the impact that X-rays have on these different oxidation states of myoglobin. An underlying goal has been to find a way to be able to determine mostly unreduced states. We have by using single-crystal light absorption spectroscopy found that the different oxidation states of myoglobin are to a different extent influenced by the X-rays (e.g. ferric Fe(III) myoglobin is faster reduced than ferryl Fe(IV)═O myoglobin). We observe that the higher oxidation states are not reduced to normal ferrous Fe(II) or ferric Fe(III) states, but end up in some intermediate and possibly artificial states. For ferric myoglobin, it seems that annealing of the radiation-induced/reduced state can reversibly more or less give the starting point (ferric myoglobin). Both scavengers and different dose-rates might influence to which extent the different states are affected by the X-rays. Our study shows that it is essential to do a time/dose monitoring of the influence X-rays have on each specific redox-state with spectroscopic techniques like single-crystal light absorption spectroscopy. This will determine to which

  5. Fusion protein-based biofilm fabrication composed of recombinant azurin–myoglobin for dual-level biomemory application

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Taek [Research Institute for Basic Science, Sogang University, Heukseok-dong, Dongjak-gu, Seoul 156-756 (Korea, Republic of); Chung, Yong-Ho; Yoon, Jinho [Department of Chemical and Biomolecular Engineering, Sogang University, Heukseok-dong, Dongjak-gu, 35 Baekbeom-ro (Sinsu-dong), Mapo-gu, Seoul 121-742 (Korea, Republic of); Min, Junhong [School of Integrative Engineering, Chung-Ang University, Heukseok-dong, Dongjak-gu, Seoul 156-756 (Korea, Republic of); Choi, Jeong-Woo, E-mail: jwchoi@sogang.ac.kr [Department of Chemical and Biomolecular Engineering, Sogang University, Heukseok-dong, Dongjak-gu, 35 Baekbeom-ro (Sinsu-dong), Mapo-gu, Seoul 121-742 (Korea, Republic of)

    2014-11-30

    Graphical abstract: - Highlights: • We developed the fusion protein-based biofilm on the inorganic surface. • For making the fusion protein, the recombinant azurin and the myoglobin was conjugated by the native chemical ligation method. • The developed fusion protein shows unique electrochemical property. • The proposed fusion protein biofilm appears to be a good method for dual-level biomemory device. - Abstract: In the present study, a fusion protein-based biofilm composed of a recombinant azurin–myoglobin (Azu-Myo) has been developed and confirmed its original electrochemical property for dual-level biomemory device application. For this purpose, the azurin was modified with cysteine residues for direct immobilization and conjugation. Then, the recombinant azurin was conjugated with the myoglobin via a sulfo-SMCC bifunctional linker using the chemical ligation method (CLM). The SDS-PAGE and UV–vis spectroscopy were performed to examine the fusion protein conjugates. The prepared Azu-Myo fusion protein was self-assembled onto Au substrate for the biofilm fabrication. Then, the atomic force microscopy (AFM) was used to confirm the immobilization and the surface-enhanced Raman spectroscopy (SERS) was carried out to the surface analysis. Also, the cyclic voltammetry (CV) was carried out to observe an electrochemical property of fabricated biofilm. As a result, the two pair of redox potential values was obtained for dual-level biomemory device application. Then, the dual-level biomemory function was verified by the multi-potential chronoamperometry (MPCA). The results indicate a new fabrication method and material combination for advances in bioelectronic device development.

  6. Fusion protein-based biofilm fabrication composed of recombinant azurin–myoglobin for dual-level biomemory application

    International Nuclear Information System (INIS)

    Lee, Taek; Chung, Yong-Ho; Yoon, Jinho; Min, Junhong; Choi, Jeong-Woo

    2014-01-01

    Graphical abstract: - Highlights: • We developed the fusion protein-based biofilm on the inorganic surface. • For making the fusion protein, the recombinant azurin and the myoglobin was conjugated by the native chemical ligation method. • The developed fusion protein shows unique electrochemical property. • The proposed fusion protein biofilm appears to be a good method for dual-level biomemory device. - Abstract: In the present study, a fusion protein-based biofilm composed of a recombinant azurin–myoglobin (Azu-Myo) has been developed and confirmed its original electrochemical property for dual-level biomemory device application. For this purpose, the azurin was modified with cysteine residues for direct immobilization and conjugation. Then, the recombinant azurin was conjugated with the myoglobin via a sulfo-SMCC bifunctional linker using the chemical ligation method (CLM). The SDS-PAGE and UV–vis spectroscopy were performed to examine the fusion protein conjugates. The prepared Azu-Myo fusion protein was self-assembled onto Au substrate for the biofilm fabrication. Then, the atomic force microscopy (AFM) was used to confirm the immobilization and the surface-enhanced Raman spectroscopy (SERS) was carried out to the surface analysis. Also, the cyclic voltammetry (CV) was carried out to observe an electrochemical property of fabricated biofilm. As a result, the two pair of redox potential values was obtained for dual-level biomemory device application. Then, the dual-level biomemory function was verified by the multi-potential chronoamperometry (MPCA). The results indicate a new fabrication method and material combination for advances in bioelectronic device development

  7. Neonatal hyper- and hypothyroidism alter the myoglobin gene expression program in adulthood

    Energy Technology Data Exchange (ETDEWEB)

    Picoli Souza, K. de [Faculdade de Ciências Biológicas e Ambientais, Universidade Federal da Grande Dourados, Dourados, MS (Brazil); Nunes, M.T. [Departamento de Fisiologia e Biofísica, Instituto de Ciências Biológicas, Universidade de São Paulo, São Paulo, SP (Brazil)

    2014-06-24

    Myoglobin acts as an oxygen store and a reactive oxygen species acceptor in muscles. We examined myoglobin mRNA in rat cardiac ventricle and skeletal muscles during the first 42 days of life and the impact of transient neonatal hypo- and hyperthyroidism on the myoglobin gene expression pattern. Cardiac ventricle and skeletal muscles of Wistar rats at 7-42 days of life were quickly removed, and myoglobin mRNA was determined by Northern blot analysis. Rats were treated with propylthiouracil (5-10 mg/100 g) and triiodothyronine (0.5-50 µg/100 g) for 5, 15, or 30 days after birth to induce hypo- and hyperthyroidism and euthanized either just after treatment or at 90 days. During postnatal (P) days 7-28, the ventricle myoglobin mRNA remained unchanged, but it gradually increased in skeletal muscle (12-fold). Triiodothyronine treatment, from days P0-P5, increased the skeletal muscle myoglobin mRNA 1.5- to 4.5-fold; a 2.5-fold increase was observed in ventricle muscle, but only when triiodothyronine treatment was extended to day P15. Conversely, hypothyroidism at P5 markedly decreased (60%) ventricular myoglobin mRNA. Moreover, transient hyperthyroidism in the neonatal period increased ventricle myoglobin mRNA (2-fold), and decreased heart rate (5%), fast muscle myoglobin mRNA (30%) and body weight (20%) in adulthood. Transient hypothyroidism in the neonatal period also permanently decreased fast muscle myoglobin mRNA (30%) and body weight (14%). These results indicated that changes in triiodothyronine supply in the neonatal period alter the myoglobin expression program in ventricle and skeletal muscle, leading to specific physiological repercussions and alterations in other parameters in adulthood.

  8. Neonatal hyper- and hypothyroidism alter the myoglobin gene expression program in adulthood

    International Nuclear Information System (INIS)

    Picoli Souza, K. de; Nunes, M.T.

    2014-01-01

    Myoglobin acts as an oxygen store and a reactive oxygen species acceptor in muscles. We examined myoglobin mRNA in rat cardiac ventricle and skeletal muscles during the first 42 days of life and the impact of transient neonatal hypo- and hyperthyroidism on the myoglobin gene expression pattern. Cardiac ventricle and skeletal muscles of Wistar rats at 7-42 days of life were quickly removed, and myoglobin mRNA was determined by Northern blot analysis. Rats were treated with propylthiouracil (5-10 mg/100 g) and triiodothyronine (0.5-50 µg/100 g) for 5, 15, or 30 days after birth to induce hypo- and hyperthyroidism and euthanized either just after treatment or at 90 days. During postnatal (P) days 7-28, the ventricle myoglobin mRNA remained unchanged, but it gradually increased in skeletal muscle (12-fold). Triiodothyronine treatment, from days P0-P5, increased the skeletal muscle myoglobin mRNA 1.5- to 4.5-fold; a 2.5-fold increase was observed in ventricle muscle, but only when triiodothyronine treatment was extended to day P15. Conversely, hypothyroidism at P5 markedly decreased (60%) ventricular myoglobin mRNA. Moreover, transient hyperthyroidism in the neonatal period increased ventricle myoglobin mRNA (2-fold), and decreased heart rate (5%), fast muscle myoglobin mRNA (30%) and body weight (20%) in adulthood. Transient hypothyroidism in the neonatal period also permanently decreased fast muscle myoglobin mRNA (30%) and body weight (14%). These results indicated that changes in triiodothyronine supply in the neonatal period alter the myoglobin expression program in ventricle and skeletal muscle, leading to specific physiological repercussions and alterations in other parameters in adulthood

  9. Neonatal hyper- and hypothyroidism alter the myoglobin gene expression program in adulthood.

    Science.gov (United States)

    Souza, K de Picoli; Nunes, M T

    2014-08-01

    Myoglobin acts as an oxygen store and a reactive oxygen species acceptor in muscles. We examined myoglobin mRNA in rat cardiac ventricle and skeletal muscles during the first 42 days of life and the impact of transient neonatal hypo- and hyperthyroidism on the myoglobin gene expression pattern. Cardiac ventricle and skeletal muscles of Wistar rats at 7-42 days of life were quickly removed, and myoglobin mRNA was determined by Northern blot analysis. Rats were treated with propylthiouracil (5-10 mg/100 g) and triiodothyronine (0.5-50 µg/100 g) for 5, 15, or 30 days after birth to induce hypo- and hyperthyroidism and euthanized either just after treatment or at 90 days. During postnatal (P) days 7-28, the ventricle myoglobin mRNA remained unchanged, but it gradually increased in skeletal muscle (12-fold). Triiodothyronine treatment, from days P0-P5, increased the skeletal muscle myoglobin mRNA 1.5- to 4.5-fold; a 2.5-fold increase was observed in ventricle muscle, but only when triiodothyronine treatment was extended to day P15. Conversely, hypothyroidism at P5 markedly decreased (60%) ventricular myoglobin mRNA. Moreover, transient hyperthyroidism in the neonatal period increased ventricle myoglobin mRNA (2-fold), and decreased heart rate (5%), fast muscle myoglobin mRNA (30%) and body weight (20%) in adulthood. Transient hypothyroidism in the neonatal period also permanently decreased fast muscle myoglobin mRNA (30%) and body weight (14%). These results indicated that changes in triiodothyronine supply in the neonatal period alter the myoglobin expression program in ventricle and skeletal muscle, leading to specific physiological repercussions and alterations in other parameters in adulthood.

  10. Neonatal hyper- and hypothyroidism alter the myoglobin gene expression program in adulthood

    Directory of Open Access Journals (Sweden)

    K. de Picoli Souza

    2014-08-01

    Full Text Available Myoglobin acts as an oxygen store and a reactive oxygen species acceptor in muscles. We examined myoglobin mRNA in rat cardiac ventricle and skeletal muscles during the first 42 days of life and the impact of transient neonatal hypo- and hyperthyroidism on the myoglobin gene expression pattern. Cardiac ventricle and skeletal muscles of Wistar rats at 7-42 days of life were quickly removed, and myoglobin mRNA was determined by Northern blot analysis. Rats were treated with propylthiouracil (5-10 mg/100 g and triiodothyronine (0.5-50 µg/100 g for 5, 15, or 30 days after birth to induce hypo- and hyperthyroidism and euthanized either just after treatment or at 90 days. During postnatal (P days 7-28, the ventricle myoglobin mRNA remained unchanged, but it gradually increased in skeletal muscle (12-fold. Triiodothyronine treatment, from days P0-P5, increased the skeletal muscle myoglobin mRNA 1.5- to 4.5-fold; a 2.5-fold increase was observed in ventricle muscle, but only when triiodothyronine treatment was extended to day P15. Conversely, hypothyroidism at P5 markedly decreased (60% ventricular myoglobin mRNA. Moreover, transient hyperthyroidism in the neonatal period increased ventricle myoglobin mRNA (2-fold, and decreased heart rate (5%, fast muscle myoglobin mRNA (30% and body weight (20% in adulthood. Transient hypothyroidism in the neonatal period also permanently decreased fast muscle myoglobin mRNA (30% and body weight (14%. These results indicated that changes in triiodothyronine supply in the neonatal period alter the myoglobin expression program in ventricle and skeletal muscle, leading to specific physiological repercussions and alterations in other parameters in adulthood.

  11. Myoglobin urine test

    Science.gov (United States)

    Urine myoglobin; Heart attack - myoglobin urine test; Myositis - myoglobin urine test; Rhabdomyolysis - myoglobin urine test ... The test involves only normal urination, which should cause no discomfort.

  12. Diagnostic strategies using myoglobin measurement in myocardial infarction.

    Science.gov (United States)

    Plebani, M; Zaninotto, M

    1998-04-06

    Myoglobin, a low molecular-weight heme protein (17800 D) present in both cardiac and skeletal muscle, is an old test with new perspectives. Advantages and disadvantages of myoglobin determination are well known. Myoglobin is the earliest known, commercially available, biochemical marker of acute myocardial infarction (AMI) and its rapid kinetics make it an early, good marker of reperfusion. However, since myoglobin is present in both skeletal and cardiac muscle, any damage to these muscle types results in its release into blood. Serum myoglobin levels are falsely elevated in conditions unrelated to AMI as skeletal muscle and neuromuscular disorders, renal failure, intramuscular injection, strenuous exercise, and after several toxins and drugs intake. New strategies for myoglobin measurement may resolve this limitation. These strategies include both the combined measurement of myoglobin and a skeletal specific marker (carbonic anhydrase III) or a cardiac specific marker (troponin I), as well as the myoglobin evaluation on serial samples. In particular, the diagnostic algorithm based on the combined measurement of myoglobin and troponin I, assuring a satisfactory analytical turnaround time, significantly improves the diagnostic efficiency of laboratory assessment of suspected AMI patients, allowing the successive monitoring of coronary reperfusion.

  13. Myoglobin chemistry and meat color.

    Science.gov (United States)

    Suman, Surendranath P; Joseph, Poulson

    2013-01-01

    Consumers rely heavily on fresh meat color as an indicator of wholesomeness at the point of sale, whereas cooked color is exploited as an indicator of doneness at the point of consumption. Deviations from the bright cherry-red color of fresh meat lead to product rejection and revenue loss. Myoglobin is the sarcoplasmic heme protein primarily responsible for the meat color, and the chemistry of myoglobin is species specific. The mechanistic interactions between myoglobin and multiple extrinsic and intrinsic factors govern the color of raw as well as cooked meats. The objective of this review is to provide an overview of the current research in meat color and how the findings are applied in the meat industry. Characterizing the fundamental basis of myoglobin's interactions with biomolecules in postmortem skeletal muscles is necessary to interpret the chemistry of meat color phenomena and to engineer innovative processing strategies to minimize meat discoloration-induced revenue loss to the agricultural economy.

  14. Myocyte specific overexpression of myoglobin impairs angiogenesis after hind-limb ischemia.

    Science.gov (United States)

    Hazarika, Surovi; Angelo, Michael; Li, Yongjun; Aldrich, Amy J; Odronic, Shelley I; Yan, Zhen; Stamler, Jonathan S; Annex, Brian H

    2008-12-01

    In preclinical models of peripheral arterial disease the angiogenic response is typically robust, though it can be impaired in conditions such as hypercholesterolemia and diabetes where the endothelium is dysfunctional. Myoglobin (Mb) is expressed exclusively in striated muscle cells. We hypothesized that myocyte specific overexpression of myoglobin attenuates ischemia-induced angiogenesis even in the presence of normal endothelium. Mb overexpressing transgenic (MbTg, n=59) and wild-type (WT, n=56) C57Bl/6 mice underwent unilateral femoral artery ligation/excision. Perfusion recovery was monitored using Laser Doppler. Ischemia-induced changes in muscle were assessed by protein and immunohistochemistry assays. Nitrite/nitrate and protein-bound NO, and vasoreactivity was measured. Vasoreactivity was similar between MbTg and WT. In ischemic muscle, at d14 postligation, MbTg increased VEGF-A, and activated eNOS the same as WT mice but nitrate/nitrite were reduced whereas protein-bound NO was higher. MbTg had attenuated perfusion recovery at d21 (0.37+/-0.03 versus 0.47+/-0.02, P<0.05), d28 (0.40+/-0.03 versus 0.50+/-0.04, P<0.05), greater limb necrosis (65.2% versus 15%, P<0.001), a lower capillary density, and greater apoptosis versus WT. Increased Mb expression in myocytes attenuates angiogenesis after hind-limb ischemia by binding NO and reducing its bioavailability. Myoglobin can modulate the angiogenic response to ischemia even in the setting of normal endothelium.

  15. Myoglobin blood test

    Science.gov (United States)

    Serum myoglobin; Heart attack - myoglobin blood test; Myositis - myoglobin blood test; Rhabdomyolysis - myoglobin blood test ... too high, it can damage the kidneys. This test is ordered when your health care provider suspects ...

  16. The role of myoglobin degradation in nephrotoxicity after rhabdomyolysis.

    Science.gov (United States)

    Zorova, Ljubava D; Pevzner, Irina B; Chupyrkina, Anastasia A; Zorov, Savva D; Silachev, Denis N; Plotnikov, Egor Y; Zorov, Dmitry B

    2016-08-25

    The fate of myoglobin in renal cells was explored in an animal model of rhabdomyolysis known as the pathology highly related to oxidative stress resulting in impairment of renal functioning. The working hypothesis was that the proper degradation of myoglobin in rhabdomyolytic kidney can activate the reparative processes in the tissue. We found that incubation of myoglobin with kidney cells causes its accumulation in the cytoplasm. In rhabdomyolytic rats, the level of heme and free iron in cytoplasm and mitochondria of kidney cells is remarkably increased while inhibition of proteolysis results in further elevation of myoglobin content in the renal tissue. Heme oxygenase and ferritin levels were found to be increased in the kidney tissue at rhabdomyolysis and simulating conditions performed by i/v injection of myoglobin. In addition, the level of peroxidized lipids was high in rhabdomyolytic kidney and became even higher after inhibition of proteolysis by aprotinin. Elevated levels of carbonylated proteins were also observed after rhabdomyolysis, however, if prior to induction of rhabdomyolysis the injection of myoglobin was done, the level of carbonylated proteins dropped versus unprimed kidney tissue thus affording protection to the kidney against oxidative stress. Injection of myoglobin to the rat results in impairment of renal functioning and inhibition of myoglobin degradation in the rhabdomyolytic animal aggravates acute renal failure, demonstrating that degradation of myoglobin is somehow beneficial although it may result in undesired release of free iron which can participate in toxic redox cycling. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  17. Immobilization of myoglobin in sodium alginate composite membranes

    Directory of Open Access Journals (Sweden)

    Katia Cecília de Souza Figueiredo

    2015-06-01

    Full Text Available AbstractThe immobilization of myoglobin in sodium alginate films was investigated with the aim of evaluating the protein stability in an ionic polymeric matrix. Myoglobin was chosen due to the resemblance to each hemoglobin tetramer. Sodium alginate, being a natural polysaccharide, was selected as the polymeric matrix because of its chemical structure and film-forming ability. To improve the mechanical resistance of sodium alginate films, the polymer was deposited over the surface of a cellulose acetate support by means of ultrafiltration. The ionic crosslink of sodium alginate was investigated by calcium ions. Composite membrane characterization comprised water swelling tests, water flux, SEM images and UV-visible spectroscopy. The electrostatic interaction between the protein and the polysaccharide did not damage the UV-visible pattern of native myoglobin. A good affinity between sodium alginate and cellulose acetate was observed. The top layer of the dense composite membrane successfully immobilized Myoglobin, retaining the native UV-visible pattern for two months.

  18. Mode of bindings of zinc oxide nanoparticles to myoglobin and horseradish peroxidase: A spectroscopic investigations

    Science.gov (United States)

    Mandal, Gopa; Bhattacharya, Sudeshna; Ganguly, Tapan

    2011-07-01

    The interactions between two heme proteins myoglobin (HMb) and horseradish peroxidase (HRP) with zinc oxide (ZnO) nanoparticles are investigated by using UV-vis absorption, steady state fluorescence, synchronous fluorescence, time-resolved fluorescence, FT-IR, atomic force microscopy (AFM) and circular dichroism (CD) techniques under physiological condition of pH˜7.4. The presence of mainly static mode in fluorescence quenching mechanism of HMb and HRP by ZnO nanoparticle indicates the possibility of formation of ground state complex. The processes of bindings of ZnO nanoparticles with the two proteins are spontaneous molecular interaction procedures. In both cases hydrogen bonding plays a major role. The circular dichroism (CD) spectra reveal that a helicity of the proteins is reduced by increasing ZnO nanoparticle concentration although the α-helical structures of HMb and HRP retain their identity. On binding to the ZnO nanoparticles the secondary structure of HRP molecules (or HMb molecules) remains unchanged while there is a substantial change in the environment of the tyrosin active site in case of HRP molecules and tryptophan active site in case of HMb molecules. Tapping mode atomic force microscopy (AFM) was applied for the investigation the structure of HRP adsorbed in the environment of nanoparticles on the silicon and on the bare silicon. HRP molecules adsorb and aggregate on the mica with ZnO nanoparticle. The aggregation indicates an attractive interaction among the adsorbed molecules. The molecules are randomly distributed on the bare silicon wafer. The adsorption of HRP in the environment of ZnO nanoparticle changes drastically the domains due to a strong interaction between HRP and ZnO nanoparticles. Similar situation is observed in case of HMb molecules. These findings demonstrate the efficacy of biomedical applications of ZnO nanoparticles as well as in elucidating their mechanisms of action as drugs in both human and plant systems.

  19. Understanding water: Molecular dynamics simulations of solubilized and crystallized myoglobin

    Energy Technology Data Exchange (ETDEWEB)

    Wei Gu; Garcia, A.E.; Schoenborn, B.P. [Los Alamos National Laboratory, NM (United States)

    1994-12-31

    Molecular dynamics simulations were performed on CO myoglobin to evaluate the stability of the bound water molecules as determined in a neutron diffraction analysis. The myoglobin structure derived from the neutron analysis provided the starting coordinate set used in the simulations. The simulations show that only a few water molecules are tightly bound to protein atoms, while most solvent molecules are labile, breaking and reforming hydrogen bonds. Comparison between myoglobin in solution and in a single crystal highlighted some of the packing effects on the solvent structure and shows that water solvent plays an indispensable role in protein dynamics and structural stability. The described observations explain some of the differences in the experimental results of protein hydration as observed in NMR, neutron and X-ray diffraction studies.

  20. Understanding water: Molecular dynamics simulations of solubilized and crystallized myoglobin

    International Nuclear Information System (INIS)

    Wei Gu; Garcia, A.E.; Schoenborn, B.P.

    1994-01-01

    Molecular dynamics simulations were performed on CO myoglobin to evaluate the stability of the bound water molecules as determined in a neutron diffraction analysis. The myoglobin structure derived from the neutron analysis provided the starting coordinate set used in the simulations. The simulations show that only a few water molecules are tightly bound to protein atoms, while most solvent molecules are labile, breaking and reforming hydrogen bonds. Comparison between myoglobin in solution and in a single crystal highlighted some of the packing effects on the solvent structure and shows that water solvent plays an indispensable role in protein dynamics and structural stability. The described observations explain some of the differences in the experimental results of protein hydration as observed in NMR, neutron and X-ray diffraction studies

  1. Dynamic Factors Affecting Gaseous Ligand Binding in an Artificial Oxygen Transport Protein‡

    Science.gov (United States)

    Zhang, Lei; Andersen, Eskil M.E.; Khajo, Abdelahad; Magliozzo, Richard S.; Koder, Ronald L.

    2013-01-01

    We report the functional analysis of an artificial hexacoordinate oxygen transport protein, HP7, which operates via a mechanism similar to that of human neuroglobin and cytoglobin: the destabilization of one of two heme-ligating histidine residues. In the case of HP7 this is the result of the coupling of histidine side chain ligation with the burial of three charged glutamate residues on the same helix. Here we compare gaseous ligand binding, including rates, affinities and oxyferrous state lifetimes, of both heme binding sites in HP7. We find that despite the identical sequence of helices in both binding sites, there are differences in oxygen affinity and oxyferrous state lifetime which may be the result of differences in the freedom of motion imposed by the candelabra fold on the two sites of the protein. We further examine the effect of mutational removal of the buried glutamates on function. Heme iron in the ferrous state of this mutant is rapidly oxidized when when exposed to oxygen. Compared to HP7, distal histidine affinity is increased by a 22-fold decrease in the histidine ligand off-rate. EPR comparison of these ferric hemoproteins demonstrates that the mutation increases disorder at the heme binding site. NMR-detected deuterium exchange demonstrates that the mutation greatly increases water penetration into the protein core. The inability of the mutant protein to bind oxygen may be due to increased water penetration, the large decrease in binding rate caused by the increase in distal histidine affinity, or a combination of the two factors. Together these data underline the importance of the control of protein dynamics in the design of functional artificial proteins. PMID:23249163

  2. Synthetic food additive dye "Tartrazine" triggers amorphous aggregation in cationic myoglobin.

    Science.gov (United States)

    Al-Shabib, Nasser Abdulatif; Khan, Javed Masood; Khan, Mohd Shahnawaz; Ali, Mohd Sajid; Al-Senaidy, Abdulrahman M; Alsenaidy, Mohammad A; Husain, Fohad Mabood; Al-Lohedan, Hamad A

    2017-05-01

    Protein aggregation, a characteristic of several neurodegenerative diseases, displays vast conformational diversity from amorphous to amyloid-like aggregates. In this study, we have explored the interaction of tartrazine with myoglobin protein at two different pHs (7.4 and 2.0). We have utilized various spectroscopic techniques (turbidity, Rayleigh light scattering (RLS), intrinsic fluorescence, Congo Red and far-UV CD) along with microscopy techniques i.e. atomic force microscopy (AFM) and transmission electron microscopy (TEM) to characterize the tartrazine-induced aggregation in myoglobin. The results showed that higher concentrations of tartrazine (2.0-10.0mM) induced amorphous aggregation in myoglobin at pH 2.0 via electrostatic interactions. However, tartrazine was not able to induce aggregation in myoglobin at pH 7.4; because of strong electrostatic repulsion between myoglobin and tartrazine at this pH. The tartrazine-induced amorphous aggregation process is kinetically very fast, and aggregation occurred without the formation of a nucleus. These results proposed that the electrostatic interaction is responsible for tartrazine-induced amorphous aggregation. This study may help in the understanding of mechanistic insight of aggregation by tartrazine. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Detection of Myoglobin with an Open-Cavity-Based Label-Free Photonic Crystal Biosensor.

    Science.gov (United States)

    Zhang, Bailin; Tamez-Vela, Juan Manuel; Solis, Steven; Bustamante, Gilbert; Peterson, Ralph; Rahman, Shafiqur; Morales, Andres; Tang, Liang; Ye, Jing Yong

    2013-01-01

    The label-free detection of one of the cardiac biomarkers, myoglobin, using a photonic-crystal-based biosensor in a total-internal-reflection configuration (PC-TIR) is presented in this paper. The PC-TIR sensor possesses a unique open optical microcavity that allows for several key advantages in biomolecular assays. In contrast to a conventional closed microcavity, the open configuration allows easy functionalization of the sensing surface for rapid biomolecular binding assays. Moreover, the properties of PC structures make it easy to be designed and engineered for operating at any optical wavelength. Through fine design of the photonic crystal structure, biochemical modification of the sensor surface, and integration with a microfluidic system, we have demonstrated that the detection sensitivity of the sensor for myoglobin has reached the clinically significant concentration range, enabling potential usage of this biosensor for diagnosis of acute myocardial infarction. The real-time response of the sensor to the myoglobin binding may potentially provide point-of-care monitoring of patients and treatment effects.

  4. Introduction of gold nanoparticles into myoglobin-Nafion film for direct electrochemistry application.

    Science.gov (United States)

    Xie, Wenting; Kong, Linlin; Kan, Meixiu; Han, Dongmei; Wang, Xueji; Zhang, Hui-Min

    2010-10-01

    An effective myoglobin-Nafion film is prepared by introducing gold nanoparticles in through a simple procedure by ion-exchange combined with electrochemical reduction. Gold nanoparticles are highly dispersed in myoglobin-Nafion film with an average size of 2.3 +/- 0.2 nm. The electrochemical behavior of myoglobin entrapped in the film has been carefully investigated with cyclic voltammetry. The results show that the introduction of gold nanoparticles into myoglobin-Nafion film makes the direct electron transfer of myoglobin efficient. A pair of well-defined redox peaks for myoglobin heme Fe(II)/Fe(III) is observed with a formal potential of -0.150 V in 0.1 M phosphate buffer (pH 7.0). The electrochemical parameters of myoglobin in the composite film are further calculated with the results of the electron-transfer rate constant (k(s)) as 0.93 s(-1) and the charge transfer coefficient (alpha) as 0.69. The experimental results also demonstrate that the immobilized myoglobin retains its electrocatalytic activity for the reduction of hydrogen peroxide and the catalytic reduction peak of myoglobin appear in a linear relationship with H2O2 concentration in the range of 10.0-235.0 microM with correlation coefficient of 0.9970. Thus fabricated Au/Mb/Nafion electrode should give a new approach for developing redox protein or enzyme-based biosensors.

  5. Rapid Myoglobin Aggregation through Glucosamine-Induced α-Dicarbonyl Formation.

    Science.gov (United States)

    Hrynets, Yuliya; Ndagijimana, Maurice; Betti, Mirko

    2015-01-01

    The extent of glycation and conformational changes of horse myoglobin (Mb) upon glycation with N-acetyl-glucosamine (GlcNAc), glucose (Glc) and glucosamine (GlcN) were investigated. Among tested sugars, the rate of glycation with GlcN was the most rapid as shown by MALDI and ESI mass spectrometries. Protein oxidation, as evaluated by the amount of carbonyl groups present on Mb, was found to increase exponentially in Mb-Glc conjugates over time, whereas in Mb-GlcN mixtures the carbonyl groups decreased significantly after maximum at 3 days of the reaction. The reaction between GlcN and Mb resulted in a significantly higher amount of α-dicarbonyl compounds, mostly glucosone and 3-deoxyglucosone, ranging from and 27 to 332 mg/L and from 14 to 304 mg/L, respectively. Already at 0.5 days, tertiary structural changes of Mb-GlcN conjugate were observed by altered tryptophan fluorescence. A reduction of metmyoglobin to deoxy-and oxymyoglobin forms was observed on the first day of reaction, coinciding with the greatest amount of glucosone produced. In contrast to native α-helical myoglobin, 41% of the glycated protein sequence was transformed into a β-sheet conformation, as determined by circular dichroism spectropolarimetry. Transmission electron microscopy demonstrated that Mb glycation with GlcN causes the formation of amorphous or fibrous aggregates, started already at 3 reaction days. These aggregates bind to an amyloid-specific dye thioflavin T. With the aid of α-dicarbonyl compounds and advanced products of reaction, this study suggests that the Mb glycation with GlcN induces the unfolding of an initially globular protein structure into amyloid fibrils comprised of a β-sheet structure.

  6. Influence of exercise on serum levels of myoglobin measured by radioimmunoassay.

    Science.gov (United States)

    Sabriá, M; Ruibal, A; Rey, C; Foz, M; Domenech, F M

    1983-01-01

    To determine the influence of exercise on serum levels of myoglobin, serum levels of this protein were determined by RIA in 90 healthy men, divided as follows: (1) Basal control (no exercise) 25 cases; (2) Moderate exercise (after subject had been working for 12 h in Medicine Emergency Service) 19 cases, and (3) Intensive exercise: (a) football professional (45-min match) 10 cases; (b) football amateur (45-min match) 10 cases; (c) basketball professional (45-min match) 10 cases, and (d) basketball professional (90-min training) 16 cases. Our results led us to the following conclusions. (1) Moderate exercise, such as the usual daily work, does not modify myoglobin levels; (2) Myoglobin serum levels after exercise increase in nearly all individuals. They are higher in untrained people; (3) There seems to be a correlation between exercise intensity and increase of myoglobin serum levels, and (4) The detection of serum myoglobin by RIA may have a wide field of application for sport medicine.

  7. Influence of exercise on serum levels of myoglobin measured by radioimmunoassay

    International Nuclear Information System (INIS)

    Sabria, M.; Rey, C.; Foz, M.; Ruibal, A.; Domenech, F.M.

    1983-01-01

    To determine the influence of exercise on serum levels of myoglobin, serum levels of this protein were determined by RIA in 90 healthy men, divided as follows: (1) Basal control (no exercise) 25 cases; (2) Moderate exercise (after subject had been working for 12 h in Medicine Emergency Service) 19 cases, and (3) Intensive exercise: (a) football professional (45-min match) 10 cases; (b) football amateur (45-min match) 10 cases; (c) basketball professional (45-min match) 10 cases, and (d) basketball professional (90-min training) 16 cases. Our results led us to the following conclusions. (1) Moderate exercise, such as the usual daily work, does not modify myoglobin levels; (2) Myoglobin serum levels after exercise increase in nearly all individuals. They are higher in untrained people; (3) There seems to be a correlation between exercise intensity and increase of myoglobin serum levels, and (4) The detection of serum myoglobin by RIA may have a wide field of application for sport medicine. (orig.)

  8. Influence of exercise on serum levels of myoglobin measured by radioimmunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Sabria, M; Rey, C; Foz, M; Ruibal, A; Domenech, F M

    1983-04-01

    To determine the influence of exercise on serum levels of myoglobin, serum levels of this protein were determined by RIA in 90 healthy men, divided as follows: (1) Basal control (no exercise) 25 cases; (2) Moderate exercise (after subject had been working for 12 h in Medicine Emergency Service) 19 cases, and (3) Intensive exercise: (a) football professional (45-min match) 10 cases; (b) football amateur (45-min match) 10 cases; (c) basketball professional (45-min match) 10 cases, and (d) basketball professional (90-min training) 16 cases. Our results led us to the following conclusions. (1) Moderate exercise, such as the usual daily work, does not modify myoglobin levels; (2) Myoglobin serum levels after exercise increase in nearly all individuals. They are higher in untrained people; (3) There seems to be a correlation between exercise intensity and increase of myoglobin serum levels, and (4) The detection of serum myoglobin by RIA may have a wide field of application for sport medicine.

  9. Studies on the optical absorption of copper-dopped myoglobin: conformational changes

    International Nuclear Information System (INIS)

    Lamy, M.T.M.

    1976-03-01

    Optical absorption changes in the visible and near U.V. spectrum of myoglobin molecules are observed when copper ions are added to the macromolecule. The heme optical transitions are investigated through a theoretical simulation of the optical absorption spectrum. A study of the absorption band in the region of 700 nm associated with the copper - myoglobin complexes indicated the existence of two kinds of metal-protein complexes: one associated with the six or eitht first added copper ions and the other related with the higher concentrations. Conformational changes caused by thermal treatment are studied in myoglobin water solutions and solutions containing copper ions. The phenomenon named pre-denaturation is observed through the optical absorption at 245 nm. It is shown that interactions between myoglobin molecules occur in the pre-denaturation phenomenon. (Author) [pt

  10. Riboflavin photosensitized oxidation of myoglobin.

    Science.gov (United States)

    Grippa, Juliana M; de Zawadzki, Andressa; Grossi, Alberto B; Skibsted, Leif H; Cardoso, Daniel R

    2014-02-05

    The reaction of the fresh meat pigment oxymyoglobin, MbFe(II)O₂, and its oxidized form metmyoglobin, MbFe(III), with triplet-state riboflavin involves the pigment protein, which is oxidatively cleaved or dimerized as shown by SDS-PAGE and Western blotting. The overall rate constant for oxidation of MbFe(II)O₂ by ³Rib is (3.0 ± 0.5) × 10⁹ L·mol⁻¹·s⁻¹ and (3.1 ± 0.4) × 10⁹ L·mol⁻¹·s⁻¹ for MbFe(III) in phosphate buffer of pH 7.4 at 25 °C as determined by laser flash photolysis. The high rates are rationalized by ground state hydrophobic interactions as detected as static quenching of fluorescence from singlet-excited state riboflavin by myoglobins using time-resolved fluorescence spectroscopy and a Stern-Volmer approach. Binding of riboflavin to MbFe(III) has K(a) = (1.2 ± 0.2) × 10⁴ mol·L⁻¹ with ΔH° = -112 ± 22 kJ·mol⁻¹ and ΔS° = -296 ± 75 J·mol⁻¹·K⁻¹. For meat, riboflavin is concluded to be a photosensitizer for protein oxidation but not for discoloration.

  11. Possible heterogeneity of centres of binding 1,8-ANS in molecules of oxygenated hemoglobin

    International Nuclear Information System (INIS)

    Parul', D.A.; Bokut', S.B.; Milyutin, A.A.; Petrov, E.P.; Nemkovich, N.A.; Sobchuk, A.N.; Dzhagarov, B.M.

    1999-01-01

    Forming of oxygenated hemoglobin is one of effects of ionizing radiation action on organism. It was revealed heterogeneity of centers of binding of 1,8-ANS in intact molecule of oxygenated hemoglobin. Two types of binding centers possible reflect the existence of two regions in protein molecule with different accessibility to molecules of water

  12. Detection of myoglobin desaturation in Mirounga angustirostris during apnea.

    Science.gov (United States)

    Ponganis, Paul J; Kreutzer, Ulrike; Sailasuta, Napapon; Knower, Torre; Hurd, Ralph; Jue, Thomas

    2002-01-01

    1H NMR solution-state study of elephant seal (Mirounga angustirostris) myoglobin (Mb) and hemoglobin (Hb) establishes the temperature-dependent chemical shifts of the proximal histidyl N(delta)H signal, which reflects the respective intracellular and vascular PO2 in vivo. Both proteins exist predominantly in one major isoform and do not exhibit any conformational heterogeneity. The Mb and Hb signals are detectable in M. angustirostris tissue in vivo. During eupnea M. angustirostris muscle maintains a well-saturated MbO2. However, during apnea, the deoxymyoglobin proximal histidyl N(delta)H signal becomes visible, reflecting a declining tissue PO2. The study establishes a firm methodological basis for using NMR to investigate the metabolic responses during sleep apnea of the elephant seal and to secure insights into oxygen regulation in diving mammals.

  13. ESR studies of heat denaturation in Cu myoglobin complexes

    International Nuclear Information System (INIS)

    Louro, S.R.W.; Ribeiro, S.C.; Bemski, G.

    1975-01-01

    An investigation is made on the behaviour of Cu2 + and Fe3 + in copper doped myoglobin, subjected to heat treatment. ESR is observed at X-band. The amplitude of the g = 5.9 line of the high spin F 3 + in met-myoglobin is studied as a function of the temperature of the heat treatment, the pH and the length of time of exposure to the heat treatment. These experiments are performed for both the pure protein and for the copper-myoglobin complex, at pH between 5 and 8.5. Results concerning the decrease of the amplitude of Fe 3+ high spin signal are discussed in comparison with Hollocher's results in hemoglobin. The Cu spectra obtained are interpreted, with the aid of a computer program, Kivelson and Neiman's results being used to analyse the authors data

  14. Significance of myoglobin as an oxygen store and oxygen transporter in the intermittently perfused human heart: a model study.

    Science.gov (United States)

    Endeward, Volker; Gros, Gerolf; Jürgens, Klaus D

    2010-07-01

    The mechanisms by which the left ventricular wall escapes anoxia during the systolic phase of low blood perfusion are investigated, especially the role of myoglobin (Mb), which can (i) store oxygen and (ii) facilitate intracellular oxygen transport. The quantitative role of these two Mb functions is studied in the maximally working human heart. Because discrimination between Mb functions has not been achieved experimentally, we use a Krogh cylinder model here. At a heart rate of 200 beats/min and a 1:1 ratio of diastole/systole, the systole lasts for 150 ms. The basic model assumption is that, with mobile Mb, the oxygen stored in the end-diastolic left ventricle wall exactly meets the demand during the 150 ms of systolic cessation of blood flow. The coronary blood flow necessary to achieve this agrees with literature data. By considering Mb immobile or setting its concentration to zero, respectively, we find that, depending on Mb concentration, Mb-facilitated O(2) transport maintains O(2) supply to the left ventricle wall during 22-34 of the 150 ms, while Mb storage function accounts for a further 12-17 ms. When Mb is completely absent, anoxia begins to develop after 116-99 ms. While Mb plays no significant role during diastole, it supplies O(2) to the left ventricular wall for < or = 50 ms of the 150 ms systole, whereas capillary haemoglobin is responsible for approximately 80 ms. Slight increases in haemoglobin concentration, blood flow, or capillary density can compensate the absence of Mb, a finding which agrees well with the observations using Mb knockout mice.

  15. Vibrational dynamics of thiocyanate and selenocyanate bound to horse heart myoglobin

    Energy Technology Data Exchange (ETDEWEB)

    Maj, Michał; Oh, Younjun; Park, Kwanghee; Lee, Jooyong; Cho, Minhaeng, E-mail: mcho@korea.ac.kr [Department of Chemistry, Korea University, Seoul 136-713 (Korea, Republic of); Kwak, Kyung-Won [Department of Chemistry, Chung-Ang University, Seoul 156-756, SouthKorea (Korea, Republic of)

    2014-06-21

    The structure and vibrational dynamics of SCN- and SeCN-bound myoglobin have been investigated using polarization-controlled IR pump-probe measurements and quantum chemistry calculations. The complexes are found to be in low and high spin states, with the dominant contribution from the latter. In addition, the Mb:SCN high spin complex exhibits a doublet feature in the thiocyanate stretch IR absorption spectra, indicating two distinct molecular conformations around the heme pocket. The binding mode of the high spin complexes was assigned to occur through the nitrogen atom, contrary to the binding through the sulfur atom that was observed in myoglobin derived from Aplysia Limacina. The vibrational energy relaxation process has been found to occur substantially faster than those of free SCN{sup −} and SeCN{sup −} ions and neutral SCN- and SeCN-derivatized molecules reported previously. This supports the N-bound configurations of MbNCS and MbNCSe, because S- and Se-bound configurations are expected to have significantly long lifetimes due to the insulation effect by heavy bridge atom like S and Se in such IR probes. Nonetheless, even though their lifetimes are much shorter than those of corresponding free ions in water, the vibrational lifetimes determined for MbNCS and MbNCSe are still fairly long compared to those of azide and cyanide myoglobin systems studied before. Thus, thiocyanate and selenocyanate can be good local probes of local electrostatic environment in the heme pocket. The globin dependence on binding mode and vibrational dynamics is also discussed.

  16. Electrochemical Aptasensor for Myoglobin-Specific Recognition Based on Porphyrin Functionalized Graphene-Conjugated Gold Nanocomposites

    Directory of Open Access Journals (Sweden)

    Guojuan Zhang

    2016-10-01

    Full Text Available In this work, a novel electrochemical aptasensor was developed for sensitive and selective detection of myoglobin based on meso-tetra (4-carboxyphenyl porphyrin-functionalized graphene-conjugated gold nanoparticles (TCPP–Gr/AuNPs. Due to its good electric conductivity, large specific surface area, and excellent mechanical properties, TCPP–Gr/AuNPs can act as an enhanced material for the electrochemical detection of myoglobin. Meanwhile, it provides an effective matrix for immobilizing myoglobin-binding aptamer (MbBA. The electrochemical aptasensor has a sensitive response to myoglobin in a linear range from 2.0 × 10−11 M to 7.7 × 10−7 M with a detection limit of 6.7 × 10−12 M (S/N = 3. Furthermore, the method has the merits of high sensitivity, low price, and high specificity. Our work will supply new horizons for the diagnostic applications of graphene-based materials in biomedicine and biosensors.

  17. The tyrosine B10 hydroxyl is crucial for oxygen avidity of Ascaris hemoglobin.

    Science.gov (United States)

    Kloek, A P; Yang, J; Mathews, F S; Frieden, C; Goldberg, D E

    1994-01-28

    The parasitic nematode Ascaris suum has a gene encoding a two-domain hemoglobin with remarkable oxygen avidity. The strong interaction with oxygen is a consequence of a particularly slow oxygen off-rate. The single polypeptide chain consists of two domains, each of which can be expressed separately in Escherichia coli as a globin-like protein exhibiting oxygen binding characteristics comparable with the native molecule. Site-directed mutagenesis was performed on the gene segment encoding domain one. The E7 position, involved in forming a hydrogen bond with the liganded oxygen in vertebrate globins, is a glutamine in both Ascaris domains. Conversion of this residue to leucine or alanine produced a hemoglobin variant with an oxygen off-rate 5- or 60-fold faster than that of unaltered domain one. Replacement of the tyrosine B10 with either phenylalanine or leucine (as found in vertebrate globins) yielded hemoglobin mutants with oxygen off-rates 280- or 570-fold faster, approaching rates found with vertebrate myoglobins. The data suggest that the distal glutamine hydrogen bonds with the liganded oxygen and that the tyrosine B10 hydroxyl contributes an additional hydrogen bond that appears substantially responsible for the extreme oxygen avidity of Ascaris hemoglobin.

  18. (R)-α-Lipoic acid inhibits fructose-induced myoglobin fructation and the formation of advanced glycation end products (AGEs) in vitro.

    Science.gov (United States)

    Ghelani, Hardik; Razmovski-Naumovski, Valentina; Pragada, Rajeswara Rao; Nammi, Srinivas

    2018-01-15

    Fructose-mediated protein glycation (fructation) has been linked to an increase in diabetic and cardiovascular complications due to over consumption of high-fructose containing diets in recent times. The objective of the present study is to evaluate the protective effect of (R)-α-lipoic acid (ALA) against fructose-induced myoglobin fructation and the formation of advanced glycation end products (AGEs) in vitro. The anti-glycation activity of ALA was determined using the formation of AGEs fluorescence intensity, iron released from the heme moiety of myoglobin and the level of fructosamine. The fructation-induced myoglobin oxidation was examined using the level of protein carbonyl content and thiol group estimation. The results showed that co-incubation of myoglobin (1 mg/mL), fructose (1 M) and ALA (1, 2 and 4 mM) significantly inhibited the formation of AGEs during the 30 day study period. ALA markedly decreased the levels of fructosamine, which is directly associated with the reduction of AGEs formation. Furthermore, ALA significantly reduced free iron release from myoglobin which is attributed to the protection of myoglobin from fructose-induced glycation. The results also demonstrated a significant protective effect of ALA on myoglobin oxidative damages, as seen from decreased protein carbonyl content and increased protein thiols. These findings provide new insights into the anti-glycation properties of ALA and emphasize that ALA supplementation is beneficial in the prevention of AGEs-mediated diabetic and cardiovascular complications.

  19. Molecular evolution of myoglobin in the Tibetan Plateau endemic schizothoracine fish (Cyprinidae, Teleostei) and tissue-specific expression changes under hypoxia.

    Science.gov (United States)

    Qi, Delin; Chao, Yan; Zhao, Yongli; Xia, Mingzhe; Wu, Rongrong

    2018-04-01

    Myoglobin (Mb) is an oxygen-binding hemoprotein that was once thought to be exclusively expressed in oxidative myocytes of skeletal and cardiac muscle where it serves in oxygen storage and facilitates intracellular oxygen diffusion. In this study, we cloned the coding sequence of the Mb gene from four species, representing three groups, of the schizothoracine fish endemic to the Qinghai-Tibetan Plateau (QTP), then conducted molecular evolution analyses. We also investigated tissue expression patterns of Mb and the expression response to moderate and severe hypoxia at the mRNA and protein levels in a representative of the highly specialized schizothoracine fish species, Schizopygopsis pylzovi. Molecular evolution analyses showed that Mb from the highly specialized schizothoracine fish have undergone positive selection and one positively selected residue (81L) was identified, which is located in the F helix, close to or in contact with the heme. We present tentative evidence that the Mb duplication event occurred in the ancestor of the schizothoracine and Cyprininae fish (common carp and goldfish), and that the Mb2 paralog was subsequently lost in the schizothoracine fish. In S. pylzovi, Mb mRNA is expressed in various tissues with the exception of the intestine and gill, but all such tissues, including the liver, muscle, kidney, brain, eye, and skin, expressed very low levels of Mb mRNA (Tibetan Plateau fish.

  20. The structure and dynamics of the Fe-CO bond in myoglobin

    International Nuclear Information System (INIS)

    Rovira, Carme

    2003-01-01

    This paper is a review of our recent work on the structure and dynamics of the Fe-CO bond in carbonmonoxy myoglobin (MbCO), performed using density functional theory, Car-Parrinello molecular dynamics and hybrid quantum mechanics/molecular mechanics approaches. The results of these investigations have served to shed light onto one of the long standing questions in myoglobin research: whether the protein discriminates the CO ligand with respect to O 2 by distorting the FeCO bond. The calculations show that both in the gas phase and in the protein the Fe-CO bond is essentially linear and therefore exclude the hypothesis that the CO in MbCO is sterically hindered. In contrast, hydrogen bonding between the O 2 ligand and the His64 residue easily explains the protein discrimination for CO

  1. Roles of the creatine kinase system and myoglobin in maintaining energetic state in the working heart

    Directory of Open Access Journals (Sweden)

    Beard Daniel A

    2009-02-01

    Full Text Available Abstract Background The heart is capable of maintaining contractile function despite a transient decrease in blood flow and increase in cardiac ATP demand during systole. This study analyzes a previously developed model of cardiac energetics and oxygen transport to understand the roles of the creatine kinase system and myoglobin in maintaining the ATP hydrolysis potential during beat-to-beat transient changes in blood flow and ATP hydrolysis rate. Results The theoretical investigation demonstrates that elimination of myoglobin only slightly increases the predicted range of oscillation of cardiac oxygenation level during beat-to-beat transients in blood flow and ATP utilization. In silico elimination of myoglobin has almost no impact on the cytoplasmic ATP hydrolysis potential (ΔGATPase. In contrast, disabling the creatine kinase system results in considerable oscillations of cytoplasmic ADP and ATP levels and seriously deteriorates the stability of ΔGATPase in the beating heart. Conclusion The CK system stabilizes ΔGATPase by both buffering ATP and ADP concentrations and enhancing the feedback signal of inorganic phosphate in regulating mitochondrial oxidative phosphorylation.

  2. Myoglobin Structure and Function: A Multiweek Biochemistry Laboratory Project

    Science.gov (United States)

    Silverstein, Todd P.; Kirk, Sarah R.; Meyer, Scott C.; Holman, Karen L. McFarlane

    2015-01-01

    We have developed a multiweek laboratory project in which students isolate myoglobin and characterize its structure, function, and redox state. The important laboratory techniques covered in this project include size-exclusion chromatography, electrophoresis, spectrophotometric titration, and FTIR spectroscopy. Regarding protein structure,…

  3. Radioimmunoassay of serum myoglobin in neuromuscular diseases

    Energy Technology Data Exchange (ETDEWEB)

    Askmark, H; Osterman, P O; Roxin, L E; Venge, P [University Hospital, Uppsala (Sweden)

    1981-01-01

    Radioimmunoassay of serum myoglobin was performed in 85 patients with muscular symptoms. Elevated levels were found in 93% of patients with myogenic myopathy, in 54% with myasthenia gravis and in 50% with neurogenic myopathy. All 11 patients with polymyositis had elevated myoglobin concentrations. In six of seven patients with polymyositis, who were followed up with repeated determinations, a clear relationship between myoglobin levels and clinical course was found. In general serum myoglobin seemed to be a more sensitive indicator of muscle disease than creatine kinase.

  4. Radioimmunoassay of serum myoglobin in neuromuscular diseases

    International Nuclear Information System (INIS)

    Askmark, H.; Osterman, P.O.; Roxin, L.-E.; Venge, P.

    1981-01-01

    Radioimmunoassay of serum myoglobin was performed in 85 patients with muscular symptoms. Elevated levels were found in 93% of patients with myogenic myopathy, in 54% with myasthenia gravis and in 50% with neurogenic myopathy. All 11 patients with polymyositis had elevated myoglobin concentrations. In six of seven patients with polymyositis, who were followed up with repeated determinations, a clear relationship between myoglobin levels and clinical course was found. In general serum myoglobin seemed to be a more sensitive indicator of muscle disease than creatine kinase. (author)

  5. Clinical usefulness of radioimmune measurement of serum myoglobin

    International Nuclear Information System (INIS)

    Ruibal, A.; Sabria, M.; Rey, C.; Fraile, M.; Foz, M.; Domenech-Torne, F.M.

    1983-01-01

    In order to ascertain the behaviour in different muscular diseases, as well as its potential clinical usefulness, we have determined by radioimmunoassay the seric levels of myoglobin in 212 patients with different muscular diseases. Our results led us to the following: 1) The myoglobin determination is an early marker and very useful during the first 24 hours in I.A.M.; 2) The myoglobin determination is useful in diagnostic of Duchenne's and Steinert's disease; 3) In healthy carriers of Duchenne's disease and limb-girdle myopathy, total CK was better than myoglobin as a biochemical marker; 4) Higher increases of seric myoglobin were shown in non-trained subjects after exercice. Then, the myoglobin determination may become a wide field of application for sport medicine

  6. Energetic costs of protein synthesis do not differ between red- and white-blooded Antarctic notothenioid fishes.

    Science.gov (United States)

    Lewis, Johanne M; Grove, Theresa J; O'Brien, Kristin M

    2015-09-01

    Antarctic icefishes (Family Channichthyidae) within the suborder Notothenioidei lack the oxygen-binding protein hemoglobin (Hb), and six of the 16 species of icefishes lack myoglobin (Mb) in heart ventricle. As iron-centered proteins, Hb and Mb can promote the formation of reactive oxygen species (ROS) that damage biological macromolecules. Consistent with this, our previous studies have shown that icefishes have lower levels of oxidized proteins and lipids in oxidative muscle compared to red-blooded notothenioids. Because oxidized proteins are usually degraded by the 20S proteasome and must be resynthesized, we hypothesized that rates of protein synthesis would be lower in icefishes compared to red-blooded notothenioids, thereby reducing the energetic costs of protein synthesis and conferring a benefit to the loss of Hb and Mb. Rates of protein synthesis were quantified in hearts, and the fraction of oxygen consumption devoted to protein synthesis was measured in isolated hepatocytes and cardiomyocytes of notothenioids differing in the expression of Hb and cardiac Mb. Neither rates of protein synthesis nor the energetic costs of protein synthesis differed among species, suggesting that red-blooded species do not degrade and replace oxidatively modified proteins at a higher rate compared to icefishes but rather, persist with higher levels of oxidized proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. The zinc-myoglobin relationships in porcine muscles

    International Nuclear Information System (INIS)

    Fogd Joergensen, P.; Wegger, I.

    1976-01-01

    Zinc and myoglobin content in muscles from pigs were studied under various conditions. Zinc concentration was considerably higher in red than in white muscles. In muscles, where the metabolic pattern changes from glycolytic to oxidative during the period from birth to weaning, a simultaneous increase in zinc content was seen. A significant positive correlation exists between myoglobin and zinc content under normal conditions. However, while myoglobin concentration decreases due to iron deficiency anaemia no changes occur in zinc content. It is concluded that no functional link seems to exist between zinc metabolism and myoglobin synthesis in porcine muscles. (author)

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

    Directory of Open Access Journals (Sweden)

    Yu-Ru Zhang

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

  9. Artificial oxygen transport protein

    Science.gov (United States)

    Dutton, P. Leslie

    2014-09-30

    This invention provides heme-containing peptides capable of binding molecular oxygen at room temperature. These compounds may be useful in the absorption of molecular oxygen from molecular oxygen-containing atmospheres. Also included in the invention are methods for treating an oxygen transport deficiency in a mammal.

  10. Microcontact imprinted surface plasmon resonance sensor for myoglobin detection

    International Nuclear Information System (INIS)

    Osman, Bilgen; Uzun, Lokman; Beşirli, Necati; Denizli, Adil

    2013-01-01

    In this study, we prepared surface plasmon resonance (SPR) sensor using the molecular imprinting technique for myoglobin detection in human serum. For this purpose, we synthesized myoglobin imprinted poly(hydroxyethyl methacrylate-N-methacryloyl-L-tryptophan methyl ester) [poly(HEMA-MATrp)] nanofilm on the surface of SPR sensor. We also synthesized non-imprinted poly(HEMA-MATrp) nanofilm without myoglobin for the control experiments. The SPR sensor was characterized with contact angle measurements, atomic force microscopy, X-ray photoelectron spectroscopy, and ellipsometry. We investigated the effectiveness of the sensor using the SPR system. We evaluated the ability of SPR sensor to sense myoglobin with myoglobin solutions (pH 7.4, phosphate buffer) in different concentration range and in the serum taken from a patient with acute myocardial infarction. We found that the Langmuir adsorption model was the most suitable for the sensor system. The detection limit was 87.6 ng/mL. In order to show the selectivity of the SPR sensor, we investigated the competitive detection of myoglobin, lysozyme, cytochrome c and bovine serum albumin. The results showed that the SPR sensor has high selectivity and sensitivity for myoglobin. - Highlights: • Micro-contact imprinted surface plasmon resonance sensor. • Real-time myoglobin detection in the serum taken from a patient with acute myocardial infarction • Reproducible results for consecutive myoglobin solution supplement • LOD and LOQ values of the SPR sensor were determined to be 26.3 and 87.6 ng/mL. • The SPR sensor has potential for myoglobin sensing during acute MI cases

  11. Competitive protein binding assay

    International Nuclear Information System (INIS)

    Kaneko, Toshio; Oka, Hiroshi

    1975-01-01

    The measurement of cyclic GMP (cGMP) by competitive protein binding assay was described and discussed. The principle of binding assay was represented briefly. Procedures of our method by binding protein consisted of preparation of cGMP binding protein, selection of 3 H-cyclic GMP on market, and measurement procedures. In our method, binding protein was isolated from the chrysalis of silk worm. This method was discussed from the points of incubation medium, specificity of binding protein, the separation of bound cGMP from free cGMP, and treatment of tissue from which cGMP was extracted. cGMP existing in the tissue was only one tenth or one scores of cGMP, and in addition, cGMP competed with cGMP in binding with binding protein. Therefore, Murad's technique was applied to the isolation of cGMP. This method provided the measurement with sufficient accuracy; the contamination by cAMP was within several per cent. (Kanao, N.)

  12. Broad Phylogenetic Occurrence of the Oxygen-Binding Hemerythrins in Bilaterians.

    Science.gov (United States)

    Costa-Paiva, Elisa M; Schrago, Carlos G; Halanych, Kenneth M

    2017-10-01

    Animal tissues need to be properly oxygenated for carrying out catabolic respiration and, as such, natural selection has presumably favored special molecules that can reversibly bind and transport oxygen. Hemoglobins, hemocyanins, and hemerythrins (Hrs) fulfill this role, with Hrs being the least studied. Knowledge of oxygen-binding proteins is crucial for understanding animal physiology. Hr genes are present in the three domains of life, Archaea, Bacteria, and Eukaryota; however, within Animalia, Hrs has been reported only in marine species in six phyla (Annelida, Brachiopoda, Priapulida, Bryozoa, Cnidaria, and Arthropoda). Given this observed Hr distribution, whether all metazoan Hrs share a common origin is circumspect. We investigated Hr diversity and evolution in metazoans, by employing in silico approaches to survey for Hrs from of 120 metazoan transcriptomes and genomes. We found 58 candidate Hr genes actively transcribed in 36 species distributed in 11 animal phyla, with new records in Echinodermata, Hemichordata, Mollusca, Nemertea, Phoronida, and Platyhelminthes. Moreover, we found that "Hrs" reported from Cnidaria and Arthropoda were not consistent with that of other metazoan Hrs. Contrary to previous suggestions that Hr genes were absent in deuterostomes, we find Hr genes present in deuterostomes and were likely present in early bilaterians, but not in nonbilaterian animal lineages. As expected, the Hr gene tree did not mirror metazoan phylogeny, suggesting that Hrs evolutionary history was complex and besides the oxygen carrying capacity, the drivers of Hr evolution may also consist of secondary functional specializations of the proteins, like immunological functions. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  13. Myoglobin solvent structure at different temperatures

    Energy Technology Data Exchange (ETDEWEB)

    Daniels, B.V.; Korszun, Z.R. [Brookhaven National Laboratory, Upton, NY (United States); Schoenborn, B.P. [Los Alamos National Laboratory, NM (United States)

    1994-12-31

    The structure of the solvent surrounding myoglobin crystals has been analyzed using neutron diffraction data, and the results indicate that the water around the protein is not disordered, but rather lies in well-defined hydration shells. We have analyzed the structure of the solvent surrounding the protein by collecting neutron diffraction data at four different temperatures, namely, 80, 130, 180, and 240K. Relative Wilson Statistics applied to low resolution data showed evidence of a phase transition in the region of 180K. A plot of the liquidity factor, B{sub sn}, versus distance from the protein surface begins with a high plateau near the surface of the protein and drops to two minima at distances from the protein surface of about 2.35{Angstrom} and 3.85{Angstrom}. Two distinct hydration shells are observed. Both hydration shells are observed to expand as the temperature is increased.

  14. Myoglobin solvent structure at different temperatures

    International Nuclear Information System (INIS)

    Daniels, B.V.; Korszun, Z.R.; Schoenborn, B.P.

    1994-01-01

    The structure of the solvent surrounding myoglobin crystals has been analyzed using neutron diffraction data, and the results indicate that the water around the protein is not disordered, but rather lies in well-defined hydration shells. We have analyzed the structure of the solvent surrounding the protein by collecting neutron diffraction data at four different temperatures, namely, 80, 130, 180, and 240K. Relative Wilson Statistics applied to low resolution data showed evidence of a phase transition in the region of 180K. A plot of the liquidity factor, B sn , versus distance from the protein surface begins with a high plateau near the surface of the protein and drops to two minima at distances from the protein surface of about 2.35 Angstrom and 3.85 Angstrom. Two distinct hydration shells are observed. Both hydration shells are observed to expand as the temperature is increased

  15. Radioimmunoassay of plasma myoglobin concentrations in normal persons

    Energy Technology Data Exchange (ETDEWEB)

    Milanov, S.; Milkov, V. (Meditsinska Akademiya, Sofia (Bulgaria). Nauchen Inst. po Rentgenologiya i Radiobiologiya)

    1982-01-01

    Plasma myoglobin concentrations by radioimmunoassay in 53 normal persons (29 women and 24 men) with ages ranging from 19 to 76 years. The average values for myoglobin concentrations in the plasma of 29 women, aged 19 to 76 years, are 4a.09 +- 4.51 ng/ml, while in 24 healthy men, aged 20 to 74 years, they are 54.39 +-4.68 ng/ml. The obtained results for the plasma myoglobin level in men are higher than the ones recorded in women at statistical significance p<0.05. In the group under study the plasma myoglobin values disclose also age-related differences, although lacking statistical significance (p>0.10).

  16. Zinc-Substituted Myoglobin Is a Naturally Occurring Photo-antimicrobial Agent with Potential Applications in Food Decontamination.

    Science.gov (United States)

    Delcanale, Pietro; Montali, Chiara; Rodríguez-Amigo, Beatriz; Abbruzzetti, Stefania; Bruno, Stefano; Bianchini, Paolo; Diaspro, Alberto; Agut, Montserrat; Nonell, Santi; Viappiani, Cristiano

    2016-11-16

    Zinc-substituted myoglobin (ZnMb) is a naturally occurring photosensitizer that generates singlet oxygen with a high quantum yield. Using a combination of photophysical and fluorescence imaging techniques, we demonstrate the interaction of ZnMb with Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. An efficient antibacterial action against S. aureus was observed, with a reduction up to 99.9999% in the number of colony-forming units, whereas no sizable effect was detected against E. coli. Because ZnMb is known to form during the maturation of additive-free not-cooked cured ham, the use of this protein as a built-in photodynamic agent may constitute a viable method for the decontamination of these food products from Gram-positive bacteria.

  17. Roles of Glutamates and Metal ions in a Rationally Designed Nitric Oxide Reductase Based on Myoglobin

    Energy Technology Data Exchange (ETDEWEB)

    Y Lin; N Yeung; Y Gao; K Miner; S Tian; H Robinson; Y Lu

    2011-12-31

    A structural and functional model of bacterial nitric oxide reductase (NOR) has been designed by introducing two glutamates (Glu) and three histidines (His) in sperm whale myoglobin. X-ray structural data indicate that the three His and one Glu (V68E) residues bind iron, mimicking the putative FeB site in NOR, while the second Glu (I107E) interacts with a water molecule and forms a hydrogen bonding network in the designed protein. Unlike the first Glu (V68E), which lowered the heme reduction potential by {approx}110 mV, the second Glu has little effect on the heme potential, suggesting that the negatively charged Glu has a different role in redox tuning. More importantly, introducing the second Glu resulted in a {approx}100% increase in NOR activity, suggesting the importance of a hydrogen bonding network in facilitating proton delivery during NOR reactivity. In addition, EPR and X-ray structural studies indicate that the designed protein binds iron, copper, or zinc in the FeB site, each with different effects on the structures and NOR activities, suggesting that both redox activity and an intermediate five-coordinate heme-NO species are important for high NOR activity. The designed protein offers an excellent model for NOR and demonstrates the power of using designed proteins as a simpler and more well-defined system to address important chemical and biological issues.

  18. Monomeric Yeast Frataxin is an Iron-Binding Protein

    International Nuclear Information System (INIS)

    Cook, J.; Bencze, K.; Jankovic, A.; Crater, A.; Busch, C.; Bradley, P.; Stemmler, A.; Spaller, M.; Stemmler, T.

    2006-01-01

    Friedreich's ataxia, an autosomal cardio- and neurodegenerative disorder that affects 1 in 50 000 humans, is caused by decreased levels of the protein frataxin. Although frataxin is nuclear-encoded, it is targeted to the mitochondrial matrix and necessary for proper regulation of cellular iron homeostasis. Frataxin is required for the cellular production of both heme and iron-sulfur (Fe-S) clusters. Monomeric frataxin binds with high affinity to ferrochelatase, the enzyme involved in iron insertion into porphyrin during heme production. Monomeric frataxin also binds to Isu, the scaffold protein required for assembly of Fe-S cluster intermediates. These processes (heme and Fe-S cluster assembly) share requirements for iron, suggesting that monomeric frataxin might function as the common iron donor. To provide a molecular basis to better understand frataxin's function, we have characterized the binding properties and metal-site structure of ferrous iron bound to monomeric yeast frataxin. Yeast frataxin is stable as an iron-loaded monomer, and the protein can bind two ferrous iron atoms with micromolar binding affinity. Frataxin amino acids affected by the presence of iron are localized within conserved acidic patches located on the surfaces of both helix-1 and strand-1. Under anaerobic conditions, bound metal is stable in the high-spin ferrous state. The metal-ligand coordination geometry of both metal-binding sites is consistent with a six-coordinate iron-(oxygen/nitrogen) based ligand geometry, surely constructed in part from carboxylate and possibly imidazole side chains coming from residues within these conserved acidic patches on the protein. On the basis of our results, we have developed a model for how we believe yeast frataxin interacts with iron

  19. Lead-Binding Proteins: A Review

    Directory of Open Access Journals (Sweden)

    Harvey C. Gonick

    2011-01-01

    Full Text Available Lead-binding proteins are a series of low molecular weight proteins, analogous to metallothionein, which segregate lead in a nontoxic form in several organs (kidney, brain, lung, liver, erythrocyte. Whether the lead-binding proteins in every organ are identical or different remains to be determined. In the erythrocyte, delta-aminolevulinic acid dehydratase (ALAD isoforms have commanded the greatest attention as proteins and enzymes that are both inhibitable and inducible by lead. ALAD-2, although it binds lead to a greater degree than ALAD-1, appears to bind lead in a less toxic form. What may be of greater significance is that a low molecular weight lead-binding protein, approximately 10 kDa, appears in the erythrocyte once blood lead exceeds 39 μg/dL and eventually surpasses the lead-binding capacity of ALAD. In brain and kidney of environmentally exposed humans and animals, a cytoplasmic lead-binding protein has been identified as thymosin β4, a 5 kDa protein. In kidney, but not brain, another lead-binding protein has been identified as acyl-CoA binding protein, a 9 kDa protein. Each of these proteins, when coincubated with liver ALAD and titrated with lead, diminishes the inhibition of ALAD by lead, verifying their ability to segregate lead in a nontoxic form.

  20. Casein kinase 1 regulates sterol regulatory element-binding protein (SREBP) to control sterol homeostasis.

    Science.gov (United States)

    Brookheart, Rita T; Lee, Chih-Yung S; Espenshade, Peter J

    2014-01-31

    Sterol homeostasis is tightly controlled by the sterol regulatory element-binding protein (SREBP) transcription factor that is highly conserved from fungi to mammals. In fission yeast, SREBP functions in an oxygen-sensing pathway to promote adaptation to decreased oxygen supply that limits oxygen-dependent sterol synthesis. Low oxygen stimulates proteolytic cleavage of the SREBP homolog Sre1, generating the active transcription factor Sre1N that drives expression of sterol biosynthetic enzymes. In addition, low oxygen increases the stability and DNA binding activity of Sre1N. To identify additional signals controlling Sre1 activity, we conducted a genetic overexpression screen. Here, we describe our isolation and characterization of the casein kinase 1 family member Hhp2 as a novel regulator of Sre1N. Deletion of Hhp2 increases Sre1N protein stability and ergosterol levels in the presence of oxygen. Hhp2-dependent Sre1N degradation by the proteasome requires Hhp2 kinase activity, and Hhp2 binds and phosphorylates Sre1N at specific residues. Our results describe a role for casein kinase 1 as a direct regulator of sterol homeostasis. Given the role of mammalian Hhp2 homologs, casein kinase 1δ and 1ε, in regulation of the circadian clock, these findings may provide a mechanism for coordinating circadian rhythm and lipid metabolism.

  1. Electrochemical detection of cardiac biomarker myoglobin using polyphenol as imprinted polymer receptor.

    Science.gov (United States)

    Ribeiro, J A; Pereira, C M; Silva, A F; Sales, M Goreti F

    2017-08-15

    An electrochemical biosensor was developed by merging the features of Molecular Imprinting technique and Screen-Printed Electrode (SPE) for the simple and fast screening of cardiac biomarker myoglobin (Myo) in point-of-care (POC). The MIP artificial receptor for Myo was prepared by electrooxidative polymerization of phenol (Ph) on a AuSPE in the presence of Myo as template molecule. The choice of the most effective protein extraction procedure from the various extraction methods tested (mildly acidic/basic solutions, pure/mixed organic solvents, solutions containing surfactants and enzymatic digestion methods), and the optimization of the thickness of the polymer film was carefully undertaken in order to improve binding characteristics of Myo to the imprinted polymer receptor and increase the sensitivity of the MIP biosensor. The film thickness was optimized by adjusting scan rate and the number of cycles during cyclic voltammetric electropolymerization of Ph. The thickness of the polyphenol nanocoating of only few nanometres (∼4.4 nm), and similar to the protein diameter, brought in significant improvements in terms of sensor sensitivity. The binding affinity of MIP receptor film was estimated by fitting the experimental data to Freundlich isotherm and a ∼8 fold increase in the binding affinity of Myo to the imprinted polymer (K F = 0.119 ± 0.002 ng -1  mL) when compared to the non-imprinted polymer (K F  = 0.015 ± 0.002 ng -1  mL) which demonstrated excellent (re)binding affinity for the imprinted protein. The incubation of the Myo MIP receptor modified electrode with increasing concentration of protein (from 0.001 ng mL -1 to 100 μg mL -1 ) resulted in a decrease of the ferro/ferricyanide redox current. LODs of 2.1 and 14 pg mL -1 were obtained from calibration curves built in neutral buffer and diluted artificial serum, respectively, using SWV technique, enabling the detection of the protein biomarker at clinically relevant levels. The

  2. Salt effects on ionization equilibria of histidines in myoglobin.

    Science.gov (United States)

    Kao, Y H; Fitch, C A; Bhattacharya, S; Sarkisian, C J; Lecomte, J T; García-Moreno E, B

    2000-09-01

    The salt dependence of histidine pK(a) values in sperm whale and horse myoglobin and in histidine-containing peptides was measured by (1)H-NMR spectroscopy. Structure-based pK(a) calculations were performed with continuum methods to test their ability to capture the effects of solution conditions on pK(a) values. The measured pK(a) of most histidines, whether in the protein or in model compounds, increased by 0.3 pH units or more between 0.02 M and 1.5 M NaCl. In myoglobin two histidines (His(48) and His(36)) exhibited a shallower dependence than the average, and one (His(113)) showed a steeper dependence. The (1)H-NMR data suggested that the salt dependence of histidine pK(a) values in the protein was determined primarily by the preferential stabilization of the charged form of histidine with increasing salt concentrations rather than by screening of electrostatic interactions. The magnitude and salt dependence of interactions between ionizable groups were exaggerated in pK(a) calculations with the finite-difference Poisson-Boltzmann method applied to a static structure, even when the protein interior was treated with arbitrarily high dielectric constants. Improvements in continuum methods for calculating salt effects on pK(a) values will require explicit consideration of the salt dependence of model compound pK(a) values used for reference in the calculations.

  3. Watching proteins function with picosecond X-ray crystallography and molecular dynamics simulations.

    Science.gov (United States)

    Anfinrud, Philip

    2006-03-01

    Time-resolved electron density maps of myoglobin, a ligand-binding heme protein, have been stitched together into movies that unveil with molecular dynamics (MD) calculations and picosecond time-resolved X-ray structures provides single-molecule insights into mechanisms of protein function. Ensemble-averaged MD simulations of the L29F mutant of myoglobin following ligand dissociation reproduce the direction, amplitude, and timescales of crystallographically-determined structural changes. This close agreement with experiments at comparable resolution in space and time validates the individual MD trajectories, which identify and structurally characterize a conformational switch that directs dissociated ligands to one of two nearby protein cavities. This unique combination of simulation and experiment unveils functional protein motions and illustrates at an atomic level relationships among protein structure, dynamics, and function. In collaboration with Friedrich Schotte and Gerhard Hummer, NIH.

  4. Photoexcitation dynamics of nitric oxide bound ferric myoglobin probed by femtosecond IR spectroscopy

    Directory of Open Access Journals (Sweden)

    Park Jaehun

    2013-03-01

    Full Text Available Time-resolved vibrational spectra show that photolysis quantum yield of NO bound ferric myoglobin is smaller than 0.86, the deligated NO geminately rebinds with subnanosecond time scale, and the rebinding kinetics depends on protein conformation.

  5. Enhancing the electrochemical response of myoglobin with carbon nanotube electrodes.

    Science.gov (United States)

    Esplandiu, M J; Pacios, M; Cyganek, L; Bartroli, J; del Valle, M

    2009-09-02

    In this paper, the electrochemical behavior of different myoglobin-modified carbon electrodes is evaluated. In particular, the performance of voltammetric biosensors made of forest-like carbon nanotubes, carbon nanotube composites and graphite composites is compared by monitoring mainly the electrocatalytic reduction of H(2)O(2) by myoglobin and their corresponding electroanalytical characteristics. Graphite composites showed the worst electroanalytical performance, exhibiting a small linear range, a limit of detection (LOD) of 9 x 10(-5) M and low sensitivity. However, it was found that the electrochemical response was enhanced with the use of carbon nanotube-based electrodes with LOD up to 5 x 10(-8) M, higher sensitivities and wider linear range response. On the one hand, in the case of the CNT epoxy composite, the improvement in the response can be mainly attributed to its more porous surface which allows the immobilization of higher amounts of the electroactive protein. On the other hand, in the case of the forest-like CNT electrodes, the enhancement is due to an increase in the electron transfer kinetics. These findings encourage the use of myoglobin-modified carbon nanotube electrodes as potential (bio)sensors of H(2)O(2) or O(2) in biology, microbiology and environmental fields.

  6. Size Polymorphism in Alleles of the Myoglobin Gene from Biomphalaria Mollusks

    Directory of Open Access Journals (Sweden)

    Marcelo M. Santoro

    2010-10-01

    Full Text Available Introns are common among all eukaryotes, while only a limited number of introns are found in prokaryotes. Globin and globin-like proteins are widely distributed in nature, being found even in prokaryotes and a wide range of patterns of intron-exon have been reported in several eukaryotic globin genes. Globin genes in invertebrates show considerable variation in the positions of introns; globins can be found without introns, with only one intron or with three introns in different positions. In this work we analyzed the introns in the myoglobin gene from Biomphalaria glabrata, B. straminea and B. tenagophila. In the Biomphalaria genus, the myoglobin gene has three introns; these were amplified by PCR and analyzed by PCR-RFLP. Results showed that the size (number or nucleotides and the nucleotide sequence of the coding gene of the myoglobin are variable in the three species. We observed the presence of size polymorphisms in intron 2 and 3; this characterizes a homozygous/heterozygous profile and it indicates the existence of two alleles which are different in size in each species of Biomphalaria. This polymorphism could be explored for specific identification of Biomphalaria individuals.

  7. IGF-1 attenuates hypoxia-induced atrophy but inhibits myoglobin expression in C2C12 skeletal muscle myotubes

    NARCIS (Netherlands)

    Peters, Eva L.; van der Linde, Sandra M.; Vogel, Ilse S.P.; Haroon, Mohammad; Offringa, Carla; de Wit, Gerard M.J.; Koolwijk, Pieter; van der Laarse, Willem J.; Jaspers, Richard T.

    2017-01-01

    Chronic hypoxia is associated with muscle wasting and decreased oxidative capacity. By contrast, training under hypoxia may enhance hypertrophy and increase oxidative capacity as well as oxygen transport to the mitochondria, by increasing myoglobin (Mb) expression. The latter may be a feasible

  8. Modelling the autoxidation of myoglobin in fresh meat under modified atmosphere packing conditions

    DEFF Research Database (Denmark)

    Tofteskov, Jon; Hansen, Jesper Schmidt; Bailey, Nicholas

    2017-01-01

    pigment concentration data is obtained, but only by substantially increasing the value of the oxygen consumption rate. Application of the model to meat stored at 5 °C shows that the metmyoglobin layer forms under the surface over a time scale of 24 h; The metmyoglobin layer forms deeper inside the meat......Modified atmosphere packing (MAP) is a technique to increase the shelf life of fresh meat. Continuing development of MAP requires better understanding of the physical and chemical processes taking place, in particular the diffusion of oxygen and its reaction with myoglobin. We model these processes...... proportional to the logarithm of the headspace oxygen partial pressure, thus improving the colour appearance of the meat....

  9. Investigation of the Copper Binding Site And the Role of Histidine As a Ligand in Riboflavin Binding Protein

    Energy Technology Data Exchange (ETDEWEB)

    Smith, S.R.; Bencze, K.Z.; Russ, K.A.; Wasiukanis, K.; Benore-Parsons, M.; Stemmler, T.L.

    2009-05-26

    Riboflavin Binding Protein (RBP) binds copper in a 1:1 molar ratio, forming a distinct well-ordered type II site. The nature of this site has been examined using X-ray absorption and pulsed electron paramagnetic resonance (EPR) spectroscopies, revealing a four coordinate oxygen/nitrogen rich environment. On the basis of analysis of the Cambridge Structural Database, the average protein bound copper-ligand bond length of 1.96 {angstrom}, obtained by extended x-ray absorption fine structure (EXAFS), is consistent with four coordinate Cu(I) and Cu(II) models that utilize mixed oxygen and nitrogen ligand distributions. These data suggest a Cu-O{sub 3}N coordination state for copper bound to RBP. While pulsed EPR studies including hyperfine sublevel correlation spectroscopy and electron nuclear double resonance show clear spectroscopic evidence for a histidine bound to the copper, inclusion of a histidine in the EXAFS simulation did not lead to any significant improvement in the fit.

  10. Successful Treatment of Severe Carbon Monoxide Poisoning and Refractory Shock Using Extracorporeal Membrane Oxygenation.

    Science.gov (United States)

    Teerapuncharoen, Krittika; Sharma, Nirmal S; Barker, Andrew B; Wille, Keith M; Diaz-Guzman, Enrique

    2015-09-01

    Carbon monoxide (CO) is the most common cause of poisoning and poisoning-related death in the United States. It is a tasteless and odorless poisonous gas produced from incomplete combustion of hydrocarbons, such as those produced by cars and heating systems. CO rapidly binds to hemoglobin to form carboxyhemoglobin, leading to tissue hypoxia, multiple-organ failure, and cardiovascular collapse. CO also binds to myocardial myoglobin, preventing oxidative phosphorylation in cardiac mitochondria and resulting in cardiac ischemia or stunning and cardiogenic pulmonary edema. Treatment of CO poisoning is mainly supportive, and supplemental oxygen remains the cornerstone of therapy, whereas hyperbaric oxygen therapy is considered for patients with evidence of neurological and myocardial injury. Extracorporeal membrane oxygenation (ECMO) has been utilized effectively in patients with respiratory failure and hemodynamic instability, but its use has rarely been reported in patients with CO poisoning. We report the successful use of venoarterial ECMO in a patient with severe CO poisoning and multiple-organ failure. Copyright © 2015 by Daedalus Enterprises.

  11. Heart-type fatty acid binding protein is an early marker of myocardial damage after radiofrequency catheter ablation.

    Science.gov (United States)

    Giannessi, Daniela; Piacenti, Marcello; Maltinti, Maristella; Rossi, Andrea; Di Cecco, Pietro; Startari, Umberto; Cabiati, Manuela; Panchetti, Luca; Del Ry, Silvia; Morales, Maria-Aurora

    2010-10-01

    Radiofrequency (RF) ablation of arrhythmias induces myocardial damage and release of biomarkers. This study aimed to assess the kinetics of heart-type fatty acid-binding protein (h-FABP), a cytosolic protein released after myocardial injury incurred by both atrial and ventricular RF ablation, compared to other markers of myocardial injury. h-FABP, cTnI, CK-MB(mass) and myoglobin were evaluated in 30 patients with atrial or ventricular tachyarrhythmias before, immediately after and at 3, 6 and 24h after the procedure. h-FABP increased immediately after the procedure in all subjects (6.6 ± 1.2 μg/L vs 2.7 ± 0.3, pvalues of time for mean power of RF application in both the entire patient cohort and in ventricular ablations. h-FABP may be an early parameter for monitoring RF-induced lesions and the site of ablation was relevant for biomarker increase. Copyright © 2010 Elsevier Inc. All rights reserved.

  12. Hydrophilic crosslinked-polymeric surface capable of effective suppression of protein adsorption

    Energy Technology Data Exchange (ETDEWEB)

    Kamon, Yuri; Inoue, Naoko; Mihara, Erika; Kitayama, Yukiya; Ooya, Tooru; Takeuchi, Toshifumi, E-mail: takeuchi@gold.kobe-u.ac.jp

    2016-08-15

    Highlights: • Three hydrophilic crosslinked polymers were examined for protein adsorption. • All polymers showed low nonspecific adsorption of negatively charged proteins. • Poly(MMPC) showed the lowest adsorption for positively charged proteins. • Poly(MMPC) is able to reduce nonspecific adsorption of a wide range of proteins. - Abstract: We investigated the nonspecific adsorption of proteins towards three hydrophilic crosslinked-polymeric thin layers prepared by surface-initiated atom transfer radical polymerization using N,N′-methylenebisacrylamide, 2-(methacryloyloxy)ethyl-[N-(2-methacryloyloxy)ethyl]phosphorylcholine (MMPC), or 6,6′-diacryloyl-trehalose crosslinkers. Protein binding experiments were performed by surface plasmon resonance with six proteins of different pI values including α-lactalbumin, bovine serum albumin (BSA), myoglobin, ribonuclease A, cytochrome C, and lysozyme in buffer solution at pH 7.4. All of the obtained crosslinked-polymeric thin layers showed low nonspecific adsorption of negatively charged proteins at pH 7.4 such as α-lactalbumin, BSA, and myoglobin. Nonspecific adsorption of positively charged proteins including ribonuclease A, cytochrome C, and lysozyme was the lowest for poly(MMPC). These results suggest poly(MMPC) can effectively reduce nonspecific adsorption of a wide range of proteins that are negatively or positively charged at pH 7.4. MMPC is a promising crosslinker for a wide range of polymeric materials requiring low nonspecific protein binding.

  13. Water-Soluble Chlorophyll Protein (WSCP) Stably Binds Two or Four Chlorophylls.

    Science.gov (United States)

    Palm, Daniel M; Agostini, Alessandro; Tenzer, Stefan; Gloeckle, Barbara M; Werwie, Mara; Carbonera, Donatella; Paulsen, Harald

    2017-03-28

    Water-soluble chlorophyll proteins (WSCPs) of class IIa from Brassicaceae form tetrameric complexes containing one chlorophyll (Chl) per apoprotein but no carotenoids. The complexes are remarkably stable toward dissociation and protein denaturation even at 100 °C and extreme pH values, and the Chls are partially protected against photooxidation. There are several hypotheses that explain the biological role of WSCPs, one of them proposing that they function as a scavenger of Chls set free upon plant senescence or pathogen attack. The biochemical properties of WSCP described in this paper are consistent with the protein acting as an efficient and flexible Chl scavenger. At limiting Chl concentrations, the recombinant WSCP apoprotein binds substoichiometric amounts of Chl (two Chls per tetramer) to form complexes that are as stable toward thermal dissociation, denaturation, and photodamage as the fully pigmented ones. If more Chl is added, these two-Chl complexes can bind another two Chls to reach the fully pigmented state. The protection of WSCP Chls against photodamage has been attributed to the apoprotein serving as a diffusion barrier for oxygen, preventing its access to triplet excited Chls and, thus, the formation of singlet oxygen. By contrast, the sequential binding of Chls by WSCP suggests a partially open or at least flexible structure, raising the question of how WSCP photoprotects its Chls without the help of carotenoids.

  14. Myocardial myoglobin release after acute myocardial infarction

    Energy Technology Data Exchange (ETDEWEB)

    Robinson, P S; Saltissi, S; Coltart, D J; Croft, D N [Saint Thomas' Hospital, London (UK)

    1980-03-01

    The magnitude and time course of myoglobin release from the myocardium following infarction was assessed by radioimmunoassay. The assay showed acceptable precision over a working range from 50 to 750 ng cm/sup -3/, provided careful control of the assay temperature was maintained. The use of this radioimmunoassay as an early diagnostic test for infarction and as a potential measure of the extent of necrosis is considered and comparison made with the release of CK-MB, the myocardial specific isoenzyme of creatine kinase. Of the twenty patients studied with myocardial infarction, all had elevated levels of serum myoglobin including those admitted within 3 hours of the onset of pain. In contrast, CK-MB was not detected in the serum within 5 hours of the onset of pain. Peak serum levels of myoglobin (mean 852 +- 365 ng cm/sup -3/) and CK-MB (mean 71 +- 25 mIU cm/sup -3/) were detected at 8-16 hours and 20-24 hours respectively after the onset of pain. A comparison of peak serum levels of myoglobin and CK-MB showed a good correlation (r = 0.84).

  15. Experimental methods in cryogenic spectroscopy: Stark effect measurements in substituted myoglobin

    Science.gov (United States)

    Moran, Bradley M.

    Dawning from well-defined tertiary structure, the active regions of enzymatic proteins exist as specifically tailored electrostatic microenvironments capable of facilitating chemical interaction. The specific influence these charge distributions have on ligand binding dynamics, and their impact on specificity, reactivity, and biological functionality, have yet to be fully understood. A quantitative determination of these intrinsic fields would offer insight towards the mechanistic aspects of protein functionality. This work seeks to investigate the internal molecular electric fields that are present at the oxygen binding site of myoglobin. Experiments are performed at 1 K on samples located within a glassy matrix, using the high-resolution technique spectral hole-burning. The internal electric field distributions can be explored by implementing a unique mathematical treatment for analyzing the effect that externally applied electric fields have on the spectral hole profiles. Precise control of the light field, the temperature, and the externally applied electric field at the site of the sample is crucial. Experimentally, the functionality of custom cryogenic temperature confocal scanning microscope was extended to allow for collection of imaging and spectral data with the ability to modulate the polarization of the light at the sample. Operation of the instrumentation was integrated into a platform allowing for seamless execution of input commands with high temporal inter-instrument resolution for collection of data streams. For the regulated control and cycling of the sample temperature. the thermal characteristics of the research Dewar were theoretically modeled to systematically predict heat flows throughout the system. A high voltage feedthrough for delivering voltages of up to 5000 V to the sample as positioned within the Dewar was developed. The burning of spectral holes with this particular experimental setup is highly repeatable. The quantum mechanical

  16. The oxygen-binding properties of hemocyanin from the mollusk Concholepas concholepas.

    Science.gov (United States)

    González, Andrea; Nova, Esteban; Del Campo, Miguel; Manubens, Augusto; De Ioannes, Alfredo; Ferreira, Jorge; Becker, María Inés

    2017-12-01

    Hemocyanins have highly conserved copper-containing active sites that bind oxygen. However, structural differences among the hemocyanins of various mollusks may affect their physicochemical properties. Here, we studied the oxygen-binding cooperativity and affinity of Concholepas concholepas hemocyanin (CCH) and its two isolated subunits over a wide range of temperatures and pH values. Considering the differences in the quaternary structures of CCH and keyhole limpet hemocyanin (KLH), we hypothesized that the heterodidecameric CCH has different oxygen-binding parameters than the homodidecameric KLH. A novel modification of the polarographic method was applied in which rat liver submitochondrial particles containing cytochrome c oxidase were introduced to totally deplete oxygen of the test solution using ascorbate as the electron donor. This method was both sensitive and reproducible. The results showed that CCH, like other hemocyanins, exhibits cooperativity, showing an inverse relationship between the oxygen-binding parameters and temperature. According to their Hill coefficients, KLH has greater cooperativity than CCH at physiological pH; however, CCH is less sensitive to pH changes than KLH. Appreciable differences in binding behavior were found between the CCH subunits: the cooperativity of CCH-A was not only almost double that of CCH-B, but it was also slightly superior to that of CCH, thus suggesting that the oxygen-binding domains of the CCH subunits are different in their primary structure. Collectively, these data suggest that CCH-A is the main oxygen-binding domain in CCH; CCH-B may play a more structural role, perhaps utilizing its surprising predisposition to form tubular polymers, unlike CCH-A, as demonstrated here using electron microscopy. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Amphitrite ornata dehaloperoxidase (DHP): investigations of structural factors that influence the mechanism of halophenol dehalogenation using "peroxidase-like" myoglobin mutants and "myoglobin-like" DHP mutants.

    Science.gov (United States)

    Du, Jing; Huang, Xiao; Sun, Shengfang; Wang, Chunxue; Lebioda, Lukasz; Dawson, John H

    2011-09-27

    Dehaloperoxidase (DHP), discovered in the marine terebellid polychaete Amphitrite ornata, is the first heme-containing globin with a peroxidase activity. The sequence and crystal structure of DHP argue that it evolved from an ancient O(2) transport and storage globin. Thus, DHP retains an oxygen carrier function but also has the ability to degrade halophenol toxicants in its living environment. Sperm whale myoglobin (Mb) in the ferric state has a peroxidase activity ∼10 times lower than that of DHP. The catalytic activity enhancement observed in DHP appears to have been generated mainly by subtle changes in the positions of the proximal and distal histidine residues that appeared during DHP evolution. Herein, we report investigations into the mechanism of action of DHP derived from examination of "peroxidase-like" Mb mutants and "Mb-like" DHP mutants. The dehalogenation ability of wild-type Mb is augmented in the peroxidase-like Mb mutants (F43H/H64L, G65T, and G65I Mb) but attenuated in the Mb-like T56G DHP variant. X-ray crystallographic data show that the distal His residues in G65T Mb and G65I are positioned ∼0.3 and ∼0.8 Å, respectively, farther from the heme iron compared to that in the wild-type protein. The H93K/T95H double mutant Mb with the proximal His shifted to the "DHP-like" position has an increased peroxidase activity. In addition, a better dehaloperoxidase (M86E DHP) was generated by introducing a negative charge near His89 to enhance the imidazolate character of the proximal His. Finally, only minimal differences in dehalogenation activities are seen among the exogenous ligand-free DHP, the acetate-bound DHP, and the distal site blocker L100F DHP mutant. Thus, we conclude that binding of halophenols in the internal binding site (i.e., distal cavity) is not essential for catalysis. This work provides a foundation for a new structure-function paradigm for peroxidases and for the molecular evolution of the dual-function enzyme DHP.

  18. Amphitrite ornata Dehaloperoxidase (DHP): Investigations of Structural Factors That Influence the Mechanism of Halophenol Dehalogenation Using ;Peroxidase-like; Myoglobin Mutants and ;Myoglobin-like; DHP Mutants

    Energy Technology Data Exchange (ETDEWEB)

    Du, Jing; Huang, Xiao; Sun, Shengfang; Wang, Chunxue; Lebioda, Lukasz; Dawson, John H. (SC)

    2012-05-14

    Dehaloperoxidase (DHP), discovered in the marine terebellid polychaete Amphitrite ornata, is the first heme-containing globin with a peroxidase activity. The sequence and crystal structure of DHP argue that it evolved from an ancient O{sub 2} transport and storage globin. Thus, DHP retains an oxygen carrier function but also has the ability to degrade halophenol toxicants in its living environment. Sperm whale myoglobin (Mb) in the ferric state has a peroxidase activity {approx}10 times lower than that of DHP. The catalytic activity enhancement observed in DHP appears to have been generated mainly by subtle changes in the positions of the proximal and distal histidine residues that appeared during DHP evolution. Herein, we report investigations into the mechanism of action of DHP derived from examination of 'peroxidase-like' Mb mutants and 'Mb-like' DHP mutants. The dehalogenation ability of wild-type Mb is augmented in the peroxidase-like Mb mutants (F43H/H64L, G65T, and G65I Mb) but attenuated in the Mb-like T56G DHP variant. X-ray crystallographic data show that the distal His residues in G65T Mb and G65I are positioned {approx}0.3 and {approx}0.8 {angstrom}, respectively, farther from the heme iron compared to that in the wild-type protein. The H93K/T95H double mutant Mb with the proximal His shifted to the 'DHP-like' position has an increased peroxidase activity. In addition, a better dehaloperoxidase (M86E DHP) was generated by introducing a negative charge near His89 to enhance the imidazolate character of the proximal His. Finally, only minimal differences in dehalogenation activities are seen among the exogenous ligand-free DHP, the acetate-bound DHP, and the distal site blocker L100F DHP mutant. Thus, we conclude that binding of halophenols in the internal binding site (i.e., distal cavity) is not essential for catalysis. This work provides a foundation for a new structure-function paradigm for peroxidases and for the

  19. Imparting albumin-binding affinity to a human protein by mimicking the contact surface of a bacterial binding protein.

    Science.gov (United States)

    Oshiro, Satoshi; Honda, Shinya

    2014-04-18

    Attachment of a bacterial albumin-binding protein module is an attractive strategy for extending the plasma residence time of protein therapeutics. However, a protein fused with such a bacterial module could induce unfavorable immune reactions. To address this, we designed an alternative binding protein by imparting albumin-binding affinity to a human protein using molecular surface grafting. The result was a series of human-derived 6 helix-bundle proteins, one of which specifically binds to human serum albumin (HSA) with adequate affinity (KD = 100 nM). The proteins were designed by transferring key binding residues of a bacterial albumin-binding module, Finegoldia magna protein G-related albumin-binding domain (GA) module, onto the human protein scaffold. Despite 13-15 mutations, the designed proteins maintain the original secondary structure by virtue of careful grafting based on structural informatics. Competitive binding assays and thermodynamic analyses of the best binders show that the binding mode resembles that of the GA module, suggesting that the contacting surface of the GA module is mimicked well on the designed protein. These results indicate that the designed protein may act as an alternative low-risk binding module to HSA. Furthermore, molecular surface grafting in combination with structural informatics is an effective approach for avoiding deleterious mutations on a target protein and for imparting the binding function of one protein onto another.

  20. Oxygen binding to partially nitrosylated hemoglobin.

    Science.gov (United States)

    Fago, Angela; Crumbliss, Alvin L; Hendrich, Michael P; Pearce, Linda L; Peterson, Jim; Henkens, Robert; Bonaventura, Celia

    2013-09-01

    Reactions of nitric oxide (NO) with hemoglobin (Hb) are important elements in protection against nitrosative damage. NO in the vasculature is depleted by the oxidative reaction with oxy Hb or by binding to deoxy Hb to generate partially nitrosylated Hb (Hb-NO). Many aspects of the formation and persistence of Hb-NO are yet to be clarified. In this study, we used a combination of EPR and visible absorption spectroscopy to investigate the interactions of partially nitrosylated Hb with O2. Partially nitrosylated Hb samples had predominantly hexacoordinate NO-heme geometry and resisted oxidation when exposed to O2 in the absence of anionic allosteric effectors. Faster oxidation occurred in the presence of 2,3-diphosphoglycerate (DPG) or inositol hexaphosphate (IHP), where the NO-heme derivatives had higher levels of pentacoordinate heme geometry. The anion-dependence of the NO-heme geometry also affected O2 binding equilibria. O2-binding curves of partially nitrosylated Hb in the absence of anions were left-shifted at low saturations, indicating destabilization of the low O2 affinity T-state of the Hb by increasing percentages of NO-heme, much as occurs with increasing levels of CO-heme. Samples containing IHP showed small decreases in O2 affinity, indicating shifts toward the low-affinity T-state and formation of inert α-NO/β-met tetramers. Most remarkably, O2-equilibria in the presence of the physiological effector DPG were essentially unchanged by up to 30% NO-heme in the samples. As will be discussed, under physiological conditions the interactions of Hb with NO provide protection against nitrosative damage without impairing O2 transport by Hb's unoccupied heme sites. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Structural studies of the 'Aplysia Brasiliana' and 'Dermochelis Coriacea' myoglobins by optical and electron paramagnetic resonance techniques

    International Nuclear Information System (INIS)

    Baffa Filho, O.

    1984-01-01

    The myoglobins of 'Applysia Brasiliana' (MbApB) and of the sea turtle 'Dermochelis Coriacea' (MbT) are studied with special attention devoted to the acid-alkalyne transition (AAT), the interaction with transition metals and temperature induced conformational changes in order to characterize structural differences in these proteins. The AAT of MbApB has a pK = 7.2 obtained from the EPR spectra of Fe 3+ at g (perpendicular) = 5.83 and a pK = 7.5 obtained from optical absorption (lambda = 590 nm). The EPR Spectrum of Fe 3+ at alkalyne pH shows a rhombic distortion of the ion crystal field which is in agreement with the absence in this protein of the distal histidine residue. The ESR lines associated with the low spin configuration are considerably broadened. This effect can be explained by fluctuations on the heme position relative to the symmetry axis. MbApB forms complexes with both Cu 2+ and Mn 2+ only one binding site is obtained for both metals in the protein. This site probably has common ligands for mN 2+ and Cu 2+ as the binding is competitive, suggesting also the at the Cu 2+ complex is more stable than the Mn 2+ one (K sub(A) sup(M) sub(n 2+ )) = (11,5 + - 0,8).10 3 M -1 . (Author) [pt

  2. Moessbauer spectroscopic evidence on the heme binding to the proximal histidine in unfolded carbonmonoxy myoglobin by guanidine hydrochloride

    Energy Technology Data Exchange (ETDEWEB)

    Harami, Taikan, E-mail: harami.taikan@jaea.go.jp [Japan Atomic Energy Agency (Japan); Kitao, Shinji; Kobayashi, Yasuhiro [Kyoto University, Research Reactor Institute (Japan); Mitsui, Takaya [Japan Atomic Energy Agency (Japan)

    2008-01-15

    The unfolded heme structure in myoglobin is controversial because of no chance of direct X-ray structure analyses. The unfolding of carbonmonoxy myoglobin (MbCO) by guanidine hydrochloride (GdnHCl) was studied by the Moessbauer spectroscopy. The spectra show the presence of a sort of spectrum in the unfolded MbCO, independent on the concentration of GdnHCl from 1 to 6 M and the increase of the fraction of unfolded MbCO, depending on the GdnHCl concentration. The isomer shift of the iron of heme in the unfolded MbCO was identified to be different from that of the native MbCO as the globin structure in Mb collapses under the unfolded conditions. This result and the existing related Moessbauer data proved that the heme in the unfolded MbCO may remain coordinated to the proximal histidine.

  3. Protoporphyrin IX-induced structural and functional changes in ...

    Indian Academy of Sciences (India)

    Unknown

    Drug-protein binding; haemoglobin; myoglobin; protoporphyrin IX; red blood cells ... haemoglobin and myoglobin are potentiated by the protein-porphyrin complexation. Possible ...... 1985 Uptake and delivery of the respiratory gases; in Best ...

  4. Diphosphoglycerate and Inosine Hexaphosphate Control of Oxygen Binding by Hemoglobin: A Theoretical Interpretation of Experimental Data*

    Science.gov (United States)

    Ling, Gilbert N.

    1970-01-01

    A theoretical equation is presented for the control of cooperative adsorption on proteins and other linear macromolecules by hormones, drugs, ATP, and other „cardinal adsorbents.” With reasonable accuracy, this equation describes quantitatively the control of oxygen binding to hemoglobin by 2,3-diphosphoglycerate and by inosine hexaphosphate. PMID:5272319

  5. Risk Mitigation during Human Electromuscular Incapacitation Research

    Science.gov (United States)

    2015-04-30

    Research Unit-San Antonio NIJ National Institute of Justice O2 Oxygen pCO2 Partial pressure of carbon dioxide PEA Pulseless electrical...Research Findings Muscle injury can occur before symptoms appear. Blood levels of the proteins , creatine kinase (CK) and myoglobin, are used to...Rhabdomyolysis occurs when the protein myoglobin is released from damaged muscle cells. Myoglobin is injurious to the kidneys. CK is another protein

  6. The occurrence of gibberellin-binding protein(s) in pea

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Z.H.

    1988-01-01

    In vitro gibberellin (GA) binding properties of a cytosol fraction from epicotyls of dwarf pea (Pisum sativum L. cv. Progress No. 9) and tall pea (Pisum sativum L. cv. Alaska) were investigated using ({sup 3}H)GA{sub 4} in a DEAE filter paper assay at 0-3 C. The binding obtained is saturable, reversible, and temperature labile in dwarf pea, and has a half-life of dissociation of 5-6 min. By varying the concentration of ({sup 3}H)GA{sub 4} in the incubation medium the Kd was estimated to be 120-140 nM in dwarf pea and 70 nM in tall pea. The number of binding sites (n) was estimated to be 0.66 and 0.43 pmole mg{sup {minus}1} soluble protein in dwarf pea and in tall pea, respectively. In competition binding assays, biologically active GAs, such as GA{sub 3} and GA{sub 4} could reduce the level of ({sup 3}H)GA{sub 4} binding much more than the biologically inactive GA{sub 4} methyl ester and epi-GA{sub 4}. Changes in gibberellin-binding protein(s) were studied during seed germination. While the Kd of the binding protein(s) for ({sup 3}H)GA{sub 4} remained the same, there was a marked increase in the number of binding sites from 24 h soaked seed to 8-day old seedlings. Also, the Kd and the number of binding sites in the GA-responsive apical part and in the nonresponsive basal part in the epicotyl were similar. The effect of light on gibberellin-binding protein in dwarf pea was also studied. The GA-binding protein in dwarf pea was partially purified by gel filtration and ion exchange chromatography.

  7. Predicting protein-binding RNA nucleotides with consideration of binding partners.

    Science.gov (United States)

    Tuvshinjargal, Narankhuu; Lee, Wook; Park, Byungkyu; Han, Kyungsook

    2015-06-01

    In recent years several computational methods have been developed to predict RNA-binding sites in protein. Most of these methods do not consider interacting partners of a protein, so they predict the same RNA-binding sites for a given protein sequence even if the protein binds to different RNAs. Unlike the problem of predicting RNA-binding sites in protein, the problem of predicting protein-binding sites in RNA has received little attention mainly because it is much more difficult and shows a lower accuracy on average. In our previous study, we developed a method that predicts protein-binding nucleotides from an RNA sequence. In an effort to improve the prediction accuracy and usefulness of the previous method, we developed a new method that uses both RNA and protein sequence data. In this study, we identified effective features of RNA and protein molecules and developed a new support vector machine (SVM) model to predict protein-binding nucleotides from RNA and protein sequence data. The new model that used both protein and RNA sequence data achieved a sensitivity of 86.5%, a specificity of 86.2%, a positive predictive value (PPV) of 72.6%, a negative predictive value (NPV) of 93.8% and Matthews correlation coefficient (MCC) of 0.69 in a 10-fold cross validation; it achieved a sensitivity of 58.8%, a specificity of 87.4%, a PPV of 65.1%, a NPV of 84.2% and MCC of 0.48 in independent testing. For comparative purpose, we built another prediction model that used RNA sequence data alone and ran it on the same dataset. In a 10 fold-cross validation it achieved a sensitivity of 85.7%, a specificity of 80.5%, a PPV of 67.7%, a NPV of 92.2% and MCC of 0.63; in independent testing it achieved a sensitivity of 67.7%, a specificity of 78.8%, a PPV of 57.6%, a NPV of 85.2% and MCC of 0.45. In both cross-validations and independent testing, the new model that used both RNA and protein sequences showed a better performance than the model that used RNA sequence data alone in

  8. Reversible and irreversible conformational transitions in myoglobin: role of hydrated amino acid ionic liquid.

    Science.gov (United States)

    Sankaranarayanan, Kamatchi; Sathyaraj, Gopal; Nair, B U; Dhathathreyan, A

    2012-04-12

    Hydrated phenylalanine ionic liquid (Phe-IL) has been used to solubilize myoglobin (Mb). Structural stability of Mb in Phe-IL analyzed using fluorescence and circular dichroism spectroscopy shows that for low levels of hydration of Phe-IL there is a large red shift in the fluorescence emission wavelength and the protein transforms to complete β sheet from its native helical conformation. Rehydration or dilution reverses the β sheet to an α helix which on aging organizes to micrometer-sized fibrils. At concentrations higher than 200 μM, the protein changes from β to a more random coiled structure. Organization of the protein in Phe-IL in a Langmuir film at the air/water interface has been investigated using the surface pressure-molecular area isotherm and shows nearly the same surface tension for both pure Mb and Mb in Phe-IL. Scanning electron microscopy of the films of Mb in Phe-IL transferred using the Langmuir-Blodgett film technique show layered morphology. This study shows that the conformation of Mb is completely reversible going from β → helix → β sheet up to 200 μM of Phe-IL. Similar surface tension values for Mb in water and in Phe-IL suggests that direct ion binding interactions with the protein coupled with the change in local viscosity from the IL seems to not only alter the secondary structure of individual proteins but also drives the self-assembly of the protein molecules leading finally to fibril formation.

  9. Lipid binding to cytoglobin leads to a change in haem co-ordination: a role for cytoglobin in lipid signalling of oxidative stress.

    Science.gov (United States)

    Reeder, Brandon J; Svistunenko, Dimitri A; Wilson, Michael T

    2011-03-15

    Cytoglobin is a recently discovered hexa-co-ordinate haemoglobin that does not appear to function as a classical oxygen-binding protein. Its function is unknown and studies on the effects of changes in its expression have not decisively determined its role within the cell. In the present paper, we report that the protein is transformed from hexa-co-ordinate to penta-co-ordinate on binding a lipid molecule. This transformation occurs with the ferric oxidation state of the protein, but not the ferrous state, indicating that this process only occurs under an oxidative environment and may thus be related to redox-linked cell signalling mechanisms. Oleate binds to the protein in a 1:1 stoichiometry and with high affinity (K(d)=0.7 μM); however, stopped-flow kinetic measurements yield a K(d) value of 110 μM. The discrepancy between these K(d) values may be rationalized by recognizing that cytoglobin is a disulfide-linked dimer and invoking co-operativity in oleate binding. The lipid-induced transformation of cytoglobin from hexa-co-ordinate to penta-co-ordinate does not occur with similar hexa-co-ordinate haemoglobins such as neuroglobin, and therefore appears to be a unique property of cytoglobin among the haemoglobin superfamily. The lipid-derived transformation may explain why cytoglobin has enhanced peroxidatic activity, converting lipids into various oxidized products, a property virtually absent from neuroglobin and much decreased in myoglobin. We propose that the binding of ferric cytoglobin to lipids and their subsequent transformation may be integral to the physiological function of cytoglobin, generating cell signalling lipid molecules under an oxidative environment.

  10. Photoaffinity labeling of the oxysterol binding protein

    International Nuclear Information System (INIS)

    Taylor, F.R.; Kandutsch, A.A.; Anzalone, L.; Spencer, T.A.

    1986-01-01

    A cytosolic receptor protein for oxygenated sterols, that is thought to be involved in the regulation of HMG-CoA reductase and cholesterol biosynthesis, can be labeled covalently by the photoactivated affinity compound [5,6- 3 H]-7,7'-azocholestane-3β,25-diol (I). Several other compounds were tested including 25-hydroxycholesta-4,6-dien-3-one, 25-azido-27-norcholest-5-en-3β-ol,3β,25-dihydroxycholest-5-en-7-one and 3β-hydroxycholesta-8(14),9(11)-dien-15-one. However, these sterols either did not bind to the receptor with adequate affinity or did not react covalently with the receptor during photolysis. Compound I binds to the receptor with very high affinity (K/sub d/ = 30 nM). After activation with long wavelength UV, two tritium labeled proteins, M/sub r/ approximately 95K and 65K daltons, are found upon SDS gel electrophoresis. No labeling occurs when the binding reaction is carried out in the presence of a large excess of 25-hydroxycholesterol. It is possible that the smaller polypeptide is a degradation product. Under the reaction conditions investigated so far labeling is relatively inefficient (< 1% of bound sterol). These results are generally consistent with previous information suggesting that the M/sub r/ of the receptor subunit is 97,000. Covalent labeling of the receptor should greatly facilitate its further purification and characterization

  11. High-level expression and deuteration of sperm whale myoglobin: A study of its solvent structure by X-ray and neutron diffraction methods

    Energy Technology Data Exchange (ETDEWEB)

    Shu, F. [State Univ. of New York, Stony Brook, NY (United States); Ramakrishnan, V. [Brookhaven National Laboratory, Upton, NY (United States); Schoenborn, B.P. [Los Alamos National Laboratory, NM (United States)

    1994-12-31

    Neutron diffraction has become one of the best ways to study light atoms, such as hydrogens. Hydrogen however has a negative coherent scattering factor, and a large incoherent scattering factor, while deuterium has virtually no incoherent scattering, but a large positive coherent scattering factor. Beside causing high background due to its incoherent scattering, the negative coherent scattering of hydrogen tends to cancel out the positive contribution from other atoms in a neutron density map. Therefore a fully deuterated sample will yield better diffraction data with stronger density in the hydrogen position. On this basis, a sperm whale myoglobin gene modified to include part of the A cII protein gene has been cloned into the T7 expression system. Milligram amounts of fully deuterated holo-myoglobin have been obtained and used for crystallization. The synthetic sperm whale myoglobin crystallized in P2{sub 1} space group isomorphous with the native protein crystal. A complete X-ray diffraction dataset at 1.5{Angstrom} has been collected. This X-ray dataset, and a neutron data set collected previously on a protonated carbon-monoxymyoglobin crystal have been used for solvent structure studies. Both X-ray and neutron data have shown that there are ordered hydration layers around the protein surface. Solvent shell analysis on the neutron data further has shown that the first hydration layer behaves differently around polar and apolar regions of the protein surface. Finally, the structure of per-deuterated myoglobin has been refined using all reflections to a R factor of 17%.

  12. High-level expression and deuteration of sperm whale myoglobin: A study of its solvent structure by X-ray and neutron diffraction methods

    International Nuclear Information System (INIS)

    Shu, F.; Ramakrishnan, V.; Schoenborn, B.P.

    1994-01-01

    Neutron diffraction has become one of the best ways to study light atoms, such as hydrogens. Hydrogen however has a negative coherent scattering factor, and a large incoherent scattering factor, while deuterium has virtually no incoherent scattering, but a large positive coherent scattering factor. Beside causing high background due to its incoherent scattering, the negative coherent scattering of hydrogen tends to cancel out the positive contribution from other atoms in a neutron density map. Therefore a fully deuterated sample will yield better diffraction data with stronger density in the hydrogen position. On this basis, a sperm whale myoglobin gene modified to include part of the A cII protein gene has been cloned into the T7 expression system. Milligram amounts of fully deuterated holo-myoglobin have been obtained and used for crystallization. The synthetic sperm whale myoglobin crystallized in P2 1 space group isomorphous with the native protein crystal. A complete X-ray diffraction dataset at 1.5 Angstrom has been collected. This X-ray dataset, and a neutron data set collected previously on a protonated carbon-monoxymyoglobin crystal have been used for solvent structure studies. Both X-ray and neutron data have shown that there are ordered hydration layers around the protein surface. Solvent shell analysis on the neutron data further has shown that the first hydration layer behaves differently around polar and apolar regions of the protein surface. Finally, the structure of per-deuterated myoglobin has been refined using all reflections to a R factor of 17%

  13. Fragment-based quantum mechanical calculation of protein-protein binding affinities.

    Science.gov (United States)

    Wang, Yaqian; Liu, Jinfeng; Li, Jinjin; He, Xiao

    2018-04-29

    The electrostatically embedded generalized molecular fractionation with conjugate caps (EE-GMFCC) method has been successfully utilized for efficient linear-scaling quantum mechanical (QM) calculation of protein energies. In this work, we applied the EE-GMFCC method for calculation of binding affinity of Endonuclease colicin-immunity protein complex. The binding free energy changes between the wild-type and mutants of the complex calculated by EE-GMFCC are in good agreement with experimental results. The correlation coefficient (R) between the predicted binding energy changes and experimental values is 0.906 at the B3LYP/6-31G*-D level, based on the snapshot whose binding affinity is closest to the average result from the molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) calculation. The inclusion of the QM effects is important for accurate prediction of protein-protein binding affinities. Moreover, the self-consistent calculation of PB solvation energy is required for accurate calculations of protein-protein binding free energies. This study demonstrates that the EE-GMFCC method is capable of providing reliable prediction of relative binding affinities for protein-protein complexes. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-07-01

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

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

    International Nuclear Information System (INIS)

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

    2014-01-01

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

  16. Radiation damage to DNA-binding proteins

    International Nuclear Information System (INIS)

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

    2003-01-01

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

  17. A structural classification of substrate-binding proteins

    NARCIS (Netherlands)

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

    2010-01-01

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

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

    Science.gov (United States)

    Piłat, Zofia; Antosiewicz, Jan M

    2008-11-27

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

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

    International Nuclear Information System (INIS)

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

    1989-01-01

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

  20. Organization of chlorophyll biosynthesis and insertion of chlorophyll into the chlorophyll-binding proteins in chloroplasts.

    Science.gov (United States)

    Wang, Peng; Grimm, Bernhard

    2015-12-01

    Oxygenic photosynthesis requires chlorophyll (Chl) for the absorption of light energy, and charge separation in the reaction center of photosystem I and II, to feed electrons into the photosynthetic electron transfer chain. Chl is bound to different Chl-binding proteins assembled in the core complexes of the two photosystems and their peripheral light-harvesting antenna complexes. The structure of the photosynthetic protein complexes has been elucidated, but mechanisms of their biogenesis are in most instances unknown. These processes involve not only the assembly of interacting proteins, but also the functional integration of pigments and other cofactors. As a precondition for the association of Chl with the Chl-binding proteins in both photosystems, the synthesis of the apoproteins is synchronized with Chl biosynthesis. This review aims to summarize the present knowledge on the posttranslational organization of Chl biosynthesis and current attempts to envision the proceedings of the successive synthesis and integration of Chl into Chl-binding proteins in the thylakoid membrane. Potential auxiliary factors, contributing to the control and organization of Chl biosynthesis and the association of Chl with the Chl-binding proteins during their integration into photosynthetic complexes, are discussed in this review.

  1. Modifications of hemoglobin and myoglobin by Maillard reaction products (MRPs).

    Science.gov (United States)

    Ioannou, Aristos; Varotsis, Constantinos

    2017-01-01

    High performance liquid chromatography (HPLC) coupled with a Fraction Collector was employed to isolate Maillard reaction products (MRPs) formed in model systems comprising of asparagine and monosaccharides in the 60-180°C range. The primary MRP which is detected at 60°C is important for Acrylamide content and color/aroma development in foods and also in the field of food biotechnology for controlling the extent of the Maillard reaction with temperature. The discrete fractions of the reaction products were reacted with Hemoglobin (Hb) and Myoglobin (Mb) at physiological conditions and the reaction adducts were monitored by UV-vis and Attenuated Total Reflection-Fourier transform infrared (FTIR) spectrophotometry. The UV-vis kinetic profiles revealed the formation of a Soret transition characteristic of a low-spin six-coordinated species and the ATR-FTIR spectrum of the Hb-MRP and Mb-MRP fractions showed modifications in the protein Amide I and II vibrations. The UV-vis and the FTIR spectra of the Hb-MRPs indicate that the six-coordinated species is a hemichrome in which the distal E7 Histidine is coordinated to the heme Fe and blocks irreversibly the ligand binding site. Although the Mb-MRPs complex is a six-coordinated species, the 1608 cm-1 FTIR band characteristic of a hemichrome was not observed.

  2. When is protein binding important?

    Science.gov (United States)

    Heuberger, Jules; Schmidt, Stephan; Derendorf, Hartmut

    2013-09-01

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

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

    DEFF Research Database (Denmark)

    Birn, Henrik; Zhai, Xiaoyue; Holm, Jan

    2005-01-01

    Folate is an essential vitamin involved in a number of biological processes. High affinity folate binding proteins (FBPs) exist both as glycosylphosphatidylinositol-linked, membrane associated folate binding proteins and as soluble FBPs in plasma and some secretory fluids such as milk, saliva...... to express high levels of megalin, is inhibitable by excess unlabeled FBP and by receptor associated protein, a known inhibitor of binding to megalin. Immortalized rat yolk sac cells, representing an established model for studying megalin-mediated uptake, reveal (125)I-labeled FBP uptake which is inhibited...

  4. Protein binding of psychotropic agents

    International Nuclear Information System (INIS)

    Hassan, H.A.

    1990-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Waqasuddin Khan

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

  6. Extracellular and intracellular steroid binding proteins

    International Nuclear Information System (INIS)

    Wagner, R.K.

    1978-01-01

    Steroid hormone binding proteins can be measured, after the removal of endogenous steroids, as specific complexes with radio-labelled hormones. In this study all the requirements for a quantitative determination of steroid hormone binding proteins are defined. For different methods, agargel electrophoresis, density gradient centrifugation, equilibrium dialysis and polyacrylamide electrophoresis have been evaluated. Agar electrophoresis at low temperature was found to be the simplest and most useful procedure. With this method the dissociation rates of high affinity complexes can be assessed and absolute binding protein concentrations can be determined. The dissociation rates of the oestradiol-oestrogen receptor complex and the R-5020-progestin receptor complex are low (1-2% per h run time.) In contrast, that of complexes between androgen receptor and dihydrotestosterone (17β-hydroxy-5α-androstan-3-one (DHT), progestin receptor and progesterone, corticosteroid binding globulin (CBG) and cortisol or progesterone and sex hormone binding globulin (SHBG) and DHT were hign (16-27% per h run time). Target tissue extracts (cytosols) contain, besides soluble tissue proteins, large amounts of plasma proteins. The extent of this plasma contamination can be determined by measuring the albumin concentration in cytosols by immunodiffusion. In cytosols of 4 different human target tissues the albumin content varied from 20-30% corresponding to an even higher whole plasma concentration. Steroid binding plasma proteins, such as CBG and SHBG are constituents of this containment. (author)

  7. Clinical significance of myoglobinuria and serum myoglobin in heroin-addicted patients

    International Nuclear Information System (INIS)

    Chen Xuehong; Zhong Ganping; Zhang Lan; Liu Jiangyan

    2001-01-01

    The authors study the relationship between myoglobinuria and acute rhabdomyolysis in heroin-addicted patients. The levels of myoglobin in serum and urine were determined by RIA in 106 heroin-addicted patients and 30 healthy volunteers who were selected as the controls. The levels of myoglobin in serum and urine increased significantly in heroin-addicted patients in 3 days after giving up heroin, and gradually decreased in 2 weeks but still higher than the levels of the controls (P 0.05). Urine myoglobin detection is a simple and effective method to find out acute rhabdomyolysis derived from heroin addiction early

  8. Evidence for glycosylation on a DNA-binding protein of Salmonella enterica

    Directory of Open Access Journals (Sweden)

    Almeida Igor C

    2007-04-01

    Full Text Available Abstract Background All organisms living under aerobic atmosphere have powerful mechanisms that confer their macromolecules protection against oxygen reactive species. Microorganisms have developed biomolecule-protecting systems in response to starvation and/or oxidative stress, such as DNA biocrystallization with Dps (DNA-binding protein from starved cells. Dps is a protein that is produced in large amounts when the bacterial cell faces harm, which results in DNA protection. In this work, we evaluated the glycosylation in the Dps extracted from Salmonella enterica serovar Typhimurium. This Dps was purified from the crude extract as an 18-kDa protein, by means of affinity chromatography on an immobilized jacalin column. Results The N-terminal sequencing of the jacalin-bound protein revealed 100% identity with the Dps of S. enterica serovar Typhimurium. Methyl-alpha-galactopyranoside inhibited the binding of Dps to jacalin in an enzyme-linked lectin assay, suggesting that the carbohydrate recognition domain (CRD of jacalin is involved in the interaction with Dps. Furthermore, monosaccharide compositional analysis showed that Dps contained mannose, glucose, and an unknown sugar residue. Finally, jacalin-binding Dps was detected in larger amounts during the bacterial earlier growth periods, whereas high detection of total Dps was verified throughout the bacterial growth period. Conclusion Taken together, these results indicate that Dps undergoes post-translational modifications in the pre- and early stationary phases of bacterial growth. There is also evidence that a small mannose-containing oligosaccharide is linked to this bacterial protein.

  9. Structural analysis of peptides capable of binding to more than one Ia antigen

    DEFF Research Database (Denmark)

    Sette, A; Buus, S; Colon, S

    1989-01-01

    The Ia binding regions were analyzed for three unrelated peptide Ag (sperm whale myoglobin 106-118, influenza hemagglutinin 130-142, and lambda repressor protein 12-26) for which binding to more than one Ia molecule has previously been demonstrated. By determining the binding profile of three...... separate series of truncated synthetic peptides, it was found that in all three cases the different Ia reactivities mapped to largely overlapping regions of the peptides; although, for two of the peptides, the regions involved in binding the different Ia specificities were distinct. Moreover, subtle...... differences were found to dramatically influence some, but not other, Ia reactivities. Using a large panel of synthetic peptides it was found that a significant correlation exists between the capacity of peptides to interact with different alleles of the same molecule (i.e., IAd and IAk), but no correlation...

  10. Partial characterization of GTP-binding proteins in Neurospora

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  11. Guardian of Genetic Messenger-RNA-Binding Proteins

    Directory of Open Access Journals (Sweden)

    Antje Anji

    2016-01-01

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

  12. Fatty acid binding protein 4 deficiency protects against oxygen-induced retinopathy in mice.

    Directory of Open Access Journals (Sweden)

    Magali Saint-Geniez

    Full Text Available Retinopathy of prematurity (ROP is a leading cause of blindness in children worldwide due to increasing survival rates of premature infants. Initial suppression, followed by increased production of the retinal vascular endothelial growth factor-A (VEGF expression are key events that trigger the pathological neovascularization in ROP. Fatty acid binding protein 4 (FABP4 is an intracellular lipid chaperone that is induced by VEGF in a subset of endothelial cells. FABP4 exhibits a pro-angiogenic function in cultured endothelial cells and in airway microvasculature, but whether it plays a role in modulation of retinal angiogenesis is not known. We hypothesized that FABP4 deficiency could ameliorate pathological retinal vascularization and investigated this hypothesis using a well-characterized mouse model of oxygen-induced retinopathy (OIR. We found that FABP4 was not expressed in retinal vessels, but was present in resident macrophages/microglial cells and endothelial cells of the hyaloid vasculature in the immature retina. While FABP4 expression was not required for normal development of retinal vessels, FABP4 expression was upregulated and localized to neovascular tufts in OIR. FABP4-/- mice demonstrated a significant decrease in neovessel formation as well as a significant improvement in physiological revascularization of the avascular retinal tissues. These alterations in retinal vasculature were accompanied by reduced endothelial cell proliferation, but no effect on apoptosis or macrophage/microglia recruitment. FABP4-/- OIR samples demonstrated decreased expression of genes involved in angiogenesis, such as Placental Growth Factor, and angiopoietin 2. Collectively, our findings suggest FABP4 as a potential target of pathologic retinal angiogenesis in proliferative retinopathies.

  13. Polymeric competitive protein binding adsorbents for radioassay

    International Nuclear Information System (INIS)

    Adams, R.J.

    1976-01-01

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

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

    Science.gov (United States)

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

    2017-09-01

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

  15. Modifications of hemoglobin and myoglobin by Maillard reaction products (MRPs.

    Directory of Open Access Journals (Sweden)

    Aristos Ioannou

    Full Text Available High performance liquid chromatography (HPLC coupled with a Fraction Collector was employed to isolate Maillard reaction products (MRPs formed in model systems comprising of asparagine and monosaccharides in the 60-180°C range. The primary MRP which is detected at 60°C is important for Acrylamide content and color/aroma development in foods and also in the field of food biotechnology for controlling the extent of the Maillard reaction with temperature. The discrete fractions of the reaction products were reacted with Hemoglobin (Hb and Myoglobin (Mb at physiological conditions and the reaction adducts were monitored by UV-vis and Attenuated Total Reflection-Fourier transform infrared (FTIR spectrophotometry. The UV-vis kinetic profiles revealed the formation of a Soret transition characteristic of a low-spin six-coordinated species and the ATR-FTIR spectrum of the Hb-MRP and Mb-MRP fractions showed modifications in the protein Amide I and II vibrations. The UV-vis and the FTIR spectra of the Hb-MRPs indicate that the six-coordinated species is a hemichrome in which the distal E7 Histidine is coordinated to the heme Fe and blocks irreversibly the ligand binding site. Although the Mb-MRPs complex is a six-coordinated species, the 1608 cm-1 FTIR band characteristic of a hemichrome was not observed.

  16. Factor VII and protein C are phosphatidic acid-binding proteins.

    Science.gov (United States)

    Tavoosi, Narjes; Smith, Stephanie A; Davis-Harrison, Rebecca L; Morrissey, James H

    2013-08-20

    Seven proteins in the human blood clotting cascade bind, via their GLA (γ-carboxyglutamate-rich) domains, to membranes containing exposed phosphatidylserine (PS), although with membrane binding affinities that vary by 3 orders of magnitude. Here we employed nanodiscs of defined phospholipid composition to quantify the phospholipid binding specificities of these seven clotting proteins. All bound preferentially to nanobilayers in which PS headgroups contained l-serine versus d-serine. Surprisingly, however, nanobilayers containing phosphatidic acid (PA) bound substantially more of two of these proteins, factor VIIa and activated protein C, than did equivalent bilayers containing PS. Consistent with this finding, liposomes containing PA supported higher proteolytic activity by factor VIIa and activated protein C toward their natural substrates (factors X and Va, respectively) than did PS-containing liposomes. Moreover, treating activated human platelets with phospholipase D enhanced the rates of factor X activation by factor VIIa in the presence of soluble tissue factor. We hypothesize that factor VII and protein C bind preferentially to the monoester phosphate of PA because of its accessibility and higher negative charge compared with the diester phosphates of most other phospholipids. We further found that phosphatidylinositol 4-phosphate, which contains a monoester phosphate attached to its myo-inositol headgroup, also supported enhanced enzymatic activity of factor VIIa and activated protein C. We conclude that factor VII and protein C bind preferentially to monoester phosphates, which may have implications for the function of these proteases in vivo.

  17. Comparison of Creatine Kinase Activity and Myoglobin Blood Level in Acute Myocardial Infarction Patients

    Directory of Open Access Journals (Sweden)

    Sabaheta Hasić

    2008-02-01

    Full Text Available The aim of this prospective study was to evaluate and compare the relative increase of serum myoglobin level and total creatine kinase(CK activity in acute myocardial infarction (AMI patients (n=36. We measured serial changes in total CK activity and myoglobin serum level in three-time periods (6-9 hours, 24 hours and 6-7 days from chest pains onset. Myoglobin peaked during the first 6-9 hours but total CK reached its peak activity after 24 hours from AMI symptoms onset. Results of this study showed that as non-specific cardiac marker myoglobin had better sensitivity and earlier rise in serum than total CK activity in AMI patients. Rapid kinetic of myoglobin level is important for its utility as marker for re-infarction diagnosis. Early myoglobin increase in serum is important for early triage of AMI patients and early "ruling out" of AMI diagnosis if there is no evidence of its elevation in circulation.

  18. GTP-binding proteins in rat liver nuclear envelopes

    International Nuclear Information System (INIS)

    Rubins, J.B.; Benditt, J.O.; Dickey, B.F.; Riedel, N.

    1990-01-01

    Nuclear transport as well as reassembly of the nuclear envelope (NE) after completion of mitosis are processes that have been shown to require GTP and ATP. To study the presence and localization of GTP-binding proteins in the NE, we have combined complementary techniques of [alpha-32P]GTP binding to Western-blotted proteins and UV crosslinking of [alpha-32P]GTP with well-established procedures for NE subfractionation. GTP binding to blotted NE proteins revealed five low molecular mass GTP-binding proteins of 26, 25, 24.5, 24, and 23 kDa, and [alpha-32P]GTP photoaffinity labeling revealed major proteins with apparent molecular masses of 140, 53, 47, 33, and 31 kDa. All GTP-binding proteins appear to localize preferentially to the inner nuclear membrane, possibly to the interface between inner nuclear membrane and lamina. Despite the evolutionary conservation between the NE and the rough endoplasmic reticulum, the GTP-binding proteins identified differed between these two compartments. Most notably, the 68- and 30-kDa GTP-binding subunits of the signal recognition particle receptor, which photolabeled with [alpha-32P]GTP in the rough endoplasmic reticulum fraction, were totally excluded from the NE fraction. Conversely, a major 53-kDa photolabeled protein in the NE was absent from rough endoplasmic reticulum. Whereas Western-blotted NE proteins bound GTP specifically, all [alpha-32P]GTP photolabeled proteins could be blocked by competition with ATP, although with a competition profile that differed from that obtained with GTP. In comparative crosslinking studies with [alpha-32P]ATP, we have identified three specific ATP-binding proteins with molecular masses of 160, 78, and 74 kDa. The localization of GTP- and ATP-binding proteins within the NE appears appropriate for their involvement in nuclear transport and in the GTP-dependent fusion of nuclear membranes

  19. Cobalamin and its binding protein in rat milk

    DEFF Research Database (Denmark)

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

    1989-01-01

    Cobalamin and its binding protein, haptocorrin, are present in rat milk throughout the lactation period. The concentration of cobalamin is approximately 0.3-times the concentration of the unsaturated binding protein. The concentration of the unsaturated cobalamin-binding protein varies between 18...

  20. Gonadal cell surface receptor for plasma retinol-binding protein

    International Nuclear Information System (INIS)

    Krishna Bhat, M.; Cama, H.R.

    1979-01-01

    A specific membrane receptor for plasma retinol-binding protein has been demonstrated in testicular cells. Prealbumin-2 did not show any specific binding to the membrane. The affinity of retinol-binding protein for receptor drastically decreases upon delivery of retinol and the retinol-binding protein does not enter the cell. The mechanism of delivery of retinol to the target cell by plasma retinol-binding protein has been investigated. The process involves two steps; direct binding of retinol-binding protein to the receptor and uptake of retinol by the target cell with a concomitant drastic reduction in the affinity of the retinol-binding protein to the receptor. Probably the second step of the process needs a cytosolic factor, possibly the cellular retinol-binding protein or an enzyme. The binding of retinol-binding protein to the receptor is saturable and reversible. The interaction shows a Ksub(d) value of 2.1x10 -10 . The specific binding of a retinol-binding protein with great affinity has been employed in the development of a method for radioassay of the receptor. The receptor level of the gonadal cell has been found to vary with the stage of differentiation. The receptor concentrations in 11-week-old birds and adult birds are comparable. Testosterone treatment of 11-week-old birds produced a substantial increase in the receptor concentration over control, while the protein content increased marginally, indicating that, probably, synthesis of the receptor is specifcally induced by testosterone during spermatogenesis, and the concentration of receptor is relatively higher before the formation of the acrosome. (Auth.)

  1. A Specific Peptide with Calcium-Binding Capacity from Defatted Schizochytrium sp. Protein Hydrolysates and the Molecular Properties

    Directory of Open Access Journals (Sweden)

    Xixi Cai

    2017-03-01

    Full Text Available Marine microorganisms have been proposed as a new kind of protein source. Efforts are needed in order to transform the protein-rich biological wastes left after lipid extraction into value-added bio-products. Thus, the utilization of protein recovered from defatted Schizochytrium sp. by-products presents an opportunity. A specific peptide Tyr-Leu (YL with calcium-binding capacity was purified from defatted Schizochytrium sp. protein hydrolysates through gel filtration chromatography and RP-HPLC. The calcium-binding activity of YL reached 126.34 ± 3.40 μg/mg. The calcium-binding mechanism was investigated through ultraviolet, fluorescence and infrared spectroscopy. The results showed that calcium ions could form dative bonds with carboxyl oxygen atoms and amino nitrogen atoms as well as the nitrogen and oxygen atoms of amide bonds. YL-Ca exhibited excellent thermal stability and solubility, which was beneficial for its absorption and transport in the basic intestinal tract of the human body. Moreover, the cellular uptake of calcium in Caco-2 cells showed that YL-Ca could enhance calcium uptake efficiency and protect calcium ions against precipitation caused by dietary inhibitors such as tannic acid, oxalate, phytate and metal ions. The findings indicate that the by-product of Schizochytrium sp. is a promising source for making peptide-calcium bio-products as algae-based functional supplements for human beings.

  2. A Specific Peptide with Calcium-Binding Capacity from Defatted Schizochytrium sp. Protein Hydrolysates and the Molecular Properties.

    Science.gov (United States)

    Cai, Xixi; Yang, Qian; Lin, Jiaping; Fu, Nanyan; Wang, Shaoyun

    2017-03-29

    Marine microorganisms have been proposed as a new kind of protein source. Efforts are needed in order to transform the protein-rich biological wastes left after lipid extraction into value-added bio-products. Thus, the utilization of protein recovered from defatted Schizochytrium sp. by-products presents an opportunity. A specific peptide Tyr-Leu (YL) with calcium-binding capacity was purified from defatted Schizochytrium sp. protein hydrolysates through gel filtration chromatography and RP-HPLC. The calcium-binding activity of YL reached 126.34 ± 3.40 μg/mg. The calcium-binding mechanism was investigated through ultraviolet, fluorescence and infrared spectroscopy. The results showed that calcium ions could form dative bonds with carboxyl oxygen atoms and amino nitrogen atoms as well as the nitrogen and oxygen atoms of amide bonds. YL-Ca exhibited excellent thermal stability and solubility, which was beneficial for its absorption and transport in the basic intestinal tract of the human body. Moreover, the cellular uptake of calcium in Caco-2 cells showed that YL-Ca could enhance calcium uptake efficiency and protect calcium ions against precipitation caused by dietary inhibitors such as tannic acid, oxalate, phytate and metal ions. The findings indicate that the by-product of Schizochytrium sp. is a promising source for making peptide-calcium bio-products as algae-based functional supplements for human beings.

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

    DEFF Research Database (Denmark)

    Mandrup, S; Hummel, R; Ravn, S

    1992-01-01

    Acyl-CoA-binding protein (ACBP) is a 10 kDa protein isolated from bovine liver by virtue of its ability to bind and induce the synthesis of medium-chain acyl-CoA esters. Surprisingly, it turned out to be identical to a protein named diazepam-binding Inhibitor (DBI) claimed to be an endogenous mod...... have molecularly cloned and characterized the ACBP/DBI gene family in rat. The rat ACBP/DBI gene family comprises one expressed gene and four processed pseudogenes of which one was shown to exist in two allelic forms. The expressed gene is organized into four exons and three introns...

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

    NARCIS (Netherlands)

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

    1993-01-01

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

  5. Neuroglobin in the rat brain: localization

    DEFF Research Database (Denmark)

    Hundahl, Christian Ansgar; Allen, Gregg C; Nyengaard, Jens Randel

    2008-01-01

    Neuroglobin (Ngb) is a neuronal hemeprotein similar to myoglobin and hemoglobin and shares their capability for oxygen binding. It has thus been proposed that Ngb acts as an oxygen reservoir or combats reactive oxygen species. In the present study, we investigated the Ngb expression pattern in th...

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

    Science.gov (United States)

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

    2003-01-01

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

  7. Cardiac Troponin I, Creatine Phosphokinase and Myoglobine Levels in Preeclampsia

    Directory of Open Access Journals (Sweden)

    Ahmet Kale

    2005-01-01

    Full Text Available To evaluate minor myocardial injury in preeclamptic pregnancies by serum markers of cardiac troponin-I, creatine phosphokinase and myoglobine. Group I consisted of 45 preeclamptic pregnancies, Group 2 consisted of uncomplicated pregnancies. The groups were compared for maternal age, parity, mean troponin–I, creatine phosphokinase and myoglobine values. Student-t test were used in statistical analyses. Significance was accepted as p<0.05. Cardiac troponin-I levels were statistically significantly higher in preeclamptic pregnancies (0,97 ± 0,11ng/ml than control groups (0,12 ± 0.09 ng/ml (p<0.001. No statistically significant difference was found with mean levels of creatine phosphokinase and myoglobin levels between two groups. Higher values of troponin-I’in preeclamptic patients is thought to be a result of myocardial injury and associated with pregnancy-induced hypertension.

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

    International Nuclear Information System (INIS)

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

    1991-01-01

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

  9. Oxygen dependency of epidermal growth factor receptor binding and DNA synthesis of rat hepatocytes

    International Nuclear Information System (INIS)

    Hirose, Tetsuro; Terajima, Hiroaki; Yamauchi, Akira

    1997-01-01

    Background/Aims: Changes in oxygen availability modulate replicative responses in several cell types, but the effects on hepatocyte replication remain unclear. We have studied the effects of transient nonlethal hypoxia on epidermal growth factor receptor binding and epidermal growth factor-induced DNA synthesis of rat hepatocytes. Methods: Lactate dehydrogenase activity in culture supernatant, intracellular adenosine triphosphate content, 125 I-epidermal growth factor specific binding, epidermal growth factor receptor protein expression, and 3 H-thymidine incorporation were compared between hepatocytes cultured in hypoxia and normoxia. Results: Hypoxia up to 3 h caused no significant increase in lactate dehydrogenase activity in the culture supernatant, while intracellular adenosine triphosphate content decreased time-dependently and was restored to normoxic levels by reoxygenation (nonlethal hypoxia). Concomitantly, 125 I-epidermal growth factor specific binding to hepatocytes decreased time-dependently (to 54.1% of normoxia) and was restored to control levels by reoxygenation, although 125 I-insulin specific binding was not affected. The decrease in 125 I-epidermal growth factor specific binding was explained by the decrease in the number or available epidermal growth factor receptors (21.37±3.08 to 12.16±1.42 fmol/10 5 cells), while the dissociation constant of the receptor was not affected. The change in the number of available receptors was not considered to be due to receptor degradation-resynthesis, since immuno-detection of the epidermal growth factor receptor revealed that the receptor protein expression did not change during hypoxia and reoxygenation, and since neither actinomycin D nor cycloheximide affected the recovery of 125 I-epidermal growth factor binding by reoxygenation. Inhibition of epidermal growth factor-induced DNA synthesis after hypoxia (to 75.4% of normoxia by 3 h hypoxia) paralleled the decrease in 125 I-epidermal growth factor binding

  10. Localization of cellular retinol-binding protein and retinol-binding protein in cells comprising the blood-brain barrier of rat and human

    International Nuclear Information System (INIS)

    MacDonald, P.N.; Ong, D.E.; Bok, D.

    1990-01-01

    Brain is not generally recognized as an organ that requires vitamin A, perhaps because no obvious histologic lesions have been observed in severely vitamin A-deficient animals. However, brain tissue does contain cellular vitamin A-binding proteins and a nuclear receptor protein for retinoic acid. In the present study, immunohistochemical techniques were used to determine the cell-specific location of cellular retinol-binding protein in human and rat brain tissue. Cellular retinol-binding protein was localized specifically within the cuboidal epithelial cells of the choroid plexus, two primary sites of the mammalian blood-brain barrier. In addition, autoradiographic procedures demonstrated binding sites for serum retinol-binding protein in the choroidal epithelium. These observations suggest that a significant movement of retinol across the blood-brain barrier may occur

  11. CC1, a novel crenarchaeal DNA binding protein.

    Science.gov (United States)

    Luo, Xiao; Schwarz-Linek, Uli; Botting, Catherine H; Hensel, Reinhard; Siebers, Bettina; White, Malcolm F

    2007-01-01

    The genomes of the related crenarchaea Pyrobaculum aerophilum and Thermoproteus tenax lack any obvious gene encoding a single-stranded DNA binding protein (SSB). SSBs are essential for DNA replication, recombination, and repair and are found in all other genomes across the three domains of life. These two archaeal genomes also have only one identifiable gene encoding a chromatin protein (the Alba protein), while most other archaea have at least two different abundant chromatin proteins. We performed a biochemical screen for novel nucleic acid binding proteins present in cell extracts of T. tenax. An assay for proteins capable of binding to a single-stranded DNA oligonucleotide resulted in identification of three proteins. The first protein, Alba, has been shown previously to bind single-stranded DNA as well as duplex DNA. The two other proteins, which we designated CC1 (for crenarchaeal chromatin protein 1), are very closely related to one another, and homologs are restricted to the P. aerophilum and Aeropyrum pernix genomes. CC1 is a 6-kDa, monomeric, basic protein that is expressed at a high level in T. tenax. This protein binds single- and double-stranded DNAs with similar affinities. These properties are consistent with a role for CC1 as a crenarchaeal chromatin protein.

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

    Directory of Open Access Journals (Sweden)

    Stormo Gary D

    2005-07-01

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

  13. Hydration behavior at the ice-binding surface of the Tenebrio molitor antifreeze protein.

    Science.gov (United States)

    Midya, Uday Sankar; Bandyopadhyay, Sanjoy

    2014-05-08

    Molecular dynamics (MD) simulations have been carried out at two different temperatures (300 and 220 K) to study the conformational rigidity of the hyperactive Tenebrio molitor antifreeze protein (TmAFP) in aqueous medium and the structural arrangements of water molecules hydrating its surface. It is found that irrespective of the temperature the ice-binding surface (IBS) of the protein is relatively more rigid than its nonice-binding surface (NIBS). The presence of a set of regularly arranged internally bound water molecules is found to play an important role in maintaining the flat rigid nature of the IBS. Importantly, the calculations reveal that the strategically located hydroxyl oxygens of the threonine (Thr) residues in the IBS influence the arrangements of five sets of ordered waters around it on two parallel planes that closely resemble the basal plane of ice. As a result, these waters can register well with the ice basal plane, thereby allowing the IBS to preferentially bind at the ice interface and inhibit its growth. This provides a possible molecular reason behind the ice-binding activity of TmAFP at the basal plane of ice.

  14. Hemoglobin and Myoglobin as Reducing Agents in Biological Systems. Redox Reactions of Globins with Copper and Iron Salts and Complexes.

    Science.gov (United States)

    Postnikova, G B; Shekhovtsova, E A

    2016-12-01

    In addition to reversible O2 binding, respiratory proteins of the globin family, hemoglobin (Hb) and myoglobin (Mb), participate in redox reactions with various metal complexes, including biologically significant ones, such as those of copper and iron. HbO 2 and MbO 2 are present in cells in large amounts and, as redox agents, can contribute to maintaining cell redox state and resisting oxidative stress. Divalent copper complexes with high redox potentials (E 0 , 200-600 mV) and high stability constants, such as [Cu(phen) 2 ] 2+ , [Cu(dmphen) 2 ] 2+ , and CuDTA oxidize ferrous heme proteins by the simple outer-sphere electron transfer mechanism through overlapping π-orbitals of the heme and the copper complex. Weaker oxidants, such as Cu2+, CuEDTA, CuNTA, CuCit, CuATP, and CuHis (E 0 ≤ 100-150 mV) react with HbO 2 and MbO 2 through preliminary binding to the protein with substitution of the metal ligands with protein groups and subsequent intramolecular electron transfer in the complex (the site-specific outer-sphere electron transfer mechanism). Oxidation of HbO 2 and MbO 2 by potassium ferricyanide and Fe(3) complexes with NTA, EDTA, CDTA, ATP, 2,3-DPG, citrate, and pyrophosphate PP i proceeds mainly through the simple outer-sphere electron transfer mechanism via the exposed heme edge. According to Marcus theory, the rate of this reaction correlates with the difference in redox potentials of the reagents and their self-exchange rates. For charged reagents, the reaction may be preceded by their nonspecific binding to the protein due to electrostatic interactions. The reactions of LbO 2 with carboxylate Fe complexes, unlike its reactions with ferricyanide, occur via the site-specific outer-sphere electron transfer mechanism, even though the same reagents oxidize structurally similar MbO 2 and cytochrome b 5 via the simple outer-sphere electron transfer mechanism. Of particular biological interest is HbO 2 and MbO 2 transformation into met-forms in the presence

  15. Efficient identification of phosphatidylserine-binding proteins by ORF phage display

    International Nuclear Information System (INIS)

    Caberoy, Nora B.; Zhou, Yixiong; Alvarado, Gabriela; Fan, Xianqun; Li, Wei

    2009-01-01

    To efficiently elucidate the biological roles of phosphatidylserine (PS), we developed open-reading-frame (ORF) phage display to identify PS-binding proteins. The procedure of phage panning was optimized with a phage clone expressing MFG-E8, a well-known PS-binding protein. Three rounds of phage panning with ORF phage display cDNA library resulted in ∼300-fold enrichment in PS-binding activity. A total of 17 PS-binding phage clones were identified. Unlike phage display with conventional cDNA libraries, all 17 PS-binding clones were ORFs encoding 13 real proteins. Sequence analysis revealed that all identified PS-specific phage clones had dimeric basic amino acid residues. GST fusion proteins were expressed for 3 PS-binding proteins and verified for their binding activity to PS liposomes, but not phosphatidylcholine liposomes. These results elucidated previously unknown PS-binding proteins and demonstrated that ORF phage display is a versatile technology capable of efficiently identifying binding proteins for non-protein molecules like PS.

  16. Characterization of a cocaine binding protein in human placenta

    International Nuclear Information System (INIS)

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

    1990-01-01

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

  17. Radioimmunoassay of serum myoglobin and its signifance for diagnosis and therapy of musculoskeletal diseases

    International Nuclear Information System (INIS)

    Kiessling, W.R.

    1982-01-01

    A commercial test kit for radioimmunologic proof of myoglobin in serum was tested with regard to its specificty, sensitivity, precision, reproducibility, recovery and use in the diagnosis and therapy of musculoskeletal diseases. In the serum of 164 healthy control persons (age: 2-79 years) the individual myoglobin concentrations ranged from 4 to 60 ng/ml. Among 300 patients with muscular diseases extreme myoglobinaemia in acute rhabdomyolysis, polymyositis and dermatomyositis and different progressive muscular dystrophies could be detected. Slightly increased myoglobin concentrations could be proved in a number of patient with amyotrophic lateral sklerosis, neural muscular atrophy and in all cases of spinal muscular atrophy of the Kugelberg-Welander type. Confirmed DMD patients exhibited in about 80% of the cases hypermyoglobinaemia, and about 11% of 43 possible DMD patients showed an increase in myoglobin. Taking acute rhabdomyolysis and myositis as an example, it was found that myoglobin correlates well with the clinical course of these diseases and permits safe inferences as to the efficiency of the therapy chosen. (orig./MG) [de

  18. Methylglyoxal-induced modification causes aggregation of myoglobin

    Science.gov (United States)

    Banerjee, Sauradipta; Maity, Subhajit; Chakraborti, Abhay Sankar

    2016-02-01

    Post-translational modification of proteins by Maillard reaction, known as glycation, is thought to be the root cause of different complications, particularly in diabetes mellitus and age-related disorders. Methylglyoxal (MG), a reactive α-oxoaldehyde, increases in diabetic condition and reacts with proteins to form advanced glycation end products (AGEs) following Maillard-like reaction. We have investigated the in vitro effect of MG (200 μM) on the monomeric heme protein myoglobin (Mb) (100 μM) in a time-dependent manner (7 to 18 days incubation at 25 °C). MG induces significant structural alterations of the heme protein, including heme loss, changes in tryptophan fluorescence, decrease of α-helicity with increased β-sheet content etc. These changes occur gradually with increased period of incubation. Incubation of Mb with MG for 7 days results in formation of the AGE adducts: carboxyethyllysine at Lys-16, carboxymethyllysine at Lys-87 and carboxyethyllysine or pyrraline-carboxymethyllysine at Lys-133. On increasing the period of incubation up to 14 days, additional AGEs namely, carboxyethyllysine at Lys-42 and hydroimidazolone or argpyrimidine at Arg-31 and Arg-139 have been detected. MG also induces aggregation of Mb, which is clearly evident with longer period of incubation (18 days), and appears to have amyloid nature. MG-derived AGEs may thus have an important role as the precursors of protein aggregation, which, in turn, may be associated with physiological complications.

  19. A phylogenomic profile of hemerythrins, the nonheme diiron binding respiratory proteins

    Directory of Open Access Journals (Sweden)

    Mizuguchi Kenji

    2008-09-01

    Full Text Available Abstract Background Hemerythrins, are the non-heme, diiron binding respiratory proteins of brachiopods, priapulids and sipunculans; they are also found in annelids and bacteria, where their functions have not been fully elucidated. Results A search for putative Hrs in the genomes of 43 archaea, 444 bacteria and 135 eukaryotes, revealed their presence in 3 archaea, 118 bacteria, several fungi, one apicomplexan, a heterolobosan, a cnidarian and several annelids. About a fourth of the Hr sequences were identified as N- or C-terminal domains of chimeric, chemotactic gene regulators. The function of the remaining single domain bacterial Hrs remains to be determined. In addition to oxygen transport, the possible functions in annelids have been proposed to include cadmium-binding, antibacterial action and immunoprotection. A Bayesian phylogenetic tree revealed a split into two clades, one encompassing archaea, bacteria and fungi, and the other comprising the remaining eukaryotes. The annelid and sipunculan Hrs share the same intron-exon structure, different from that of the cnidarian Hr. Conclusion The phylogenomic profile of Hrs demonstrated a limited occurrence in bacteria and archaea and a marked absence in the vast majority of multicellular organisms. Among the metazoa, Hrs have survived in a cnidarian and in a few protostome groups; hence, it appears that in metazoans the Hr gene was lost in deuterostome ancestor(s after the radiata/bilateria split. Signal peptide sequences in several Hirudinea Hrs suggest for the first time, the possibility of extracellular localization. Since the α-helical bundle is likely to have been among the earliest protein folds, Hrs represent an ancient family of iron-binding proteins, whose primary function in bacteria may have been that of an oxygen sensor, enabling aerophilic or aerophobic responses. Although Hrs evolved to function as O2 transporters in brachiopods, priapulids and sipunculans, their function in

  20. Mechanism of the G-protein mimetic nanobody binding to a muscarinic G-protein-coupled receptor.

    Science.gov (United States)

    Miao, Yinglong; McCammon, J Andrew

    2018-03-20

    Protein-protein binding is key in cellular signaling processes. Molecular dynamics (MD) simulations of protein-protein binding, however, are challenging due to limited timescales. In particular, binding of the medically important G-protein-coupled receptors (GPCRs) with intracellular signaling proteins has not been simulated with MD to date. Here, we report a successful simulation of the binding of a G-protein mimetic nanobody to the M 2 muscarinic GPCR using the robust Gaussian accelerated MD (GaMD) method. Through long-timescale GaMD simulations over 4,500 ns, the nanobody was observed to bind the receptor intracellular G-protein-coupling site, with a minimum rmsd of 2.48 Å in the nanobody core domain compared with the X-ray structure. Binding of the nanobody allosterically closed the orthosteric ligand-binding pocket, being consistent with the recent experimental finding. In the absence of nanobody binding, the receptor orthosteric pocket sampled open and fully open conformations. The GaMD simulations revealed two low-energy intermediate states during nanobody binding to the M 2 receptor. The flexible receptor intracellular loops contribute remarkable electrostatic, polar, and hydrophobic residue interactions in recognition and binding of the nanobody. These simulations provided important insights into the mechanism of GPCR-nanobody binding and demonstrated the applicability of GaMD in modeling dynamic protein-protein interactions.

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

    Science.gov (United States)

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

    2010-01-01

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

  2. Improved detection of calcium-binding proteins in polyacrylamide gels

    International Nuclear Information System (INIS)

    Anthony, F.A.; Babitch, J.A.

    1984-01-01

    The authors refined the method of Schibeci and Martonosi (1980) to enhance detection of calcium-binding proteins in polyacrylamide gels using 45 Ca 2+ . Their efforts have produced a method which is shorter, has 40-fold greater sensitivity over the previous method, and will detect 'EF hand'-containing calcium-binding proteins in polyacrylamide gels below the 0.5 μg level. In addition this method will detect at least one example from every described class of calcium-binding protein, including lectins and γ-carboxyglutamic acid containing calcium-binding proteins. The method should be useful for detecting calcium-binding proteins which may trigger neurotransmitter release. (Auth.)

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

    Directory of Open Access Journals (Sweden)

    Huiying Zhao

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

  4. Clinical relevance of drug binding to plasma proteins

    Science.gov (United States)

    Ascenzi, Paolo; Fanali, Gabriella; Fasano, Mauro; Pallottini, Valentina; Trezza, Viviana

    2014-12-01

    Binding to plasma proteins highly influences drug efficacy, distribution, and disposition. Serum albumin, the most abundant protein in plasma, is a monomeric multi-domain macromolecule that displays an extraordinary ligand binding capacity, providing a depot and carrier for many endogenous and exogenous compounds, such as fatty acids and most acidic drugs. α-1-Acid glycoprotein, the second main plasma protein, is a glycoprotein physiologically involved in the acute phase reaction and is the main carrier for basic and neutral drugs. High- and low-density lipoproteins play a limited role in drug binding and are natural drug delivery system only for few lipophilic drugs or lipid-based formulations. Several factors influence drug binding to plasma proteins, such as pathological conditions, concurrent administration of drugs, sex, and age. Any of these factors, in turn, influences drug efficacy and toxicity. Here, biochemical, biomedical, and biotechnological aspects of drug binding to plasma proteins are reviewed.

  5. Tracking evolution of myoglobin stability in cetaceans using experimentally calibrated computational methods that account for generic protein relaxation

    DEFF Research Database (Denmark)

    Holm, Jeppe; Dasmeh, Pouria; Kepp, Kasper Planeta

    2016-01-01

    The evolution of cetaceans (whales, dolphins, and porpoises) from land to water is one of the most spectacular events in mammal evolution. It has been suggested that selection for higher myoglobin stability (ΔG of folding) allowed whales to conquer the deep-diving niche. The stability of multi-si...

  6. Characterization of binding of N'-nitrosonornicotine to protein

    International Nuclear Information System (INIS)

    Hughes, M.F.

    1986-01-01

    The NADPH-dependent activation of the carcinogenic nitrosamine, N'-nitrosonornicotine (NNN) to a reactive intermediate which binds covalently to protein was assessed using male Sprague-Dawley rat liver and lung microsomes. The NADPH-dependent covalent binding of [ 14 C]NNN to liver and lung microsomes was linear with time up to 90 and 45 min, respectively and was also linear with protein concentrations up to 3.0 and 2.0 mg/ml, respectively. The apparent K/sub m/ and V/sub max/ of the NADPH-dependent binding to liver microsomes were determined from the initial velocities. Addition of the thiols glutathione, cystein, N-acetylcysteine or 2-mercapthoethanol significantly decreased the non-NADPH-dependent binding to liver microsomal protein, but did not affect the NADPH-dependent binding. Glutathione was required in order to observe any NADPH-dependent binding to lung microsomal protein. In lung microsomes, SKF-525A significantly decreased the NADPH-dependent binding by 79%. Replacement of an air atmosphere with N 2 or CO:O 2 (8:2) significantly decreased the NADPH-dependent binding of [ 14 C]NNN to liver microsomal protein by 40% or 27% respectively. Extensive covalent binding of [ 14 C]NNN to liver and muscle microsomal protein occurred in the absence of an NADPH-generating system, in the presence of 50% methanol and also to bovine serum albumin, indicating a nonenzymatic reaction. These data indicate that cytochrome P-450 is at least in part responsible for the metabolic activation of the carcinogen NNN, but also suggest additional mechanisms of activation

  7. DNA Damage: Quantum Mechanics/Molecular Mechanics Study on the Oxygen Binding and Substrate Hydroxylation Step in AlkB Repair Enzymes

    Science.gov (United States)

    Quesne, Matthew G; Latifi, Reza; Gonzalez-Ovalle, Luis E; Kumar, Devesh; de Visser, Sam P

    2014-01-01

    AlkB repair enzymes are important nonheme iron enzymes that catalyse the demethylation of alkylated DNA bases in humans, which is a vital reaction in the body that heals externally damaged DNA bases. Its mechanism is currently controversial and in order to resolve the catalytic mechanism of these enzymes, a quantum mechanics/molecular mechanics (QM/MM) study was performed on the demethylation of the N1-methyladenine fragment by AlkB repair enzymes. Firstly, the initial modelling identified the oxygen binding site of the enzyme. Secondly, the oxygen activation mechanism was investigated and a novel pathway was found, whereby the catalytically active iron(IV)–oxo intermediate in the catalytic cycle undergoes an initial isomerisation assisted by an Arg residue in the substrate binding pocket, which then brings the oxo group in close contact with the methyl group of the alkylated DNA base. This enables a subsequent rate-determining hydrogen-atom abstraction on competitive σ-and π-pathways on a quintet spin-state surface. These findings give evidence of different locations of the oxygen and substrate binding channels in the enzyme and the origin of the separation of the oxygen-bound intermediates in the catalytic cycle from substrate. Our studies are compared with small model complexes and the effect of protein and environment on the kinetics and mechanism is explained. PMID:24339041

  8. Substrate-Triggered Exosite Binding: Synergistic Dendrimer/Folic Acid Action for Achieving Specific, Tight-Binding to Folate Binding Protein.

    Science.gov (United States)

    Chen, Junjie; van Dongen, Mallory A; Merzel, Rachel L; Dougherty, Casey A; Orr, Bradford G; Kanduluru, Ananda Kumar; Low, Philip S; Marsh, E Neil G; Banaszak Holl, Mark M

    2016-03-14

    Polymer-ligand conjugates are designed to bind proteins for applications as drugs, imaging agents, and transport scaffolds. In this work, we demonstrate a folic acid (FA)-triggered exosite binding of a generation five poly(amidoamine) (G5 PAMAM) dendrimer scaffold to bovine folate binding protein (bFBP). The protein exosite is a secondary binding site on the protein surface, separate from the FA binding pocket, to which the dendrimer binds. Exosite binding is required to achieve the greatly enhanced binding constants and protein structural change observed in this study. The G5Ac-COG-FA1.0 conjugate bound tightly to bFBP, was not displaced by a 28-fold excess of FA, and quenched roughly 80% of the initial fluorescence. Two-step binding kinetics were measured using the intrinsic fluorescence of the FBP tryptophan residues to give a KD in the low nanomolar range for formation of the initial G5Ac-COG-FA1.0/FBP* complex, and a slow conversion to the tight complex formed between the dendrimer and the FBP exosite. The extent of quenching was sensitive to the choice of FA-dendrimer linker chemistry. Direct amide conjugation of FA to G5-PAMAM resulted in roughly 50% fluorescence quenching of the FBP. The G5Ac-COG-FA, which has a longer linker containing a 1,2,3-triazole ring, exhibited an ∼80% fluorescence quenching. The binding of the G5Ac-COG-FA1.0 conjugate was compared to poly(ethylene glycol) (PEG) conjugates of FA (PEGn-FA). PEG2k-FA had a binding strength similar to that of FA, whereas other PEG conjugates with higher molecular weight showed weaker binding. However, no PEG conjugates gave an increased degree of total fluorescence quenching.

  9. Human plasminogen binding protein tetranectin

    DEFF Research Database (Denmark)

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

    1997-01-01

    The recombinant human plasminogen binding protein tetranectin (TN) and the C-type lectin CRD of this protein (TN3) have been crystallized. TN3 crystallizes in the tetragonal space group P4(2)2(1)2 with cell dimensions a = b = 64.0, c = 75.7 A and with one molecule per asymmetric unit. The crystals...... to at least 2.5 A. A full data set has been collected to 3.0 A. The asymmetric unit contains one monomer of TN. Molecular replacement solutions for TN3 and TN have been obtained using the structure of the C-type lectin CRD of rat mannose-binding protein as search model. The rhombohedral space group indicates...

  10. Determining Membrane Protein-Lipid Binding Thermodynamics Using Native Mass Spectrometry.

    Science.gov (United States)

    Cong, Xiao; Liu, Yang; Liu, Wen; Liang, Xiaowen; Russell, David H; Laganowsky, Arthur

    2016-04-06

    Membrane proteins are embedded in the biological membrane where the chemically diverse lipid environment can modulate their structure and function. However, the thermodynamics governing the molecular recognition and interaction of lipids with membrane proteins is poorly understood. Here, we report a method using native mass spectrometry (MS), to determine thermodynamics of individual ligand binding events to proteins. Unlike conventional methods, native MS can resolve individual ligand binding events and, coupled with an apparatus to control the temperature, determine binding thermodynamic parameters, such as for protein-lipid interactions. We validated our approach using three soluble protein-ligand systems (maltose binding protein, lysozyme, and nitrogen regulatory protein) and obtained similar results to those using isothermal titration calorimetry and surface plasmon resonance. We also determined for the first time the thermodynamics of individual lipid binding to the ammonia channel (AmtB), an integral membrane protein from Escherichia coli. Remarkably, we observed distinct thermodynamic signatures for the binding of different lipids and entropy-enthalpy compensation for binding lipids of variable chain length. Additionally, using a mutant form of AmtB that abolishes a specific phosphatidylglycerol (PG) binding site, we observed distinct changes in the thermodynamic signatures for binding PG, implying these signatures can identify key residues involved in specific lipid binding and potentially differentiate between specific lipid binding sites.

  11. Lack of conventional oxygen-linked proton and anion binding sites does not impair allosteric regulation of oxygen binding in dwarf caiman hemoglobin

    Science.gov (United States)

    Fago, Angela; Malte, Hans; Storz, Jay F.; Gorr, Thomas A.

    2013-01-01

    In contrast to other vertebrate hemoglobins (Hbs) whose high intrinsic O2 affinities are reduced by red cell allosteric effectors (mainly protons, CO2, organic phosphates, and chloride ions), crocodilian Hbs exhibit low sensitivity to organic phosphates and high sensitivity to bicarbonate (HCO3−), which is believed to augment Hb-O2 unloading during diving and postprandial alkaline tides when blood HCO3− levels and metabolic rates increase. Examination of α- and β-globin amino acid sequences of dwarf caiman (Paleosuchus palpebrosus) revealed a unique combination of substitutions at key effector binding sites compared with other vertebrate and crocodilian Hbs: β82Lys→Gln, β143His→Val, and β146His→Tyr. These substitutions delete positive charges and, along with other distinctive changes in residue charge and polarity, may be expected to disrupt allosteric regulation of Hb-O2 affinity. Strikingly, however, P. palpebrosus Hb shows a strong Bohr effect, and marked deoxygenation-linked binding of organic phosphates (ATP and DPG) and CO2 as carbamate (contrasting with HCO3− binding in other crocodilians). Unlike other Hbs, it polymerizes to large complexes in the oxygenated state. The highly unusual properties of P. palpebrosus Hb align with a high content of His residues (potential sites for oxygenation-linked proton binding) and distinctive surface Cys residues that may form intermolecular disulfide bridges upon polymerization. On the basis of its singular properties, P. palpebrosus Hb provides a unique opportunity for studies on structure-function coupling and the evolution of compensatory mechanisms for maintaining tissue O2 delivery in Hbs that lack conventional effector-binding residues. PMID:23720132

  12. Engineering cofactor and ligand binding in an artificial neuroglobin

    Science.gov (United States)

    Zhang, Lei

    HP-7 is one artificial mutated oxygen transport protein, which operates via a mechanism akin to human neuroglobin and cytoglobin. This protein destabilizes one of two heme-ligating histidine residues by coupling histidine side chain ligation with the burial of three charged glutamate residues on the same helix. Replacement of these glutamate residues with alanine, which has a neutral hydrophobicity, slows gaseous ligand binding 22-fold, increases the affinity of the distal histidine ligand by a factor of thirteen, and decreases the binding affinity of carbon monoxide, a nonreactive oxygen analogue, three-fold. Paradoxically, it also decreases heme binding affinity by a factor of three in the reduced state and six in the oxidized state. Application of a two-state binding model, in which an initial pentacoordinate binding event is followed by a protein conformational change to hexacoordinate, provides insight into the mechanism of this seemingly counterintuitive result: the initial pentacoordinate encounter complex is significantly destabilized by the loss of the glutamate side chains, and the increased affinity for the distal histidine only partially compensates. These results point to the importance of considering each oxidation and conformational state in the design of functional artificial proteins. We have also examined the effects these mutations have on function. The K d of the nonnreactive oxygen analogue carbon monoxide (CO) is only decreased three-fold, despite the large increase in distal histidine affinity engendered by the 22-fold decrease in the histidine ligand off-rate. This is a result of the four-fold increase in affinity for CO binding to the pentacoordinate state. Oxygen binds to HP7 with a Kd of 117 µM, while the mutant rapidly oxidizes when exposed to oxygen. EPR analysis of both ferric hemoproteins demonstrates that the mutation increases disorder at the heme binding site. NMR-detected deuterium exchange demonstrates that the mutation causes a

  13. The RNA-binding protein repertoire of Arabidopsis thaliana

    KAUST Repository

    Marondedze, Claudius; Thomas, Ludivine; Serano, Natalia Lorena Gorron; Lilley, Kathryn S.; Gehring, Christoph A

    2016-01-01

    RNA-binding proteins (RBPs) have essential roles in determining the fate of RNA from synthesis to decay and have been studied on a protein-by-protein basis, or computationally based on a number of well-characterised RNA-binding domains. Recently

  14. Identification of FUSE-binding proteins as interacting partners of TIA proteins

    International Nuclear Information System (INIS)

    Rothe, Francoise; Gueydan, Cyril; Bellefroid, Eric; Huez, Georges; Kruys, Veronique

    2006-01-01

    TIA-1 and TIAR are closely related RNA-binding proteins involved in several mechanisms of RNA metabolism, including alternative hnRNA splicing and mRNA translation regulation. In particular, TIA-1 represses tumor necrosis factor (TNF) mRNA translation by binding to the AU-rich element (ARE) present in the mRNA 3' untranslated region. Here, we demonstrate that TIA proteins interact with FUSE-binding proteins (FBPs) and that fbp genes are co-expressed with tia genes during Xenopus embryogenesis. FBPs participate in various steps of RNA processing and degradation. In Cos cells, FBPs co-localize with TIA proteins in the nucleus and migrate into TIA-enriched cytoplasmic granules upon oxidative stress. Overexpression of FBP2-KH3 RNA-binding domain fused to EGFP induces the specific sequestration of TIA proteins in cytoplasmic foci, thereby precluding their nuclear accumulation. In cytosolic RAW 264.7 macrophage extracts, FBPs are found associated in EMSA to the TIA-1/TNF-ARE complex. Together, our results indicate that TIA and FBP proteins may thus be relevant biological involved in common events of RNA metabolism occurring both in the nucleus and the cytoplasm

  15. Identification of actin binding protein, ABP-280, as a binding partner of human Lnk adaptor protein.

    Science.gov (United States)

    He, X; Li, Y; Schembri-King, J; Jakes, S; Hayashi, J

    2000-08-01

    Human Lnk (hLnk) is an adaptor protein with multiple functional domains that regulates T cell activation signaling. In order to identify cellular Lnk binding partners, a yeast two-hybrid screening of human spleen cDNA library was carried out using human hLnk as bait. A polypeptide sequence identical to the C-terminal segment of the actin binding protein (ABP-280) was identified as a hLnk binding protein. The expressed hLnk and the FLAG tagged C-terminal 673 amino acid residues of ABP-280 or the endogenous ABP-280 in COS-7 cells could be co-immunoprecipitated using antibodies either to hLnk, FLAG or ABP-280, respectively. Furthermore, immunofluorescence confocal microscope showed that hLnk and ABP-280 co-localized at the plasma membrane and at juxtanuclear region of COS-7 cells. In Jurkat cells, the endogenous hLnk also associates with the endogenous ABP-280 indicating that the association of these two proteins is physiological. The interacting domains of both proteins were mapped using yeast two-hybrid assays. Our results indicate that hLnk binds to the residues 2006-2454 (repeats 19-23C) of ABP-280. The domain in hLnk that associates with ABP-280 was mapped to an interdomain region of 56 amino acids between pleckstrin homology and Src homology 2 domains. These results suggest that hLnk may exert its regulatory role through its association with ABP-280.

  16. The RNA-Binding Site of Poliovirus 3C Protein Doubles as a Phosphoinositide-Binding Domain.

    Science.gov (United States)

    Shengjuler, Djoshkun; Chan, Yan Mei; Sun, Simou; Moustafa, Ibrahim M; Li, Zhen-Lu; Gohara, David W; Buck, Matthias; Cremer, Paul S; Boehr, David D; Cameron, Craig E

    2017-12-05

    Some viruses use phosphatidylinositol phosphate (PIP) to mark membranes used for genome replication or virion assembly. PIP-binding motifs of cellular proteins do not exist in viral proteins. Molecular-docking simulations revealed a putative site of PIP binding to poliovirus (PV) 3C protein that was validated using nuclear magnetic resonance spectroscopy. The PIP-binding site was located on a highly dynamic α helix, which also functions in RNA binding. Broad PIP-binding activity was observed in solution using a fluorescence polarization assay or in the context of a lipid bilayer using an on-chip, fluorescence assay. All-atom molecular dynamics simulations of the 3C protein-membrane interface revealed PIP clustering and perhaps PIP-dependent conformations. PIP clustering was mediated by interaction with residues that interact with the RNA phosphodiester backbone. We conclude that 3C binding to membranes will be determined by PIP abundance. We suggest that the duality of function observed for 3C may extend to RNA-binding proteins of other viruses. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Variability in the leptin, leptin receptor and heart fatty acid binding protein genes in relationship with meat quality traits in pigs

    Directory of Open Access Journals (Sweden)

    Renata Mikolášová

    2005-01-01

    Full Text Available The leptin (LEP-HinfI, leptin receptor (LEPR-HpaII and heart fatty acid binding protein (H-FABP-HinfI genes and their genotypes combination (LEP-HinfI *LEPR-HpaII were tested for associations with the pH1, pH24, myoglobin content (mg/100 g, intramuscular fat content (% and remission (%. The genotypes were determined in Large White, Landrace and Duroc breeds (n = 106, 56 and 4, respectively. The allele frequencies were: LEP-HinfI: C = 0.133 T = 0.867; LEPR-HpaII: A = 0.331 B = 0.669; H-FABP-HinfI: H = 0.745 h = 0.255. The populations of breeds were in the genetic equilibrium according to the χ2 test in the tested loci. The combinations of LEP-HinfI and LEPR-HpaII were significantly associated with the pH24 and remission. The H-FABP-HinfI locus was significantly associated with intramuscular fat content.

  18. Human proteins that specifically bind to 8-oxoguanine-containing RNA and their responses to oxidative stress

    International Nuclear Information System (INIS)

    Hayakawa, Hiroshi; Fujikane, Aya; Ito, Riyoko; Matsumoto, Masaki; Nakayama, Keiichi I.; Sekiguchi, Mutsuo

    2010-01-01

    Research highlights: → We performed comprehensive survey for proteins that bind to oxidized RNA. → HNRNPD and HNRNPC proteins were identified as oxidized RNA binding proteins. → Knockdown of HNRNPD/C expression caused increased sensitivity to H 2 O 2 . → Amounts of HNRNPD protein rapidly decreased when cells were exposed to H 2 O 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.

  19. DNA-binding proteins essential for protein-primed bacteriophage ø29 DNA replication

    Directory of Open Access Journals (Sweden)

    Margarita Salas

    2016-08-01

    Full Text Available Bacillus subtilis phage Φ29 has a linear, double-stranded DNA 19 kb long with an inverted terminal repeat of 6 nucleotides and a protein covalently linked to the 5’ ends of the DNA. This protein, called terminal protein (TP, is the primer for the initiation of replication, a reaction catalyzed by the viral DNA polymerase at the two DNA ends. The DNA polymerase further elongates the nascent DNA chain in a processive manner, coupling strand displacement with elongation. The viral protein p5 is a single-stranded DNA binding protein (SSB that binds to the single strands generated by strand displacement during the elongation process. Viral protein p6 is a double-stranded DNA binding protein (DBP that preferentially binds to the origins of replication at the Φ29 DNA ends and is required for the initiation of replication. Both SSB and DBP are essential for Φ29 DNA amplification. This review focuses on the role of these phage DNA-binding proteins in Φ29 DNA replication both in vitro and in vivo, as well as on the implication of several B. subtilis DNA-binding proteins in different processes of the viral cycle. We will revise the enzymatic activities of the Φ29 DNA polymerase: TP-deoxynucleotidylation, processive DNA polymerization coupled to strand displacement, 3’-5’ exonucleolysis and pyrophosphorolysis. The resolution of the Φ29 DNA polymerase structure has shed light on the translocation mechanism and the determinants responsible for processivity and strand displacement. These two properties have made Φ29 DNA polymerase one of the main enzymes used in the current DNA amplification technologies. The determination of the structure of Φ29 TP revealed the existence of three domains: the priming domain, where the primer residue Ser232, as well as Phe230, involved in the determination of the initiating nucleotide, are located, the intermediate domain, involved in DNA polymerase binding, and the N-terminal domain, responsible for DNA binding

  20. Aluminium fluoride and magnesium, activators of heterotrimeric GTP-binding proteins, affect high-affinity binding of the fungal toxin fusicoccin to the fusicoccin-binding protein in oat root plasma membranes.

    NARCIS (Netherlands)

    de Boer, A.H.; Van der Molen, G.W.; Prins, H.B.A.; Korthout, H.A.A.J.; van der Hoeven, P.C.J.

    1994-01-01

    The fusicoccin-binding protein was solubilised from purified oat root plasma membranes. The solubilised protein retained full binding activity, provided that protease inhibitors were included. Sodium fluoride reduced the high-affinity [H-3]fusicoccin binding to almost zero in a

  1. Drosophila DNA-Binding Proteins in Polycomb Repression

    Directory of Open Access Journals (Sweden)

    Maksim Erokhin

    2018-01-01

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

  2. The roles of tissue nitrate reductase activity and myoglobin in securing nitric oxide availability in deeply hypoxic crucian carp

    DEFF Research Database (Denmark)

    Hansen, Marie Niemann; Lundberg, Jon O; Filice, Mariacristina

    2016-01-01

    . We also tested whether liver, muscle and heart tissue possess nitrate reductase activity that supplies nitrite to the tissues during severe hypoxia. Crucian carp exposed to deep hypoxia (1nitrite in red musculature to more than double the value in normoxic fish......In mammals, treatment with low doses of nitrite has a cytoprotective effect in ischemia/reperfusion events, as a result of nitric oxide formation and S-nitrosation of proteins. Interestingly, anoxia-tolerant lower vertebrates possess an intrinsic ability to increase intracellular nitrite...... concentration during anoxia in tissues with high myoglobin and mitochondria content, such as the heart. Here, we tested the hypothesis that red and white skeletal muscles develop different nitrite levels in crucian carp exposed to deep hypoxia and assessed whether this correlates with myoglobin concentration...

  3. De novo design and engineering of functional metal and porphyrin-binding protein domains

    Science.gov (United States)

    Everson, Bernard H.

    In this work, I describe an approach to the rational, iterative design and characterization of two functional cofactor-binding protein domains. First, a hybrid computational/experimental method was developed with the aim of algorithmically generating a suite of porphyrin-binding protein sequences with minimal mutual sequence information. This method was explored by generating libraries of sequences, which were then expressed and evaluated for function. One successful sequence is shown to bind a variety of porphyrin-like cofactors, and exhibits light- activated electron transfer in mixed hemin:chlorin e6 and hemin:Zn(II)-protoporphyrin IX complexes. These results imply that many sophisticated functions such as cofactor binding and electron transfer require only a very small number of residue positions in a protein sequence to be fixed. Net charge and hydrophobic content are important in determining protein solubility and stability. Accordingly, rational modifications were made to the aforementioned design procedure in order to improve its overall success rate. The effects of these modifications are explored using two `next-generation' sequence libraries, which were separately expressed and evaluated. Particular modifications to these design parameters are demonstrated to effectively double the purification success rate of the procedure. Finally, I describe the redesign of the artificial di-iron protein DF2 into CDM13, a single chain di-Manganese four-helix bundle. CDM13 acts as a functional model of natural manganese catalase, exhibiting a kcat of 0.08s-1 under steady-state conditions. The bound manganese cofactors have a reduction potential of +805 mV vs NHE, which is too high for efficient dismutation of hydrogen peroxide. These results indicate that as a high-potential manganese complex, CDM13 may represent a promising first step toward a polypeptide model of the Oxygen Evolving Complex of the photosynthetic enzyme Photosystem II.

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

    Science.gov (United States)

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

    2008-04-01

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

  5. Conformational change of spin labelled myoglobin

    International Nuclear Information System (INIS)

    Wajnberg, E.; Ribeiro, P.C.; Nascimento, O.R.; Bemski, G.

    1978-01-01

    A conformational change of spin labelled myoglobin have been followed by measuring the spin label's (isothiocyanate) correlation time for temperatures between 18 0 C and 44 0 C. The correlation time was calculated from Electrom Paramagnetic Ressonance Spectra using the components of the espectroscopic and hiperfine tensors obtained by fitting the powder spectra using Lefebvre and Maruani's program- [pt

  6. Saccharomyces cerevisiae SSB1 protein and its relationship to nucleolar RNA-binding proteins.

    Science.gov (United States)

    Jong, A Y; Clark, M W; Gilbert, M; Oehm, A; Campbell, J L

    1987-08-01

    To better define the function of Saccharomyces cerevisiae SSB1, an abundant single-stranded nucleic acid-binding protein, we determined the nucleotide sequence of the SSB1 gene and compared it with those of other proteins of known function. The amino acid sequence contains 293 amino acid residues and has an Mr of 32,853. There are several stretches of sequence characteristic of other eucaryotic single-stranded nucleic acid-binding proteins. At the amino terminus, residues 39 to 54 are highly homologous to a peptide in calf thymus UP1 and UP2 and a human heterogeneous nuclear ribonucleoprotein. Residues 125 to 162 constitute a fivefold tandem repeat of the sequence RGGFRG, the composition of which suggests a nucleic acid-binding site. Near the C terminus, residues 233 to 245 are homologous to several RNA-binding proteins. Of 18 C-terminal residues, 10 are acidic, a characteristic of the procaryotic single-stranded DNA-binding proteins and eucaryotic DNA- and RNA-binding proteins. In addition, examination of the subcellular distribution of SSB1 by immunofluorescence microscopy indicated that SSB1 is a nuclear protein, predominantly located in the nucleolus. Sequence homologies and the nucleolar localization make it likely that SSB1 functions in RNA metabolism in vivo, although an additional role in DNA metabolism cannot be excluded.

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

    Science.gov (United States)

    Takemura, Kazuhiro; Matubayasi, Nobuyuki; Kitao, Akio

    2018-03-14

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

  8. Factors Affecting the Binding of a Recombinant Heavy Metal-Binding Domain (CXXC motif Protein to Heavy Metals

    Directory of Open Access Journals (Sweden)

    Kamala Boonyodying

    2012-06-01

    Full Text Available A number of heavy metal-binding proteins have been used to study bioremediation. CXXC motif, a metal binding domain containing Cys-X-X-Cys motif, has been identified in various organisms. These proteins are capable of binding various types of heavy metals. In this study, heavy metal binding domain (CXXC motif recombinant protein encoded from mcsA gene of S. aureus were cloned and overexpressed in Escherichia coli. The factors involved in the metal-binding activity were determined in order to analyze the potential of recombinant protein for bioremediation. A recombinant protein can be bound to Cd2+, Co2+, Cu2+ and Zn2+. The thermal stability of a recombinant protein was tested, and the results showed that the metal binding activity to Cu2+ and Zn2+ still exist after treating the protein at 85ºC for 30 min. The temperature and pH that affected the metal binding activity was tested and the results showed that recombinant protein was still bound to Cu2+ at 65ºC, whereas a pH of 3-7 did not affect the metal binding E. coli harboring a pRset with a heavy metal-binding domain CXXC motif increased the resistance of heavy metals against CuCl2 and CdCl2. This study shows that metal binding domain (CXXC motif recombinant protein can be effectively bound to various types of heavy metals and may be used as a potential tool for studying bioremediation.

  9. Purification and binding analysis of the nitrogen fixation regulatory NifA protein from Azospirillum brasilense

    Directory of Open Access Journals (Sweden)

    L.M.P. Passaglia

    1998-11-01

    Full Text Available NifA protein activates transcription of nitrogen fixation operons by the alternative sigma54 holoenzyme form of RNA polymerase. This protein binds to a well-defined upstream activator sequence (UAS located at the -200/-100 position of nif promoters with the consensus motif TGT-N10-ACA. NifA of Azospirillum brasilense was purified in the form of a glutathione-S-transferase (GST-NifA fusion protein and proteolytic release of GST yielded inactive and partially soluble NifA. However, the purified NifA was able to induce the production of specific anti-A. brasilense NifA-antiserum that recognized NifA from A. brasilense but not from K. pneumoniae. Both GST-NifA and NifA expressed from the E. coli tac promoter are able to activate transcription from the nifHDK promoter but only in an A. brasilense background. In order to investigate the mechanism that regulates NifA binding capacity we have used E. coli total protein extracts expressing A. brasilense nifA in mobility shift assays. DNA fragments carrying the two overlapping, wild-type or mutated UAS motifs present in the nifH promoter region revealed a retarded band of related size. These data show that the binding activity present in the C-terminal domain of A. brasilense NifA protein is still functional even in the presence of oxygen.

  10. Predicting the binding patterns of hub proteins: a study using yeast protein interaction networks.

    Directory of Open Access Journals (Sweden)

    Carson M Andorf

    Full Text Available Protein-protein interactions are critical to elucidating the role played by individual proteins in important biological pathways. Of particular interest are hub proteins that can interact with large numbers of partners and often play essential roles in cellular control. Depending on the number of binding sites, protein hubs can be classified at a structural level as singlish-interface hubs (SIH with one or two binding sites, or multiple-interface hubs (MIH with three or more binding sites. In terms of kinetics, hub proteins can be classified as date hubs (i.e., interact with different partners at different times or locations or party hubs (i.e., simultaneously interact with multiple partners.Our approach works in 3 phases: Phase I classifies if a protein is likely to bind with another protein. Phase II determines if a protein-binding (PB protein is a hub. Phase III classifies PB proteins as singlish-interface versus multiple-interface hubs and date versus party hubs. At each stage, we use sequence-based predictors trained using several standard machine learning techniques.Our method is able to predict whether a protein is a protein-binding protein with an accuracy of 94% and a correlation coefficient of 0.87; identify hubs from non-hubs with 100% accuracy for 30% of the data; distinguish date hubs/party hubs with 69% accuracy and area under ROC curve of 0.68; and SIH/MIH with 89% accuracy and area under ROC curve of 0.84. Because our method is based on sequence information alone, it can be used even in settings where reliable protein-protein interaction data or structures of protein-protein complexes are unavailable to obtain useful insights into the functional and evolutionary characteristics of proteins and their interactions.We provide a web server for our three-phase approach: http://hybsvm.gdcb.iastate.edu.

  11. Singlet oxygen-mediated damage to proteins and its consequences

    DEFF Research Database (Denmark)

    Davies, Michael Jonathan

    2003-01-01

    by the transfer of energy to ground state (triplet) molecular oxygen by either protein-bound, or other, chromophores. Singlet oxygen can also be generated by a range of other enzymatic and non-enzymatic reactions including processes mediated by heme proteins, lipoxygenases, and activated leukocytes, as well...... the absorption of UV radiation by the protein, or bound chromophore groups, thereby generating excited states (singlet or triplets) or radicals via photo-ionisation. The second major process involves indirect oxidation of the protein via the formation and subsequent reactions of singlet oxygen generated...... as radical termination reactions. This paper reviews the data available on singlet oxygen-mediated protein oxidation and concentrates primarily on the mechanisms by which this excited state species brings about changes to both the side-chains and backbone of amino acids, peptides, and proteins. Recent work...

  12. Intramuscular variation in mitochondrial functionality of beef ...

    African Journals Online (AJOL)

    Suman, Surendranath P

    2017-08-16

    Aug 16, 2017 ... Myoglobin is the sarcoplasmic heme protein responsible for meat color, ... formation was induced in the samples by submerging them in a solution of 0.3% sodium nitrite for 20 min at ..... Dependence on the electron transport chain. ... Myoglobin-enhanced oxygen delivery to isolated cardiac mitochondria.

  13. Exploring the binding sites and binding mechanism for hydrotrope encapsulated griseofulvin drug on γ-tubulin protein.

    Directory of Open Access Journals (Sweden)

    Shubhadip Das

    Full Text Available The protein γ-tubulin plays an important role in centrosomal clustering and this makes it an attractive therapeutic target for treating cancers. Griseofulvin, an antifungal drug, has recently been used to inhibit proliferation of various types of cancer cells. It can also affect the microtubule dynamics by targeting the γ-tubulin protein. So far, the binding pockets of γ-tubulin protein are not properly identified and the exact mechanism by which the drug binds to it is an area of intense speculation and research. The aim of the present study is to investigate the binding mechanism and binding affinity of griseofulvin on γ-tubulin protein using classical molecular dynamics simulations. Since the drug griseofulvin is sparingly soluble in water, here we also present a promising approach for formulating and achieving delivery of hydrophobic griseofulvin drug via hydrotrope sodium cumene sulfonate (SCS cluster. We observe that the binding pockets of γ-tubulin protein are mainly formed by the H8, H9 helices and S7, S8, S14 strands and the hydrophobic interactions between the drug and γ-tubulin protein drive the binding process. The release of the drug griseofulvin from the SCS cluster is confirmed by the coordination number analysis. We also find hydrotrope-induced alteration of the binding sites of γ-tubulin protein and the weakening of the drug-protein interactions.

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

    Science.gov (United States)

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

    2015-12-29

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

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

    Directory of Open Access Journals (Sweden)

    Anders Gerd

    2008-01-01

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

  16. Trans-acting translational regulatory RNA binding proteins.

    Science.gov (United States)

    Harvey, Robert F; Smith, Tom S; Mulroney, Thomas; Queiroz, Rayner M L; Pizzinga, Mariavittoria; Dezi, Veronica; Villenueva, Eneko; Ramakrishna, Manasa; Lilley, Kathryn S; Willis, Anne E

    2018-05-01

    The canonical molecular machinery required for global mRNA translation and its control has been well defined, with distinct sets of proteins involved in the processes of translation initiation, elongation and termination. Additionally, noncanonical, trans-acting regulatory RNA-binding proteins (RBPs) are necessary to provide mRNA-specific translation, and these interact with 5' and 3' untranslated regions and coding regions of mRNA to regulate ribosome recruitment and transit. Recently it has also been demonstrated that trans-acting ribosomal proteins direct the translation of specific mRNAs. Importantly, it has been shown that subsets of RBPs often work in concert, forming distinct regulatory complexes upon different cellular perturbation, creating an RBP combinatorial code, which through the translation of specific subsets of mRNAs, dictate cell fate. With the development of new methodologies, a plethora of novel RNA binding proteins have recently been identified, although the function of many of these proteins within mRNA translation is unknown. In this review we will discuss these methodologies and their shortcomings when applied to the study of translation, which need to be addressed to enable a better understanding of trans-acting translational regulatory proteins. Moreover, we discuss the protein domains that are responsible for RNA binding as well as the RNA motifs to which they bind, and the role of trans-acting ribosomal proteins in directing the translation of specific mRNAs. This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes Translation > Translation Regulation Translation > Translation Mechanisms. © 2018 Medical Research Council and University of Cambridge. WIREs RNA published by Wiley Periodicals, Inc.

  17. Oxygen binding properties of hemoglobin from the white rhinoceros (beta 2-GLU) and the tapir.

    Science.gov (United States)

    Baumann, R; Mazur, G; Braunitzer, G

    1984-04-01

    The beta-chain of rhinoceros hemoglobin contains glutamic acid at position beta 2, and important site for the binding of organic phosphates. We have investigated the oxygen binding properties of this hemoglobin and its interaction with ATP, 2,3-diphosphoglycerate, CO2 and chloride. The results show that the presence of GLU at position beta 2 nearly abolishes the effect of organic phosphates and CO2, whereas the oxygen-linked binding of chloride is not affected. Thus rhinoceros hemoglobin has only protons and chloride anions as major allosteric effectors for the control of its oxygen affinity. From the results obtained with hemoglobin solutions it can be calculated that the blood oxygen affinity of the rhinoceros must be rather high with a P50 of about 20 torr at pH 7.4 and 37 degrees C, which conforms with observations obtained for other large mammals.

  18. Characterization of cap binding proteins associated with the nucleus

    International Nuclear Information System (INIS)

    Patzelt, E.

    1986-04-01

    Eucaryotic mRNAs a carry 7-methylguanosine triphosphate residue (called cap structure) at their 5' terminus. The cap plays an important role in RNA recognition. Cap binding proteins (CBP) of HeLa cells were identified by photoaffinity labelling using the cap analogue γ-( 32 P)-(4-(benzoyl-phenyl)methylamido)-7-methylguanosine-5'-triphosphate (BP-m 7 GTP). Photoreaction of this cap analogue with HeLa cell initiation factors resulted in specific labelling of two polypeptides of Msub(r) 37000 and 26000. The latter was also labelled in crude initiation factors prepared from reticulocytes and is identical to the cap binding protein CBP I previously identified. These cap binding proteins were also affinity labelled in poliovirus infected cell extracts. Photoaffinity reaction with BP-m 7 GTP of whole HeLa cell homogenate showed three additional polypeptides with Msub(r) 120000, 89000 and 80000. These cap binding proteins were found to be associated with the nucleus and are therefore referred to as nuclear cap binding proteins, i.e. NCBP 1, NCBP 2 and NCBP 3. They were also present in splicing extracts. Photoaffinity labelling in these nuclear extracts was differentially inhibited by various cap analogues and capped mRNAs. Affinity chromatography on immobilized globin mRNA led to a partial separation of the three nuclear cap binding proteins. Chromatography on m 7 GTP-Sepharose resulted in a specific binding of NCBP 3. The different behaviour of the cap binding proteins suggests that they are functionally distinct and that they might be involved in different processes requiring cap recognition. (Author)

  19. Allosteric Regulation of Proteins

    Indian Academy of Sciences (India)

    interactions with other proteins, or binding of small molecules. Covalent .... vealed through structural elucidation of the protein in free and oxygen-bound forms .... stance, molecular dynamic simulation of glutamine binding pro- tein shows that ...

  20. Probing protein phosphatase substrate binding

    DEFF Research Database (Denmark)

    Højlys-Larsen, Kim B.; Sørensen, Kasper Kildegaard; Jensen, Knud Jørgen

    2012-01-01

    Proteomics and high throughput analysis for systems biology can benefit significantly from solid-phase chemical tools for affinity pull-down of proteins from complex mixtures. Here we report the application of solid-phase synthesis of phosphopeptides for pull-down and analysis of the affinity...... profile of the integrin-linked kinase associated phosphatase (ILKAP), a member of the protein phosphatase 2C (PP2C) family. Phosphatases can potentially dephosphorylate these phosphopeptide substrates but, interestingly, performing the binding studies at 4 °C allowed efficient binding to phosphopeptides......, without the need for phosphopeptide mimics or phosphatase inhibitors. As no proven ILKAP substrates were available, we selected phosphopeptide substrates among known PP2Cδ substrates including the protein kinases: p38, ATM, Chk1, Chk2 and RSK2 and synthesized directly on PEGA solid supports through a BAL...

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

    Directory of Open Access Journals (Sweden)

    M.A.M. Marques

    2001-04-01

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

  2. An overview of the prediction of protein DNA-binding sites.

    Science.gov (United States)

    Si, Jingna; Zhao, Rui; Wu, Rongling

    2015-03-06

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

  3. Endogenous myoglobin in human breast cancer is a hallmark of luminal cancer phenotype.

    Science.gov (United States)

    Kristiansen, G; Rose, M; Geisler, C; Fritzsche, F R; Gerhardt, J; Lüke, C; Ladhoff, A-M; Knüchel, R; Dietel, M; Moch, H; Varga, Z; Theurillat, J-P; Gorr, T A; Dahl, E

    2010-06-08

    We aimed to clarify the incidence and the clinicopathological value of non-muscle myoglobin (Mb) in a large cohort of non-invasive and invasive breast cancer cases. Matched pairs of breast tissues from 10 patients plus 17 breast cell lines were screened by quantitative PCR for Mb mRNA. In addition, 917 invasive and 155 non-invasive breast cancer cases were analysed by immunohistochemistry for Mb expression and correlated to clinicopathological parameters and basal molecular characteristics including oestrogen receptor-alpha (ERalpha)/progesteron receptor (PR)/HER2, fatty acid synthase (FASN), hypoxia-inducible factor-1alpha (HIF-1alpha), HIF-2alpha, glucose transporter 1 (GLUT1) and carbonic anhydrase IX (CAIX). The spatial relationship of Mb and ERalpha or FASN was followed up by double immunofluorescence. Finally, the effects of estradiol treatment and FASN inhibition on Mb expression in breast cancer cells were analysed. Myoglobin mRNA was found in a subset of breast cancer cell lines; in microdissected tumours Mb transcript was markedly upregulated. In all, 71% of tumours displayed Mb protein expression in significant correlation with a positive hormone receptor status and better prognosis. In silico data mining confirmed higher Mb levels in luminal-type breast cancer. Myoglobin was also correlated to FASN, HIF-2alpha and CAIX, but not to HIF-1alpha or GLUT1, suggesting hypoxia to participate in its regulation. Double immunofluorescence showed a cellular co-expression of ERalpha or FASN and Mb. In addition, Mb levels were modulated on estradiol treatment and FASN inhibition in a cell model. We conclude that in breast cancer, Mb is co-expressed with ERalpha and co-regulated by oestrogen signalling and can be considered a hallmark of luminal breast cancer phenotype. This and its possible new role in fatty acid metabolism may have fundamental implications for our understanding of Mb in solid tumours.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    NARCIS (Netherlands)

    Koppelman, S.J.

    1995-01-01

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

  6. Computational analysis of protein-protein interfaces involving an alpha helix: insights for terphenyl-like molecules binding.

    Science.gov (United States)

    Isvoran, Adriana; Craciun, Dana; Martiny, Virginie; Sperandio, Olivier; Miteva, Maria A

    2013-06-14

    Protein-Protein Interactions (PPIs) are key for many cellular processes. The characterization of PPI interfaces and the prediction of putative ligand binding sites and hot spot residues are essential to design efficient small-molecule modulators of PPI. Terphenyl and its derivatives are small organic molecules known to mimic one face of protein-binding alpha-helical peptides. In this work we focus on several PPIs mediated by alpha-helical peptides. We performed computational sequence- and structure-based analyses in order to evaluate several key physicochemical and surface properties of proteins known to interact with alpha-helical peptides and/or terphenyl and its derivatives. Sequence-based analysis revealed low sequence identity between some of the analyzed proteins binding alpha-helical peptides. Structure-based analysis was performed to calculate the volume, the fractal dimension roughness and the hydrophobicity of the binding regions. Besides the overall hydrophobic character of the binding pockets, some specificities were detected. We showed that the hydrophobicity is not uniformly distributed in different alpha-helix binding pockets that can help to identify key hydrophobic hot spots. The presence of hydrophobic cavities at the protein surface with a more complex shape than the entire protein surface seems to be an important property related to the ability of proteins to bind alpha-helical peptides and low molecular weight mimetics. Characterization of similarities and specificities of PPI binding sites can be helpful for further development of small molecules targeting alpha-helix binding proteins.

  7. Renal myoglobin in drug addicts: occurrence and significance in a postmortem study

    DEFF Research Database (Denmark)

    Kock, Kirsten Friis; Simonsen, Kirsten Wiese

    1994-01-01

    In a 3-year period (1989–1991) a non-selected, consecutive series of 62 deaths in drug addicts was autopsied at the Forensic Institute in Odense. The kidney sections from these addicts were examined for the presence of renal myoglobin using immunohistochemical methods. A reference group consisting......, immobilization, hypovolemia). In sufficient amounts, renal myoglobin may be of importance as a cause of death or a contributing factor to death in both drug addicts and non-addicts....

  8. Ion Binding Energies Determining Functional Transport of ClC Proteins

    Science.gov (United States)

    Yu, Tao; Guo, Xu; Zou, Xian-Wu; Sang, Jian-Ping

    2014-06-01

    The ClC-type proteins, a large family of chloride transport proteins ubiquitously expressed in biological organisms, have been extensively studied for decades. Biological function of ClC proteins can be reflected by analyzing the binding situation of Cl- ions. We investigate ion binding properties of ClC-ec1 protein with the atomic molecular dynamics simulation approach. The calculated electrostatic binding energy results indicate that Cl- at the central binding site Scen has more binding stability than the internal binding site Sint. Quantitative comparison between the latest experimental heat release data isothermal titration calorimetry (ITC) and our calculated results demonstrates that chloride ions prefer to bind at Scen than Sint in the wild-type ClC-ec1 structure and prefer to bind at Sext and Scen than Sint in mutant E148A/E148Q structures. Even though the chloride ions make less contribution to heat release when binding to Sint and are relatively unstable in the Cl- pathway, they are still part contributors for the Cl- functional transport. This work provides a guide rule to estimate the importance of Cl- at the binding sites and how chloride ions have influences on the function of ClC proteins.

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

    DEFF Research Database (Denmark)

    Nordhoff, E.; Korgsdam, A.-M.; Jørgensen, H.F.

    1999-01-01

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

  10. Protein Binding Capacity of Different Forages Tannin

    Science.gov (United States)

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

    2018-02-01

    Eight forages of tannin sources(Leucaena leucocephala, Arachis hypogaea, Mimosa pudica, Morus alba L, Swietenia mahagoni, Manihot esculenta, Gliricidia sepium, and Bauhinia purpurea)were evaluated their tannin content and protein binding capacity. The protein binding capacity of tannin were determined using precipitation of bovine serum albumin (BSA). Swietenia mahagonihas higest total tannin level and condensed tannin (CT) compared with other forages (P<0.01). The Leucaena leucocephala has highest hydrolysable tannin (HT) level (P<0.01). The total and condensed tannin content of Swietenia mahagoni were 11.928±0.04 mg/100 mg and 9.241±0.02mg/100mg dry matter (DM) of leaves. The hydrolysable tannin content of Leucaena leucocephala was 5.338±0.03 mg/100 mg DM of leaves. Binding capacity was highest in Swietenia mahagoni and Leucaena leucocephala compared to the other forages (P<0.01). The optimum binding of BSA to tannin in Leucaena leucocephala and Swietenia mahagoniwere1.181±0.44 and 1.217±0.60mg/mg dry matter of leaves. The present study reports that Swietenia mahagoni has highest of tannin content and Leucaena leucocephala and Swietenia mahagoni capacity of protein binding.

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

    Science.gov (United States)

    Ma, Xin; Guo, Jing; Sun, Xiao

    2016-01-01

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

  12. IGF binding proteins.

    Science.gov (United States)

    Bach, Leon A

    2017-12-18

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

  13. Ligand Binding Domain Protein in Tetracycline-Inducible Expression

    African Journals Online (AJOL)

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

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

    Directory of Open Access Journals (Sweden)

    Jingna Si

    2015-03-01

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

  15. STRUCTURAL FEATURES OF PLANT CHITINASES AND CHITIN-BINDING PROTEINS

    NARCIS (Netherlands)

    BEINTEMA, JJ

    1994-01-01

    Structural features of plant chitinases and chitin-binding proteins are discussed. Many of these proteins consist of multiple domains,of which the chitin-binding hevein domain is a predominant one. X-ray and NMR structures of representatives of the major classes of these proteins are available now,

  16. A tool for calculating binding-site residues on proteins from PDB structures

    Directory of Open Access Journals (Sweden)

    Hu Jing

    2009-08-01

    Full Text Available Abstract Background In the research on protein functional sites, researchers often need to identify binding-site residues on a protein. A commonly used strategy is to find a complex structure from the Protein Data Bank (PDB that consists of the protein of interest and its interacting partner(s and calculate binding-site residues based on the complex structure. However, since a protein may participate in multiple interactions, the binding-site residues calculated based on one complex structure usually do not reveal all binding sites on a protein. Thus, this requires researchers to find all PDB complexes that contain the protein of interest and combine the binding-site information gleaned from them. This process is very time-consuming. Especially, combing binding-site information obtained from different PDB structures requires tedious work to align protein sequences. The process becomes overwhelmingly difficult when researchers have a large set of proteins to analyze, which is usually the case in practice. Results In this study, we have developed a tool for calculating binding-site residues on proteins, TCBRP http://yanbioinformatics.cs.usu.edu:8080/ppbindingsubmit. For an input protein, TCBRP can quickly find all binding-site residues on the protein by automatically combining the information obtained from all PDB structures that consist of the protein of interest. Additionally, TCBRP presents the binding-site residues in different categories according to the interaction type. TCBRP also allows researchers to set the definition of binding-site residues. Conclusion The developed tool is very useful for the research on protein binding site analysis and prediction.

  17. Crystal Structure of the Zorbamycin-Binding Protein ZbmA, the Primary Self-Resistance Element in Streptomyces flavoviridis ATCC21892

    Energy Technology Data Exchange (ETDEWEB)

    Rudolf, Jeffrey D. [Scripps Research Inst., Jupiter, FL (United States); Bigelow, Lance [Argonne National Lab. (ANL), Argonne, IL (United States); Chang, Changsoo [Argonne National Lab. (ANL), Argonne, IL (United States); Cuff, Marianne E. [Argonne National Lab. (ANL), Argonne, IL (United States); Lohman, Jeremy R. [Scripps Research Inst., Jupiter, FL (United States); Chang, Chin-Yuan [Scripps Research Inst., Jupiter, FL (United States); Ma, Ming [Scripps Research Inst., Jupiter, FL (United States); Yang, Dong [Scripps Research Inst., Jupiter, FL (United States); Clancy, Shonda [Argonne National Lab. (ANL), Argonne, IL (United States); Babnigg, Gyorgy [Argonne National Lab. (ANL), Argonne, IL (United States); Joachimiak, Andrzej [Argonne National Lab. (ANL), Argonne, IL (United States); Phillips, George N. [Rice Univ., Houston, TX (United States); Shen, Ben [Scripps Research Inst., Jupiter, FL (United States)

    2015-11-17

    The bleomycins (BLMs), tallysomycins (TLMs), phleomycin, and zorbamycin (ZBM) are members of the BLM family of glycopeptide-derived antitumor antibiotics. The BLM-producing Streptomyces verticillus ATCC15003 and the TLM-producing Streptoalloteichus hindustanus E465-94 ATCC31158 both possess at least two self-resistance elements, an N-acetyltransferase and a binding protein. The N-acetyltransferase provides resistance by disrupting the metal-binding domain of the antibiotic that is required for activity, while the binding protein confers resistance by sequestering the metal-bound antibiotic and preventing drug activation via molecular oxygen. We recently established that the ZBM producer, Streptomyces flavoviridis ATCC21892, lacks the N-acetyltransferase resistance gene and that the ZBM-binding protein, ZbmA, is sufficient to confer resistance in the producing strain. To investigate the resistance mechanism attributed to ZbmA, we determined the crystal structures of apo and Cu(II)-ZBM-bound ZbmA at high resolutions of 1.90 and 1.65 angstrom, respectively. A comparison and contrast with other structurally characterized members of the BLM-binding protein family revealed key differences in the protein ligand binding environment that fine-tunes the ability of ZbmA to sequester metal-bound ZBM and supports drug sequestration as the primary resistance mechanism in the producing organisms of the BLM family of antitumor antibiotics.

  18. Plant RNA binding proteins for control of RNA virus infection

    Directory of Open Access Journals (Sweden)

    Sung Un eHuh

    2013-12-01

    Full Text Available Plant RNA viruses have effective strategies to infect host plants through either direct or indirect interactions with various host proteins, thus suppressing the host immune system. When plant RNA viruses enter host cells exposed RNAs of viruses are recognized by the host immune system through processes such as siRNA-dependent silencing. Interestingly, some host RNA binding proteins have been involved in the inhibition of RNA virus replication, movement, and translation through RNA-specific binding. Host plants intensively use RNA binding proteins for defense against viral infections in nature. In this mini review, we will summarize the function of some host RNA binding proteins which act in a sequence-specific binding manner to the infecting virus RNA. It is important to understand how plants effectively suppresses RNA virus infections via RNA binding proteins, and this defense system can be potentially developed as a synthetic virus defense strategy for use in crop engineering.

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

    Science.gov (United States)

    Chen, Eileen; Joseph, Simpson

    2015-07-01

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

  20. Oxygen infrared spectra of oxyhemoglobins and oxymyoglobins. Evidence of two major liganded O2 structures

    International Nuclear Information System (INIS)

    Potter, W.T.; Tucker, M.P.; Houtchens, R.A.; Caughey, W.S.

    1987-01-01

    The dioxygen stretch bands in infrared spectra for solutions of oxy species of human hemoglobin A and its separated subunits, human mutant hemoglobin Zurich (β63His to Arg), rabbit hemoglobin, lamprey, hemoglobin, sperm whale myoglobin, bovine myoglobin, and a sea worm chlorocruorin are examined. Each protein exhibits multiple isotope-sensitive bands between 1160 and 1060 cm -1 for the liganded 16 O 2 , 17 O 2 , and 18 O 2 . The O-O stretch bands for each of the mammalian myoglobins and hemoglobins are similar, with frequencies that differ between proteins by only 3-5 cm -1 . The spectra for the lamprey and sea worm hemoglobins exhibit greater diversity. For all proteins an O-O stretch band expected to occur near 1125 cm -1 for 16 O 2 and 17 O 2 , but not 18 O 2 , appears split by ∼25 cm -1 due to an unidentified perturbation. The spectrum for each dioxygen isotope, if unperturbed, would contain two strong bands for the mammalian myoglobins (1150 and 1120 cm -1 ) and hemoglobins (1155 and 1125 cm -1 ). Two strong bands separated by ∼30 cm -1 for each oxy heme protein subunit indicate that two major protein conformations (structure) that differ substantially in O 2 bonding are present. The two dioxygen structures can result from a combination of dynamic distal and proximal effects upon the O 2 ligand bound in a bent-end-on stereochemistry

  1. Detection and properties of A-factor-binding protein from Streptomyces griseus

    International Nuclear Information System (INIS)

    Miyake, K.; Horinouchi, S.; Yoshida, M.; Chiba, N.; Mori, K.; Nogawa, N.; Morikawa, N.; Beppu, T.

    1989-01-01

    The optically active form of tritium-labeled A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone), a pleiotropic autoregulator responsible for streptomycin production, streptomycin resistance, and sporulation in Streptomyces griseus, was chemically synthesized. By using the radioactive A-factor, a binding protein for A-factor was detected in the cytoplasmic fraction of this organism. The binding protein had an apparent molecular weight of approximately 26,000, as determined by gel filtration. Scatchard analysis suggested that A-factor bound the protein in the molar ratio of 1:1 with a binding constant, Kd, of 0.7 nM. The number of the binding protein was roughly estimated to be 37 per genome. The inducing material virginiae butanolide C (VB-C), which has a structure very similar to that of A-factor and is essential for virginiamycin production in Streptomyces virginiae, did not inhibit binding. In addition, no protein capable of specifically binding 3 H-labeled VB-C was found in S. griseus. Together with the observation that VB-C had almost no biological activity on the restoration of streptomycin production or sporulation in an A-factor-deficient mutant of S. griseus, these results indicated that the binding protein had a strict ligand specificity. Examination for an A-factor-binding protein in Streptomyces coelicolor A3(2) and Streptomyces lividans showed the absence of any specifically binding protein

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-07-27

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

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

    Science.gov (United States)

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

    2017-09-15

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

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

    Science.gov (United States)

    Messina, Troy; Talaga, David

    2006-03-01

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

  5. Functional Advantages of Conserved Intrinsic Disorder in RNA-Binding Proteins.

    Science.gov (United States)

    Varadi, Mihaly; Zsolyomi, Fruzsina; Guharoy, Mainak; Tompa, Peter

    2015-01-01

    Proteins form large macromolecular assemblies with RNA that govern essential molecular processes. RNA-binding proteins have often been associated with conformational flexibility, yet the extent and functional implications of their intrinsic disorder have never been fully assessed. Here, through large-scale analysis of comprehensive protein sequence and structure datasets we demonstrate the prevalence of intrinsic structural disorder in RNA-binding proteins and domains. We addressed their functionality through a quantitative description of the evolutionary conservation of disordered segments involved in binding, and investigated the structural implications of flexibility in terms of conformational stability and interface formation. We conclude that the functional role of intrinsically disordered protein segments in RNA-binding is two-fold: first, these regions establish extended, conserved electrostatic interfaces with RNAs via induced fit. Second, conformational flexibility enables them to target different RNA partners, providing multi-functionality, while also ensuring specificity. These findings emphasize the functional importance of intrinsically disordered regions in RNA-binding proteins.

  6. Functional Advantages of Conserved Intrinsic Disorder in RNA-Binding Proteins.

    Directory of Open Access Journals (Sweden)

    Mihaly Varadi

    Full Text Available Proteins form large macromolecular assemblies with RNA that govern essential molecular processes. RNA-binding proteins have often been associated with conformational flexibility, yet the extent and functional implications of their intrinsic disorder have never been fully assessed. Here, through large-scale analysis of comprehensive protein sequence and structure datasets we demonstrate the prevalence of intrinsic structural disorder in RNA-binding proteins and domains. We addressed their functionality through a quantitative description of the evolutionary conservation of disordered segments involved in binding, and investigated the structural implications of flexibility in terms of conformational stability and interface formation. We conclude that the functional role of intrinsically disordered protein segments in RNA-binding is two-fold: first, these regions establish extended, conserved electrostatic interfaces with RNAs via induced fit. Second, conformational flexibility enables them to target different RNA partners, providing multi-functionality, while also ensuring specificity. These findings emphasize the functional importance of intrinsically disordered regions in RNA-binding proteins.

  7. A theoretical model for oxygen transport in skeletal muscle under conditions of high oxygen demand.

    Science.gov (United States)

    McGuire, B J; Secomb, T W

    2001-11-01

    Oxygen transport from capillaries to exercising skeletal muscle is studied by use of a Krogh-type cylinder model. The goal is to predict oxygen consumption under conditions of high demand, on the basis of a consideration of transport processes occurring at the microvascular level. Effects of the decline in oxygen content of blood flowing along capillaries, intravascular resistance to oxygen diffusion, and myoglobin-facilitated diffusion are included. Parameter values are based on human skeletal muscle. The dependence of oxygen consumption on oxygen demand, perfusion, and capillary density are examined. When demand is moderate, the tissue is well oxygenated and consumption is slightly less than demand. When demand is high, capillary oxygen content declines rapidly with axial distance and radial oxygen transport is limited by diffusion resistance within the capillary and the tissue. Under these conditions, much of the tissue is hypoxic, consumption is substantially less than demand, and consumption is strongly dependent on capillary density. Predicted consumption rates are comparable with experimentally observed maximal rates of oxygen consumption.

  8. Motions and electrostatic interactions in natural and semisynthetic myoglobins: a carbon-13 nuclear magnetic resonance study

    International Nuclear Information System (INIS)

    Maskalick, D.G.

    1984-01-01

    It is expected that the internal motions of amino acid side chains and protein backbone segments influence and are in turn affected by charge-charge and related interactions, steric constraints, hydrophobic forces, and hydrogen bonding. As an initial test of this theory 13 C-enriched glycine, alanine, and isoleucine have been substituted for the amino terminal valine of sperm whale myoglobin using semisynthetic techniques. 13 C-NMR has been used to analyze the motions of the side chain and the protonation state of the alpha amino group as a function of pH. The addition of a single methyl group to the side chain can alter the alpha amino pK value by as much as 0.3 pH units indicating a delicately balanced set of change-charge interactions between the alpha amino group and the rest of the protein. Further evidence in support of the state theory was found upon examination of the internal motions of seven of nine isoleucine vectors. These motions were extracted from natural abundance 13 C-NMR relaxation data. The results suggest a strong possibility that concerted motions are important. Also, an increase in temperature from 32 0 C to 52 0 C leads to an electrostatically driven tightening of the myoglobin structure as evidenced by no significant increase in motion amplitude of most of the vectors

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

    International Nuclear Information System (INIS)

    Matsumoto, Hideaki; Takahashi, Eiichi

    1976-01-01

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

  10. Agitation down-regulates immunoglobulin binding protein EibG expression in Shiga toxin-producing Escherichia coli (STEC.

    Directory of Open Access Journals (Sweden)

    Thorsten Kuczius

    Full Text Available Shiga toxin (Stx-producing Escherichia coli (STEC carrying eibG synthesize Escherichia coli immunoglobulin binding protein (EibG. EibG nonspecifically binds to immunoglobulins and tends to aggregate in multimers but is poorly expressed in wild-type strains. To study synthesis of the proteins and their regulation in the pathogens, we identified natural growth conditions that increased EibG synthesis. EibG proteins as well as corresponding mRNA were highly expressed under static growth conditions while shearing stress created by agitation during growth repressed protein synthesis. Further regulation effects were driven by reduced oxygen tension, and pH up-regulated EibG expression, but to a lesser extent than growth conditions while decreased temperature down-regulated EibG. Bacteria with increased EibG expression during static growth conditions showed a distinct phenotype with chain formation and biofilm generation, which disappeared with motion. High and low EibG expression was reversible indicating a process with up- and down-regulation of the protein expression. Our findings indicate that shear stress represses EibG expression and might reduce bacterial attachments to cells and surfaces.

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

    Science.gov (United States)

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

    2016-01-29

    We use evolutionary conservation derived from structure alignment of polypeptide sequences along with structural and physicochemical attributes of protein-RNA interfaces to probe the binding hot spots at protein-RNA recognition sites. We find that the degree of conservation varies across the RNA binding proteins; some evolve rapidly compared to others. Additionally, irrespective of the structural class of the complexes, residues at the RNA binding sites are evolutionary better conserved than those at the solvent exposed surfaces. For recognitions involving duplex RNA, residues interacting with the major groove are better conserved than those interacting with the minor groove. We identify multi-interface residues participating simultaneously in protein-protein and protein-RNA interfaces in complexes where more than one polypeptide is involved in RNA recognition, and show that they are better conserved compared to any other RNA binding residues. We find that the residues at water preservation site are better conserved than those at hydrated or at dehydrated sites. Finally, we develop a Random Forests model using structural and physicochemical attributes for predicting binding hot spots. The model accurately predicts 80% of the instances of experimental ΔΔG values in a particular class, and provides a stepping-stone towards the engineering of protein-RNA recognition sites with desired affinity. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. Relationship between hot spot residues and ligand binding hot spots in protein-protein interfaces.

    Science.gov (United States)

    Zerbe, Brandon S; Hall, David R; Vajda, Sandor; Whitty, Adrian; Kozakov, Dima

    2012-08-27

    In the context of protein-protein interactions, the term "hot spot" refers to a residue or cluster of residues that makes a major contribution to the binding free energy, as determined by alanine scanning mutagenesis. In contrast, in pharmaceutical research, a hot spot is a site on a target protein that has high propensity for ligand binding and hence is potentially important for drug discovery. Here we examine the relationship between these two hot spot concepts by comparing alanine scanning data for a set of 15 proteins with results from mapping the protein surfaces for sites that can bind fragment-sized small molecules. We find the two types of hot spots are largely complementary; the residues protruding into hot spot regions identified by computational mapping or experimental fragment screening are almost always themselves hot spot residues as defined by alanine scanning experiments. Conversely, a residue that is found by alanine scanning to contribute little to binding rarely interacts with hot spot regions on the partner protein identified by fragment mapping. In spite of the strong correlation between the two hot spot concepts, they fundamentally differ, however. In particular, while identification of a hot spot by alanine scanning establishes the potential to generate substantial interaction energy with a binding partner, there are additional topological requirements to be a hot spot for small molecule binding. Hence, only a minority of hot spots identified by alanine scanning represent sites that are potentially useful for small inhibitor binding, and it is this subset that is identified by experimental or computational fragment screening.

  13. Characterization of a Chitin-Binding Protein from Bacillus thuringiensis HD-1.

    Directory of Open Access Journals (Sweden)

    Naresh Arora

    Full Text Available Strains of Bacillus thuringiensis produce insecticidal proteins. These strains have been isolated from diverse ecological niches, such as soil, phylloplane, insect cadavers and grain dust. To effectively propagate, these strains produce a range of molecules that facilitate its multiplication in a competing environment. In this report, we have examined synthesis of a chitin-binding protein and evaluated its effect on fungi encountered in environment and its interaction with insecticidal proteins synthesized by B. thuringiensis. The gene encoding chitin-binding protein has been cloned and expressed. The purified protein has been demonstrated to interact with Cry insecticidal protein, Cry1Ac by Circular Dichrosim spectroscopy (CD and in vitro pull down assays. The chitin-binding protein potentiates insecticidal activity of bacillar insecticidal protein, Cry1Ac. Further, chitin-binding protein was fungistatic against several soil fungi. The chitin binding protein is expressed in spore mother cell and deposited along with insecticidal protein, Cry1Ac. It interacts with Cry1Ac to potentiate its insecticidal activity and facilitate propagation of Bacillus strain in environment by inhibiting growth of certain fungi.

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

    Directory of Open Access Journals (Sweden)

    Vesa Kirjavainen

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

  15. Characterization and Expression of the Lucina pectinata Oxygen and Sulfide Binding Hemoglobin Genes

    Science.gov (United States)

    López-Garriga, Juan; Cadilla, Carmen L.

    2016-01-01

    The clam Lucina pectinata lives in sulfide-rich muds and houses intracellular symbiotic bacteria that need to be supplied with hydrogen sulfide and oxygen. This clam possesses three hemoglobins: hemoglobin I (HbI), a sulfide-reactive protein, and hemoglobin II (HbII) and III (HbIII), which are oxygen-reactive. We characterized the complete gene sequence and promoter regions for the oxygen reactive hemoglobins and the partial structure and promoters of the HbI gene from Lucina pectinata. We show that HbI has two mRNA variants, where the 5’end had either a sequence of 96 bp (long variant) or 37 bp (short variant). The gene structure of the oxygen reactive Hbs is defined by having 4-exons/3-introns with conservation of intron location at B12.2 and G7.0 and the presence of pre-coding introns, while the partial gene structure of HbI has the same intron conservation but appears to have a 5-exon/ 4-intron structure. A search for putative transcription factor binding sites (TFBSs) was done with the promoters for HbII, HbIII, HbI short and HbI long. The HbII, HbIII and HbI long promoters showed similar predicted TFBSs. We also characterized MITE-like elements in the HbI and HbII gene promoters and intronic regions that are similar to sequences found in other mollusk genomes. The gene expression levels of the clam Hbs, from sulfide-rich and sulfide-poor environments showed a significant decrease of expression in the symbiont-containing tissue for those clams in a sulfide-poor environment, suggesting that the sulfide concentration may be involved in the regulation of these proteins. Gene expression evaluation of the two HbI mRNA variants indicated that the longer variant is expressed at higher levels than the shorter variant in both environments. PMID:26824233

  16. Echinococcus granulosus fatty acid binding proteins subcellular localization.

    Science.gov (United States)

    Alvite, Gabriela; Esteves, Adriana

    2016-05-01

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

  17. Calcium phosphate–gold nanoparticles nanocomposite for protein adsorption and mediator-free H2O2 biosensor construction

    International Nuclear Information System (INIS)

    Xu Qin; Lu Guiju; Bian XiaoJun; Jin Gendi; Wang Wei; Hu Xiaoya; Wang Yang; Yang Zhanjun

    2012-01-01

    This work reports a new method for the preparation and application of a kind of biocompatible calcium phosphate–gold nanoparticles (Ca 3 (PO 4 ) 2 –AuNPs) nanocomposite. UV–vis spectroscopy and transmittance electron microscopy (TEM) have been used to monitor the formation process of the nanocomposite and to examine the interaction between calcium phosphate and gold nanoparticles (AuNPs). The nanocomposite has multiple sites and improved conductivity which make it suitable for the binding of proteins to construct electrochemical sensors. Myoglobin (Mb) adsorbed on the nanocomposite retained its native structure which was proved by Fourier transform infrared spectroscopy (FTIR). Direct electron transfer between the adsorbed Mb and the electrode was observed. Further results demonstrated that the adsorbed Mb has good electrocatalytic activity towards the reduction of H 2 O 2 in the absence of any mediator. Highlights: ► Using gelatin modified gold nanoparticles to prepare needle-like calcium phosphate. ► Calcium phosphate provides multiple sites for protein adsorption. ► Gold nanoparticles act as electron tunneling. ► Myoglobin adsorbed on the material showed direct electrochemistry and good catalysis.

  18. Quantifying drug-protein binding in vivo

    International Nuclear Information System (INIS)

    Buchholz, B; Bench, G; Keating III, G; Palmblad, M; Vogel, J; Grant, P G; Hillegonds, D

    2004-01-01

    Accelerator mass spectrometry (AMS) provides precise quantitation of isotope labeled compounds that are bound to biological macromolecules such as DNA or proteins. The sensitivity is high enough to allow for sub-pharmacological (''micro-'') dosing to determine macromolecular targets without inducing toxicities or altering the system under study, whether it is healthy or diseased. We demonstrated an application of AMS in quantifying the physiologic effects of one dosed chemical compound upon the binding level of another compound in vivo at sub-toxic doses [4].We are using tissues left from this study to develop protocols for quantifying specific binding to isolated and identified proteins. We also developed a new technique to quantify nanogram to milligram amounts of isolated protein at precisions that are comparable to those for quantifying the bound compound by AMS

  19. Myoglobin production in emperor penguins.

    Science.gov (United States)

    Ponganis, P J; Welch, T J; Welch, L S; Stockard, T K

    2010-06-01

    Increased oxygen storage is essential to the diving capacities of marine mammals and seabirds. However, the molecular mechanisms underlying this adaptation are unknown. Myoglobin (Mb) and Mb mRNA concentrations were analyzed in emperor penguin (Aptenodytes forsteri) adults and chicks with spectrophotometric and RNase protection assays to evaluate production of their large Mb-bound O(2) stores. Mean pectoral Mb concentration and Mb mRNA content increased throughout the pre-fledging period and were 15-fold and 3-fold greater, respectively, in adults than in 3.5 month old chicks. Mean Mb concentration in 5.9 month old juveniles was 2.7+/-0.4 g 100 g(-1) muscle (44% that of wild adults), and in adults that had been captive all their lives it was 3.7+/-0.1 g 100 g(-1) muscle. The Mb and Mb mRNA data are consistent with regulation of Mb production at the level of transcription as in other animals. Significant Mb and Mb mRNA production occurred in chicks and young juveniles even without any diving activity. The further increase in adult Mb concentrations appears to require the exercise/hypoxia of diving because Mb concentration in captive, non-diving adults only reached 60% of that of wild adults. The much greater relative increase in Mb concentration than in Mb mRNA content between young chicks and adults suggests that there is not a simple 1:1 relationship between Mb mRNA content and Mb concentration. Nutritional limitation in young chicks and post-transcriptional regulation of Mb concentration may also be involved.

  20. Identification and characterization of riboflavin-binding proteins in human circulation

    International Nuclear Information System (INIS)

    Innis-Whitehouse, W.S.A.

    1988-01-01

    Riboflavin binding by plasma proteins from healthy human subjects was examined by equilibrium dialysis and binding was observed to vary over a greater than 10-fold range. Upon fractionation of plasma by gel filtration, the major riboflavin-binding components eluted with albumin and gamma-globulins. Albumin was purified and found to bind riboflavin only very weakly, although FMN and photo-chemical degradation products were more tightly bound. Most of the binding occurred in the gamma-globulin fraction and was attributed to immunoglobulins because the binding proteins and immunoglobulins copurified using various methods, were removed by treatment of plasma with protein A-agarose, and were coincident upon immuno-electrophoresis followed by autoradiography to detect [2- 14 C]-riboflavin. Binding differences among plasma samples were reflected in the binding recovered with the immunoglobulin fractions; however, there was not a direct relationship between the amount of immunoglobulin and the amount of [2- 14 C]riboflavin bound. Hence, it appeared that the binding was due to a subfraction of immunoglobulins

  1. NifI inhibits nitrogenase by competing with Fe protein for binding to the MoFe protein

    International Nuclear Information System (INIS)

    Dodsworth, Jeremy A.; Leigh, John A.

    2007-01-01

    Reduction of substrate by nitrogenase requires direct electron transfer from the Fe protein to the MoFe protein. Inhibition of nitrogenase activity in Methanococcus maripaludis occurs when the regulatory protein NifI 1,2 binds the MoFe protein. This inhibition is relieved by 2-oxoglutarate. Here we present evidence that NifI 1,2 binding prevents association of the two nitrogenase components. Increasing amounts of Fe protein competed with NifI 1,2 , decreasing its inhibitory effect. NifI 1,2 prevented the co-purification of MoFe protein with a mutant form of the Fe protein that forms a stable complex with the MoFe protein, and NifI 1,2 was unable to bind to an AlF 4 - -stabilized Fe protein:MoFe protein complex. NifI 1,2 inhibited ATP- and MoFe protein-dependent oxidation of the Fe protein, and 2OG relieved this inhibition. These results support a model where NifI 1,2 competes with the Fe protein for binding to MoFe protein and prevents electron transfer

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

    Science.gov (United States)

    Khorshid, Mohsen; Rodak, Christoph; Zavolan, Mihaela

    2011-01-01

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

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

    Science.gov (United States)

    Lam, Pui-Man; Zhen, Yi

    2011-04-01

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

  4. [Determination of plasma protein binding rate of arctiin and arctigenin with ultrafiltration].

    Science.gov (United States)

    Han, Xue-Ying; Wang, Wei; Tan, Ri-Qiu; Dou, De-Qiang

    2013-02-01

    To determine the plasma protein binding rate of arctiin and arctigenin. The ultrafiltration combined with HPLC was employed to determine the plasma protein binding rate of arctiin and arctigenin as well as rat plasma and healthy human plasma proteins. The plasma protein binding rate of arctiin with rat plasma at the concentrations of 64. 29, 32.14, 16.07 mg x L(-1) were (71.2 +/- 2.0)%, (73.4 +/- 0.61)%, (78.2 +/- 1.9)%, respectively; while the plasma protein binding rate of arctiin with healthy human plasma at the above concentrations were (64.8 +/- 3.1)%, (64.5 +/- 2.5)%, (77.5 +/- 1.7)%, respectively. The plasma protein binding rate of arctigenin with rat plasma at the concentrations of 77.42, 38.71, 19.36 mg x L(-1) were (96.7 +/- 0.41)%, (96.8 +/- 1.6)%, (97.3 +/- 0.46)%, respectively; while the plasma protein binding rate of arctigenin with normal human plasma at the above concentrations were (94.7 +/- 3.1)%, (96.8 +/- 1.6)%, (97.9 +/- 1.3)%, respectively. The binding rate of arctiin with rat plasma protein was moderate, which is slightly higher than the binding rate of arctiin with healthy human plasma protein. The plasma protein binding rates of arctigenin with both rat plasma and healthy human plasma are very high.

  5. Bridging the gap between chemistry, physiology, and evolution: Quantifying the functionality of sperm whale myoglobin mutants

    DEFF Research Database (Denmark)

    Dasmeh, Pouria; Kepp, Kasper Planeta

    2011-01-01

    This work merges a large set of previously reported thermochemical data for myoglobin (Mb) mutants with a physiological model of O2-transport and -storage. The model allows a quantification of the functional proficiency of myoglobin (Mb) mutants under various physiological conditions, i.e. O2-con...

  6. A hydrogen-donating monohydroxamate scavenges ferryl myoglobin radicals

    DEFF Research Database (Denmark)

    Cooper, C E; Green, E S; Rice-Evans, C A

    1994-01-01

    The addition of 25 microM hydrogen peroxide to 20 microM metmyoglobin produces ferryl (FeIV = O) myoglobin. Optical spectroscopy shows that the ferryl species reaches a maximum concentration (60-70% of total haem) after 10 minutes and decays slowly (hours). Low temperature EPR spectroscopy of the...

  7. Predicting binding affinities of protein ligands from three-dimensional models: application to peptide binding to class I major histocompatibility proteins

    DEFF Research Database (Denmark)

    Rognan, D; Lauemoller, S L; Holm, A

    1999-01-01

    A simple and fast free energy scoring function (Fresno) has been developed to predict the binding free energy of peptides to class I major histocompatibility (MHC) proteins. It differs from existing scoring functions mainly by the explicit treatment of ligand desolvation and of unfavorable protein...... coordinates of the MHC-bound peptide have first been determined with an accuracy of about 1-1.5 A. Furthermore, it may be easily recalibrated for any protein-ligand complex.......) and of a series of 16 peptides to H-2K(k). Predictions were more accurate for HLA-A2-binding peptides as the training set had been built from experimentally determined structures. The average error in predicting the binding free energy of the test peptides was 3.1 kJ/mol. For the homology model-derived equation...

  8. The RNA-binding protein repertoire of Arabidopsis thaliana

    KAUST Repository

    Marondedze, Claudius

    2016-07-11

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

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

    Science.gov (United States)

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

    2012-02-01

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

  10. Analysis of electric moments of RNA-binding proteins: implications for mechanism and prediction

    Directory of Open Access Journals (Sweden)

    Sarai Akinori

    2011-02-01

    Full Text Available Abstract Background Protein-RNA interactions play important role in many biological processes such as gene regulation, replication, protein synthesis and virus assembly. Although many structures of various types of protein-RNA complexes have been determined, the mechanism of protein-RNA recognition remains elusive. We have earlier shown that the simplest electrostatic properties viz. charge, dipole and quadrupole moments, calculated from backbone atomic coordinates of proteins are biased relative to other proteins, and these quantities can be used to identify DNA-binding proteins. Closely related, RNA-binding proteins are investigated in this study. In particular, discrimination between various types of RNA-binding proteins, evolutionary conservation of these bulk electrostatic features and effect of conformational changes by complex formation are investigated. Basic binding mechanism of a putative RNA-binding protein (HI1333 from Haemophilus influenza is suggested as a potential application of this study. Results We found that similar to DNA-binding proteins (DBPs, RNA-binding proteins (RBPs also show significantly higher values of electric moments. However, higher moments in RBPs are found to strongly depend on their functional class: proteins binding to ribosomal RNA (rRNA constitute the only class with all three of the properties (charge, dipole and quadrupole moments being higher than control proteins. Neural networks were trained using leave-one-out cross-validation to predict RBPs from control data as well as pair-wise classification capacity between proteins binding to various RNA types. RBPs and control proteins reached up to 78% accuracy measured by the area under the ROC curve. Proteins binding to rRNA are found to be best distinguished (AUC = 79%. Changes in dipole and quadrupole moments between unbound and bound structures were small and these properties are found to be robust under complex formation. Conclusions Bulk electric

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

    Directory of Open Access Journals (Sweden)

    Yu Zhu

    2015-11-01

    Full Text Available Chitin-binding proteins (CBPs are present in many species and they act in a variety of biological processes. We analyzed a Pteromalus puparum venom apparatus proteome and transcriptome and identified a partial gene encoding a possible CBP. Here, we report cloning a full-length cDNA of a sequence encoding a chitin-binding-like protein (PpCBP from P. puparum, a pupal endoparasitoid of Pieris rapae. The cDNA encoded a 96-amino-acid protein, including a secretory signal peptide and a chitin-binding peritrophin-A domain. Phylogenetic analysis of chitin binding domains (CBDs of cuticle proteins and peritrophic matrix proteins in selected insects revealed that the CBD of PpCBP clustered with the CBD of Nasonia vitripennis. The PpCBP is specifically expressed in the venom apparatus of P. puparum, mostly in the venom gland. PpCBP expression was highest at day one after adult eclosion and much lower for the following five days. We produced a recombinant PpCBP and binding assays showed the recombinant protein selectively binds chitin but not cellulose in vitro. We infer that PpCBP serves a structural role in the venom reservoir, or may be injected into the host to help wound healing of the host exoskeleton.

  12. Identification of Arsenic Direct-Binding Proteins in Acute Promyelocytic Leukaemia Cells

    Directory of Open Access Journals (Sweden)

    Tao Zhang

    2015-11-01

    Full Text Available The identification of arsenic direct-binding proteins is essential for determining the mechanism by which arsenic trioxide achieves its chemotherapeutic effects. At least two cysteines close together in the amino acid sequence are crucial to the binding of arsenic and essential to the identification of arsenic-binding proteins. In the present study, arsenic binding proteins were pulled down with streptavidin and identified using a liquid chromatograph-mass spectrometer (LC-MS/MS. More than 40 arsenic-binding proteins were separated, and redox-related proteins, glutathione S-transferase P1 (GSTP1, heat shock 70 kDa protein 9 (HSPA9 and pyruvate kinase M2 (PKM2, were further studied using binding assays in vitro. Notably, PKM2 has a high affinity for arsenic. In contrast to PKM2, GSTP1and HSPA9 did not combine with arsenic directly in vitro. These observations suggest that arsenic-mediated acute promyelocytic leukaemia (APL suppressive effects involve PKM2. In summary, we identified several arsenic binding proteins in APL cells and investigated the therapeutic mechanisms of arsenic trioxide for APL. Further investigation into specific signal pathways by which PKM2 mediates APL developments may lead to a better understanding of arsenic effects on APL.

  13. DMPD: LPS-binding proteins and receptors. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 9665271 LPS-binding proteins and receptors. Fenton MJ, Golenbock DT. J Leukoc Biol.... 1998 Jul;64(1):25-32. (.png) (.svg) (.html) (.csml) Show LPS-binding proteins and receptors. PubmedID 9665271 Title LPS-binding prot...eins and receptors. Authors Fenton MJ, Golenbock DT. Publication J Leukoc Biol. 199

  14. Effects of Fiber Type and Size on the Heterogeneity of Oxygen Distribution in Exercising Skeletal Muscle

    Science.gov (United States)

    Liu, Gang; Mac Gabhann, Feilim; Popel, Aleksander S.

    2012-01-01

    The process of oxygen delivery from capillary to muscle fiber is essential for a tissue with variable oxygen demand, such as skeletal muscle. Oxygen distribution in exercising skeletal muscle is regulated by convective oxygen transport in the blood vessels, oxygen diffusion and consumption in the tissue. Spatial heterogeneities in oxygen supply, such as microvascular architecture and hemodynamic variables, had been observed experimentally and their marked effects on oxygen exchange had been confirmed using mathematical models. In this study, we investigate the effects of heterogeneities in oxygen demand on tissue oxygenation distribution using a multiscale oxygen transport model. Muscles are composed of different ratios of the various fiber types. Each fiber type has characteristic values of several parameters, including fiber size, oxygen consumption, myoglobin concentration, and oxygen diffusivity. Using experimentally measured parameters for different fiber types and applying them to the rat extensor digitorum longus muscle, we evaluated the effects of heterogeneous fiber size and fiber type properties on the oxygen distribution profile. Our simulation results suggest a marked increase in spatial heterogeneity of oxygen due to fiber size distribution in a mixed muscle. Our simulations also suggest that the combined effects of fiber type properties, except size, do not contribute significantly to the tissue oxygen spatial heterogeneity. However, the incorporation of the difference in oxygen consumption rates of different fiber types alone causes higher oxygen heterogeneity compared to control cases with uniform fiber properties. In contrast, incorporating variation in other fiber type-specific properties, such as myoglobin concentration, causes little change in spatial tissue oxygenation profiles. PMID:23028531

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

    DEFF Research Database (Denmark)

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

    1996-01-01

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

  16. Increasing Protein Charge State When Using Laser Electrospray Mass Spectrometry

    Science.gov (United States)

    Karki, Santosh; Flanigan, Paul M.; Perez, Johnny J.; Archer, Jieutonne J.; Levis, Robert J.

    2015-05-01

    Femtosecond (fs) laser vaporization is used to transfer cytochrome c, myoglobin, lysozyme, and ubiquitin from the condensed phase into an electrospray (ES) plume consisting of a mixture of a supercharging reagent, m-nitrobenzyl alcohol ( m-NBA), and trifluoroacetic acid (TFA), acetic acid (AA), or formic acid (FA). Interaction of acid-sensitive proteins like cytochrome c and myoglobin with the highly charged ES droplets resulted in a shift to higher charge states in comparison with acid-stable proteins like lysozyme and ubiquitin. Laser electrospray mass spectrometry (LEMS) measurements showed an increase in both the average charge states (Zavg) and the charge state with maximum intensity (Zmode) for acid-sensitive proteins compared with conventional electrospray ionization mass spectrometry (ESI-MS) under equivalent solvent conditions. A marked increase in ion abundance of higher charge states was observed for LEMS in comparison with conventional electrospray for cytochrome c (ranging from 19+ to 21+ versus 13+ to 16+) and myoglobin (ranging from 19+ to 26+ versus 18+ to 21+) using an ES solution containing m-NBA and TFA. LEMS measurements as a function of electrospray flow rate yielded increasing charge states with decreasing flow rates for cytochrome c and myoglobin.

  17. Genome-wide identification, sequence characterization, and protein-protein interaction properties of DDB1 (damaged DNA binding protein-1)-binding WD40-repeat family members in Solanum lycopersicum.

    Science.gov (United States)

    Zhu, Yunye; Huang, Shengxiong; Miao, Min; Tang, Xiaofeng; Yue, Junyang; Wang, Wenjie; Liu, Yongsheng

    2015-06-01

    One hundred DDB1 (damaged DNA binding protein-1)-binding WD40-repeat domain (DWD) family genes were identified in the S. lycopersicum genome. The DWD genes encode proteins presumably functioning as the substrate recognition subunits of the cullin4-ring ubiquitin E3 ligase complex. These findings provide candidate genes and a research platform for further gene functionality and molecular breeding study. A subclass of DDB1 (damaged DNA binding protein-1)-binding WD40-repeat domain (DWD) family proteins has been demonstrated to function as the substrate recognition subunits of the cullin4-ring ubiquitin E3 ligase complex. However, little information is available about the cognate subfamily genes in tomato (S. lycopersicum). In this study, based on the recently released tomato genome sequences, 100 tomato genes encoding DWD proteins that potentially interact with DDB1 were identified and characterized, including analyses of the detailed annotations, chromosome locations and compositions of conserved amino acid domains. In addition, a phylogenetic tree, which comprises of three main groups, of the subfamily genes was constructed. The physical interaction between tomato DDB1 and 14 representative DWD proteins was determined by yeast two-hybrid and co-immunoprecipitation assays. The subcellular localization of these 14 representative DWD proteins was determined. Six of them were localized in both nucleus and cytoplasm, seven proteins exclusively in cytoplasm, and one protein either in nucleus and cytoplasm, or exclusively in cytoplasm. Comparative genomic analysis demonstrated that the expansion of these subfamily members in tomato predominantly resulted from two whole-genome triplication events in the evolution history.

  18. Serotonin binding in vitro by releasable proteins from human blood platelets

    International Nuclear Information System (INIS)

    Heemstra, V.L.

    1983-11-01

    Among the substances released from human blood platelets are serotonin and various proteins. It was hypothesized that one of these proteins binds serotonin and that serotonin might be important to the protein's function or that the protein might be important to serotonin's function. Two platelet-specific proteins, platelet factor 4 (PF4) and β-thromboglobulin (βTG) were found to bind serotonin in vitro. Endogenous PF4 was isolated by serotonin-affinity chromatography and was identified by radioimmunoassay. Purified [ 125 I] -PF4 and native PF4 bound to and eluted from a serotonin-affinity column similarly. Ultrafiltration of the homologous protein, βTG, with [ 14 C]-serotonin demonstrated binding of about 8 moles serotonin per mole tetrameric βTG with a dissociation constant of about 4 X 10(sup-8) M. Equilibrium dialysis of PF4 with radiolabelled serotonin was attempted, but no binding constant values were obtained because serotonin apparently bound to the dialysis membrane. Since EDTA was one of the two agents that eluted PF4 from the serotonin-affinity gel, calcium binding by PF4 was investigated by equilibrium dialysis. Evidence was obtained for positively cooperative binding of calcium ions by PF4. It is concluded that PF4 and βTG bind serotonin in vitro, that they may also bind in vivo when platelets undergo release, and that the functions of serotonin, PF4 and βTG may be mediated in part by serotonin-protein associations

  19. Differential plasma protein binding to metal oxide nanoparticles

    International Nuclear Information System (INIS)

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

    2009-01-01

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

  20. The expression of selenium-binding protein 1 is decreased in uterine leiomyoma

    Directory of Open Access Journals (Sweden)

    Quddus M Ruhul

    2010-12-01

    Full Text Available Abstract Background Selenium has been shown to inhibit cancer development and growth through the mediation of selenium-binding proteins. Decreased expression of selenium-binding protein 1 has been reported in cancers of the prostate, stomach, colon, and lungs. No information, however, is available concerning the roles of selenium-binding protein 1 in uterine leiomyoma. Methods Using Western Blot analysis and immunohistochemistry, we examined the expression of selenium-binding protein 1 in uterine leiomyoma and normal myometrium in 20 patients who had undergone hysterectomy for uterine leiomyoma. Results and Discussion The patient age ranged from 34 to 58 years with a mean of 44.3 years. Proliferative endometrium was seen in 8 patients, secretory endometrium in 7 patients, and atrophic endometrium in 5 patients. Two patients showed solitary leiomyoma, and eighteen patients revealed 2 to 5 tumors. Tumor size ranged from 1 to 15.5 cm with a mean of 4.3 cm. Both Western Blot analysis and immunohistochemistry showed a significant lower level of selenium-binding protein 1 in leiomyoma than in normal myometrium. Larger tumors had a tendency to show a lower level of selenium-binding protein 1 than smaller ones, but the difference did not reach a statistical significance. The expression of selenium-binding protein 1 was the same among patients with proliferative, secretory, and atrophic endometrium in either leiomyoma or normal myometrium. Also, we did not find a difference of selenium-binding protein 1 level between patients younger than 45 years and older patients in either leiomyoma or normal myometrium. Conclusions Decreased expression of selenium-binding protein 1 in uterine leiomyoma may indicate a role of the protein in tumorigenesis. Our findings may provide a basis for future studies concerning the molecular mechanisms of selenium-binding protein 1 in tumorigenesis as well as the possible use of selenium in prevention and treatment of uterine

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

    Science.gov (United States)

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

    2017-03-24

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

  2. Interfacial hydration, dynamics and electron transfer: multi-scale ET modeling of the transient [myoglobin, cytochrome b5] complex.

    Science.gov (United States)

    Keinan, Shahar; Nocek, Judith M; Hoffman, Brian M; Beratan, David N

    2012-10-28

    Formation of a transient [myoglobin (Mb), cytochrome b(5) (cyt b(5))] complex is required for the reductive repair of inactive ferri-Mb to its functional ferro-Mb state. The [Mb, cyt b(5)] complex exhibits dynamic docking (DD), with its cyt b(5) partner in rapid exchange at multiple sites on the Mb surface. A triple mutant (Mb(3M)) was designed as part of efforts to shift the electron-transfer process to the simple docking (SD) regime, in which reactive binding occurs at a restricted, reactive region on the Mb surface that dominates the docked ensemble. An electrostatically-guided brownian dynamics (BD) docking protocol was used to generate an initial ensemble of reactive configurations of the complex between unrelaxed partners. This ensemble samples a broad and diverse array of heme-heme distances and orientations. These configurations seeded all-atom constrained molecular dynamics simulations (MD) to generate relaxed complexes for the calculation of electron tunneling matrix elements (T(DA)) through tunneling-pathway analysis. This procedure for generating an ensemble of relaxed complexes combines the ability of BD calculations to sample the large variety of available conformations and interprotein distances, with the ability of MD to generate the atomic level information, especially regarding the structure of water molecules at the protein-protein interface, that defines electron-tunneling pathways. We used the calculated T(DA) values to compute ET rates for the [Mb(wt), cyt b(5)] complex and for the complex with a mutant that has a binding free energy strengthened by three D/E → K charge-reversal mutations, [Mb(3M), cyt b(5)]. The calculated rate constants are in agreement with the measured values, and the mutant complex ensemble has many more geometries with higher T(DA) values than does the wild-type Mb complex. Interestingly, water plays a double role in this electron-transfer system, lowering the tunneling barrier as well as inducing protein interface

  3. Saccharomyces cerevisiae SSB1 protein and its relationship to nucleolar RNA-binding proteins.

    OpenAIRE

    Jong, A Y; Clark, M W; Gilbert, M; Oehm, A; Campbell, J L

    1987-01-01

    To better define the function of Saccharomyces cerevisiae SSB1, an abundant single-stranded nucleic acid-binding protein, we determined the nucleotide sequence of the SSB1 gene and compared it with those of other proteins of known function. The amino acid sequence contains 293 amino acid residues and has an Mr of 32,853. There are several stretches of sequence characteristic of other eucaryotic single-stranded nucleic acid-binding proteins. At the amino terminus, residues 39 to 54 are highly ...

  4. USING BIOPOLYMERS TO STABILIZE THE PROTEIN OXYGEN FOAM

    Directory of Open Access Journals (Sweden)

    N. V. Nepovinnyh

    2013-01-01

    Full Text Available The cottage cheese whey as an oxygen cocktail foaming base and natural juices as a flavoring ingredient are analyzed. The lifetime of foam generated by the serum proteins is not long: foam falls off rapidly; because from the foam liquid is released (syneresis. The effects of plant polysaccharides on the stabilization of the protein foam oxygen cocktail is studied. It was shown that the use of plant polysaccharides (guar gum, high methoxyl citrus pectin, locust been gum prolong the life of the foam up to 20 times, compared with conventional blowing agents. It was found that oxygen foam properties depend on the molecular weight of guar gum.

  5. A novel signal transduction protein: Combination of solute binding and tandem PAS-like sensor domains in one polypeptide chain: Periplasmic Ligand Binding Protein Dret_0059

    Energy Technology Data Exchange (ETDEWEB)

    Wu, R. [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Biosciences Division, Argonne National Laboratory, Argonne Illinois 60439; Wilton, R. [Biosciences Division, Argonne National Laboratory, Argonne Illinois 60439; Cuff, M. E. [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Biosciences Division, Argonne National Laboratory, Argonne Illinois 60439; Structural Biology Center, Argonne National Laboratory, Argonne Illinois 60439; Endres, M. [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Babnigg, G. [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Biosciences Division, Argonne National Laboratory, Argonne Illinois 60439; Edirisinghe, J. N. [Mathematics and Computer Science Division, Argonne National Laboratory, Argonne Illinois 60439; Computation Institute, University of Chicago, Chicago Illinois 60637; Henry, C. S. [Mathematics and Computer Science Division, Argonne National Laboratory, Argonne Illinois 60439; Computation Institute, University of Chicago, Chicago Illinois 60637; Joachimiak, A. [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Biosciences Division, Argonne National Laboratory, Argonne Illinois 60439; Structural Biology Center, Argonne National Laboratory, Argonne Illinois 60439; Department of Biochemistry and Molecular Biology, University of Chicago, Chicago Illinois 60637; Schiffer, M. [Biosciences Division, Argonne National Laboratory, Argonne Illinois 60439; Pokkuluri, P. R. [Biosciences Division, Argonne National Laboratory, Argonne Illinois 60439

    2017-03-06

    We report the structural and biochemical characterization of a novel periplasmic ligand-binding protein, Dret_0059, from Desulfohalobium retbaense DSM 5692, an organism isolated from the Salt Lake Retba in Senegal. The structure of the protein consists of a unique combination of a periplasmic solute binding protein (SBP) domain at the N-terminal and a tandem PAS-like sensor domain at the C-terminal region. SBP domains are found ubiquitously and their best known function is in solute transport across membranes. PAS-like sensor domains are commonly found in signal transduction proteins. These domains are widely observed as parts of many protein architectures and complexes but have not been observed previously within the same polypeptide chain. In the structure of Dret_0059, a ketoleucine moiety is bound to the SBP, whereas a cytosine molecule is bound in the distal PAS-like domain of the tandem PAS-like domain. Differential scanning flourimetry support the binding of ligands observed in the crystal structure. There is significant interaction between the SBP and tandem PAS-like domains, and it is possible that the binding of one ligand could have an effect on the binding of the other. We uncovered three other proteins with this structural architecture in the non-redundant sequence data base, and predict that they too bind the same substrates. The genomic context of this protein did not offer any clues for its function. We did not find any biological process in which the two observed ligands are coupled. The protein Dret_0059 could be involved in either signal transduction or solute transport.

  6. Protein Attachment on Nanodiamonds.

    Science.gov (United States)

    Lin, Chung-Lun; Lin, Cheng-Huang; Chang, Huan-Cheng; Su, Meng-Chih

    2015-07-16

    A recent advance in nanotechnology is the scale-up production of small and nonaggregated diamond nanoparticles suitable for biological applications. Using detonation nanodiamonds (NDs) with an average diameter of ∼4 nm as the adsorbents, we have studied the static attachment of three proteins (myoglobin, bovine serum albumin, and insulin) onto the nanoparticles by optical spectroscopy, mass spectrometry, and dynamic light scattering, and electrophoretic zeta potential measurements. Results show that the protein surface coverage is predominantly determined by the competition between protein-protein and protein-ND interactions, giving each protein a unique and characteristic structural configuration in its own complex. Specifically, both myoglobin and bovine serum albumin show a Langmuir-type adsorption behavior, forming 1:1 complexes at saturation, whereas insulin folds into a tightly bound multimer before adsorption. The markedly different adsorption patterns appear to be independent of the protein concentration and are closely related to the affinity of the individual proteins for the NDs. The present study provides a fundamental understanding for the use of NDs as a platform for nanomedical drug delivery.

  7. Binding Direction-Based Two-Dimensional Flattened Contact Area Computing Algorithm for Protein-Protein Interactions.

    Science.gov (United States)

    Kang, Beom Sik; Pugalendhi, GaneshKumar; Kim, Ku-Jin

    2017-10-13

    Interactions between protein molecules are essential for the assembly, function, and regulation of proteins. The contact region between two protein molecules in a protein complex is usually complementary in shape for both molecules and the area of the contact region can be used to estimate the binding strength between two molecules. Although the area is a value calculated from the three-dimensional surface, it cannot represent the three-dimensional shape of the surface. Therefore, we propose an original concept of two-dimensional contact area which provides further information such as the ruggedness of the contact region. We present a novel algorithm for calculating the binding direction between two molecules in a protein complex, and then suggest a method to compute the two-dimensional flattened area of the contact region between two molecules based on the binding direction.

  8. Coupling ligand recognition to protein folding in an engineered variant of rabbit ileal lipid binding protein.

    Science.gov (United States)

    Kouvatsos, Nikolaos; Meldrum, Jill K; Searle, Mark S; Thomas, Neil R

    2006-11-28

    We have engineered a variant of the beta-clam shell protein ILBP which lacks the alpha-helical motif that caps the central binding cavity; the mutant protein is sufficiently destabilised that it is unfolded under physiological conditions, however, it unexpectedly binds its natural bile acid substrates with high affinity forming a native-like beta-sheet rich structure and demonstrating strong thermodynamic coupling between ligand binding and protein folding.

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

    DEFF Research Database (Denmark)

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

    2011-01-01

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

  10. Singlet oxygen-mediated formation of protein peroxides within cells

    International Nuclear Information System (INIS)

    Wright, A.; Policarpio, V.

    2003-01-01

    Full text: Singlet oxygen is generated by a number of cellular, enzymatic and chemical reactions as well as by exposure to UV, or visible light in the presence of a sensitizer; as a consequence this oxidant has been proposed as a damaging agent in a number of pathologies including photo-aging and skin cancer. Proteins are major targets for singlet oxygen as a result of their abundance and high rate constants for reaction. In this study it is shown that illumination of viable, sensitizer-loaded, THP-1 (human monocyte-like) cells with visible light gives rise to intra-cellular protein-derived peroxides. The peroxide yield increases with illumination time, requires the presence of the sensitizer, is enhanced in D 2 O, and decreased by azide; these data are consistent with the mediation of singlet oxygen. The concentration of peroxides detected, which is not affected by glucose or ascorbate loading of the cells, corresponds to ca. 1.5 nmoles peroxide per 10 6 cells using rose bengal as sensitizer, or 10 nmoles per mg cell protein and account for up to ca. 15% of the O 2 consumed by the cells. Similar peroxides have been detected on isolated cellular proteins exposed to light in the presence of rose bengal and oxygen. After cessation of illumination, the cellular protein peroxide levels decreases with t 1/2 ca. 4 hrs at 37 deg C, and this is associated with increased cell lysis. Decomposition of protein peroxides formed within cells, or on isolated cellular proteins, by metal ions, gives rise to radicals as detected by EPR spin trapping. These protein peroxides, and radicals derived from them, can inactivate key cellular enzymes (including caspases, GAPDH and glutathione reductase) and induce DNA base oxidation, strand breaks and DNA-protein cross-links. These studies demonstrate that exposure of intact cells to visible light in the presence of a sensitizer gives rise to novel long-lived, but reactive, intra-cellular protein peroxides via singlet oxygen

  11. Cellulose binding domain proteins

    Science.gov (United States)

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc; Doi, Roy

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  12. Three-state combinatorial switch models as applied to the binding of oxygen by human hemoglobin.

    Science.gov (United States)

    Straume, M; Johnson, M L

    1988-02-23

    We have generated a series of all 6561 unique, discrete three-state combinatorial switch models to describe the partitioning of the cooperative oxygen-binding free change among the 10 variously ligated forms of human hemoglobin tetramers. These models were inspired by the experimental observation of Smith and Ackers that the cooperative free energy of the intersubunit contact regions of the 10 possible ligated forms of human hemoglobin tetramers can be represented by a particular distribution of three distinct energy levels [Smith, F. R., & Ackers, G. K. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5347-5351]. A statistical thermodynamic formulation accounting for both dimer-tetramer equilibria and ligand binding properties of hemoglobin solutions as a function of oxygen and protein concentrations was utilized to exhaustively test these thermodynamic models. In this series of models each of the 10 ligated forms of the hemoglobin tetramer can exist in one, and only one, of three possible energy levels; i.e., each ligated form was assumed to be associated with a discrete energy state. This series of models includes all possible ways that the 10 ligation states of hemoglobin can be distributed into three distinct cooperative energy levels. The mathematical models, as presented here, do not permit equilibria between energy states to exist for any of the 10 unique ligated forms of hemoglobin tetramers. These models were analyzed by nonlinear least-squares estimation of the free energy parameters characteristic of this statistical thermodynamic development.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Functional Advantages of Conserved Intrinsic Disorder in RNA-Binding Proteins

    OpenAIRE

    Varadi, Mihaly; Zsolyomi, Fruzsina; Guharoy, Mainak; Tompa, Peter

    2015-01-01

    Proteins form large macromolecular assemblies with RNA that govern essential molecular processes. RNA-binding proteins have often been associated with conformational flexibility, yet the extent and functional implications of their intrinsic disorder have never been fully assessed. Here, through large-scale analysis of comprehensive protein sequence and structure datasets we demonstrate the prevalence of intrinsic structural disorder in RNA-binding proteins and domains. We addressed their func...

  14. Does protein binding modulate the effect of angiotensin II receptor antagonists?

    Directory of Open Access Journals (Sweden)

    Marc P Maillard

    2001-03-01

    Full Text Available IntroductionAngiotensin II AT 1-receptor antagonists are highly bound to plasma proteins (≥ 99%. With some antagonists, such as DuP-532, the protein binding was such that no efficacy of the drug could be demonstrated clinically. Whether protein binding interferes with the efficacy of other antagonists is not known. We have therefore investigated in vitro how plasma proteins may affect the antagonistic effect of different AT1-receptor antagonists.MethodsA radio-receptor binding assay was used to analyse the interaction between proteins and the ability of various angiotensin II (Ang II antagonists to block AT1-receptors. In addition, the Biacore technology, a new technique which enables the real-time monitoring of binding events between two molecules, was used to evaluate the dissociation rate constants of five AT1-receptor antagonists from human serum albumin.ResultsThe in vitro AT 1-antagonistic effects of different Ang II receptor antagonists were differentially affected by the presence of human plasma, with rightward shifts of the IC50 ranging from one to several orders of magnitude. The importance of the shift correlates with the dissociation rate constants of these drugs from albumin. Our experiments also show that the way that AT1-receptor antagonists bind to proteins differs from one compound to another. These results suggest that the interaction with plasma proteins appears to modulate the efficacy of some Ang II antagonists.ConclusionAlthough the high binding level of Ang II receptor antagonist to plasma proteins appears to be a feature common to this class of compounds, the kinetics and characteristics of this binding is of great importance. With some antagonists, protein binding interferes markedly with their efficacy to block AT1-receptors.

  15. SECRET domain of variola virus CrmB protein can be a member of poxviral type II chemokine-binding proteins family.

    Science.gov (United States)

    Antonets, Denis V; Nepomnyashchikh, Tatyana S; Shchelkunov, Sergei N

    2010-10-27

    Variola virus (VARV) the causative agent of smallpox, eradicated in 1980, have wide spectrum of immunomodulatory proteins to evade host immunity. Recently additional biological activity was discovered for VARV CrmB protein, known to bind and inhibit tumour necrosis factor (TNF) through its N-terminal domain homologous to cellular TNF receptors. Besides binding TNF, this protein was also shown to bind with high affinity several chemokines which recruit B- and T-lymphocytes and dendritic cells to sites of viral entry and replication. Ability to bind chemokines was shown to be associated with unique C-terminal domain of CrmB protein. This domain named SECRET (Smallpox virus-Encoded Chemokine Receptor) is unrelated to the host proteins and lacks significant homology with other known viral chemokine-binding proteins or any other known protein. De novo modelling of VARV-CrmB SECRET domain spatial structure revealed its apparent structural homology with cowpox virus CC-chemokine binding protein (vCCI) and vaccinia virus A41 protein, despite low sequence identity between these three proteins. Potential ligand-binding surface of modelled VARV-CrmB SECRET domain was also predicted to bear prominent electronegative charge which is characteristic to known orthopoxviral chemokine-binding proteins. Our results suggest that SECRET should be included into the family of poxviral type II chemokine-binding proteins and that it might have been evolved from the vCCI-like predecessor protein.

  16. Evaluating the binding efficiency of pheromone binding protein with its natural ligand using molecular docking and fluorescence analysis

    Science.gov (United States)

    Ilayaraja, Renganathan; Rajkumar, Ramalingam; Rajesh, Durairaj; Muralidharan, Arumugam Ramachandran; Padmanabhan, Parasuraman; Archunan, Govindaraju

    2014-06-01

    Chemosignals play a crucial role in social and sexual communication among inter- and intra-species. Chemical cues are bound with protein that is present in the pheromones irrespective of sex are commonly called as pheromone binding protein (PBP). In rats, the pheromone compounds are bound with low molecular lipocalin protein α2u-globulin (α2u). We reported farnesol is a natural endogenous ligand (compound) present in rat preputial gland as a bound volatile compound. In the present study, an attempt has been made through computational method to evaluating the binding efficiency of α2u with the natural ligand (farnesol) and standard fluorescent molecule (2-naphthol). The docking analysis revealed that the binding energy of farnesol and 2-naphthol was almost equal and likely to share some binding pocket of protein. Further, to extrapolate the results generated through computational approach, the α2u protein was purified and subjected to fluorescence titration and binding assay. The results showed that the farnesol is replaced by 2-naphthol with high hydrophobicity of TYR120 in binding sites of α2u providing an acceptable dissociation constant indicating the binding efficiency of α2u. The obtained results are in corroboration with the data made through computational approach.

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

    Science.gov (United States)

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

    2017-04-04

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

  18. Nickel electrodes as a cheap and versatile platform for studying structure and function of immobilized redox proteins

    Energy Technology Data Exchange (ETDEWEB)

    Han, Xiao Xia [State Key Laboratory of Supramolecular Structure and Materials, Jilin University, 2699 Qianjin Street, Changchun 130012 (China); Institut für Chemie, Technische Universität Berlin, Sekr. PC14, Strasse des 17. Juni 135, D-10623 Berlin (Germany); Li, Junbo [State Key Laboratory of Supramolecular Structure and Materials, Jilin University, 2699 Qianjin Street, Changchun 130012 (China); Öner, Ibrahim Halil [Institut für Chemie, Technische Universität Berlin, Sekr. PC14, Strasse des 17. Juni 135, D-10623 Berlin (Germany); Zhao, Bing [State Key Laboratory of Supramolecular Structure and Materials, Jilin University, 2699 Qianjin Street, Changchun 130012 (China); Leimkühler, Silke [Institut für Biochemie und Biologie, Universität Potsdam, Karl-Liebknecht Straße 24-25, H. 25, Golm D-14476 (Germany); Hildebrandt, Peter [Institut für Chemie, Technische Universität Berlin, Sekr. PC14, Strasse des 17. Juni 135, D-10623 Berlin (Germany); Weidinger, Inez M., E-mail: i.weidinger@mailbox.tu-berlin.de [Institut für Chemie, Technische Universität Berlin, Sekr. PC14, Strasse des 17. Juni 135, D-10623 Berlin (Germany)

    2016-10-19

    Practical use of many bioelectronic and bioanalytical devices is limited by the need of expensive materials and time consuming fabrication. Here we demonstrate the use of nickel electrodes as a simple and cheap solid support material for bioelectronic applications. The naturally nanostructured electrodes showed a surprisingly high electromagnetic surface enhancement upon light illumination such that immobilization and electron transfer reactions of the model redox proteins cytochrome b{sub 5} (Cyt b{sub 5}) and cytochrome c (Cyt c) could be followed via surface enhanced resonance Raman spectroscopy. It could be shown that the nickel surface, when used as received, promotes a very efficient binding of the proteins upon preservation of their native structure. The immobilized redox proteins could efficiently exchange electrons with the electrode and could even act as an electron relay between the electrode and solubilized myoglobin. Our results open up new possibility for nickel electrodes as an exceptional good support for bioelectronic devices and biosensors on the one hand and for surface enhanced spectroscopic investigations on the other hand. - Highlights: • Nickel electrodes were used without further functionalization as supports for various redox proteins. • It was possible to monitor the immobilized proteins via surface enhanced Raman spectroscopy. • The native structure of the immobilized proteins was preserved and they could exchange electrons with the Ni electrode. • The immobilized redox proteins worked as an electron relay between electrode and solubilized myoglobin.

  19. Actin, actin-binding proteins, and actin-related proteins in the nucleus.

    Science.gov (United States)

    Kristó, Ildikó; Bajusz, Izabella; Bajusz, Csaba; Borkúti, Péter; Vilmos, Péter

    2016-04-01

    Extensive research in the past decade has significantly broadened our view about the role actin plays in the life of the cell and added novel aspects to actin research. One of these new aspects is the discovery of the existence of nuclear actin which became evident only recently. Nuclear activities including transcriptional activation in the case of all three RNA polymerases, editing and nuclear export of mRNAs, and chromatin remodeling all depend on actin. It also became clear that there is a fine-tuned equilibrium between cytoplasmic and nuclear actin pools and that this balance is ensured by an export-import system dedicated to actin. After over half a century of research on conventional actin and its organizing partners in the cytoplasm, it was also an unexpected finding that the nucleus contains more than 30 actin-binding proteins and new classes of actin-related proteins which are not able to form filaments but had evolved nuclear-specific functions. The actin-binding and actin-related proteins in the nucleus have been linked to RNA transcription and processing, nuclear transport, and chromatin remodeling. In this paper, we attempt to provide an overview of the wide range of information that is now available about actin, actin-binding, and actin-related proteins in the nucleus.

  20. Characterization of fatty acid binding by the P2 myelin protein

    International Nuclear Information System (INIS)

    Gudaitis, P.G.; Weise, M.J.

    1987-01-01

    In recent years, significant sequence homology has been found between the P2 protein of peripheral myelin and intracellular retinoid- and fatty acid-binding proteins. They have found that salt extracts of bovine intradural nerve roots contain the P2 basic protein in association with free fatty acid. Preliminary results from quantitative analyses showed a ratio of 0.4-1.1 fatty acid (mainly oleate and palmitate) per P2 molecule. P2/ligand interactions were partially characterized using ( 3 H)-oleate in gel permeation assays and binding studies using lipidex to separated bound and free fatty acid. Methyloleate was found to displace ( 3 H)-oleate from P2, indicating that ligand binding interactions are predominantly hydrophobic in nature. On the other hand, myristic acid and retinol did not inhibit the binding of oleate to the protein, results consistent with a decided affinity for long chain fatty acids but not for the retinoids. The binding between P2 and oleic acid showed an apparent Kd in the micromolar range, a value comparable to those found for other fatty acid-binding proteins. From these results they conclude that P2 shares not only structural homology with certain fatty acid binding proteins but also an ability to bind long chain fatty acids. Although the significance of these similarities is not yet clear, they may, by analogy, expect P2 to have a role in PNS lipid metabolism

  1. A sequence-based dynamic ensemble learning system for protein ligand-binding site prediction

    KAUST Repository

    Chen, Peng

    2015-12-03

    Background: Proteins have the fundamental ability to selectively bind to other molecules and perform specific functions through such interactions, such as protein-ligand binding. Accurate prediction of protein residues that physically bind to ligands is important for drug design and protein docking studies. Most of the successful protein-ligand binding predictions were based on known structures. However, structural information is not largely available in practice due to the huge gap between the number of known protein sequences and that of experimentally solved structures

  2. A sequence-based dynamic ensemble learning system for protein ligand-binding site prediction

    KAUST Repository

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

    2015-01-01

    Background: Proteins have the fundamental ability to selectively bind to other molecules and perform specific functions through such interactions, such as protein-ligand binding. Accurate prediction of protein residues that physically bind to ligands is important for drug design and protein docking studies. Most of the successful protein-ligand binding predictions were based on known structures. However, structural information is not largely available in practice due to the huge gap between the number of known protein sequences and that of experimentally solved structures

  3. Simultaneous Blood–Tissue Exchange of Oxygen, Carbon Dioxide, Bicarbonate, and Hydrogen Ion

    Science.gov (United States)

    Dash, Ranjan K.; Bassingthwaighte, James B.

    2014-01-01

    A detailed nonlinear four-region (red blood cell, plasma, interstitial fluid, and parenchymal cell) axially distributed convection-diffusion-permeation-reaction-binding computational model is developed to study the simultaneous transport and exchange of oxygen (O2) and carbon dioxide (CO2) in the blood–tissue exchange system of the heart. Since the pH variation in blood and tissue influences the transport and exchange of O2 and CO2 (Bohr and Haldane effects), and since most CO2 is transported as HCO3- (bicarbonate) via the CO2 hydration (buffering) reaction, the transport and exchange of HCO3- and H+ are also simulated along with that of O2 and CO2. Furthermore, the model accounts for the competitive nonlinear binding of O2 and CO2 with the hemoglobin inside the red blood cells (nonlinear O2–CO2 interactions, Bohr and Haldane effects), and myoglobin-facilitated transport of O2 inside the parenchymal cells. The consumption of O2 through cytochrome-c oxidase reaction inside the parenchymal cells is based on Michaelis–Menten kinetics. The corresponding production of CO2 is determined by respiratory quotient (RQ), depending on the relative consumption of carbohydrate, protein, and fat. The model gives a physiologically realistic description of O2 transport and metabolism in the microcirculation of the heart. Furthermore, because model solutions for tracer transients and steady states can be computed highly efficiently, this model may be the preferred vehicle for routine data analysis where repetitive solutions and parameter optimization are required, as is the case in PET imaging for estimating myocardial O2 consumption. PMID:16775761

  4. 2-Oxoglutarate levels control adenosine nucleotide binding by Herbaspirillum seropedicae PII proteins.

    Science.gov (United States)

    Oliveira, Marco A S; Gerhardt, Edileusa C M; Huergo, Luciano F; Souza, Emanuel M; Pedrosa, Fábio O; Chubatsu, Leda S

    2015-12-01

    Nitrogen metabolism in Proteobacteria is controlled by the Ntr system, in which PII proteins play a pivotal role, controlling the activity of target proteins in response to the metabolic state of the cell. Characterization of the binding of molecular effectors to these proteins can provide information about their regulation. Here, the binding of ATP, ADP and 2-oxoglutarate (2-OG) to the Herbaspirillum seropedicae PII proteins, GlnB and GlnK, was characterized using isothermal titration calorimetry. Results show that these proteins can bind three molecules of ATP, ADP and 2-OG with homotropic negative cooperativity, and 2-OG binding stabilizes the binding of ATP. Results also show that the affinity of uridylylated forms of GlnB and GlnK for nucleotides is significantly lower than that of the nonuridylylated proteins. Furthermore, fluctuations in the intracellular concentration of 2-OG in response to nitrogen availability are shown. Results suggest that under nitrogen-limiting conditions, PII proteins tend to bind ATP and 2-OG. By contrast, after an ammonium shock, a decrease in the 2-OG concentration is observed causing a decrease in the affinity of PII proteins for ATP. This phenomenon may facilitate the exchange of ATP for ADP on the ligand-binding pocket of PII proteins, thus it is likely that under low ammonium, low 2-OG levels would favor the ADP-bound state. © 2015 FEBS.

  5. Identification of arsenite-and arsenic diglutathione-binding proteins in human hepatocarcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Mizumura, Ayano; Watanabe, Takayuki [Graduate School of Pharmaceutical Sciences, Chiba University, Yayoi, Inage, Chiba 263-8522 (Japan); Kobayashi, Yayoi [Graduate School of Pharmaceutical Sciences, Chiba University, Yayoi, Inage, Chiba 263-8522 (Japan); Environmental Health Sciences Division, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506 (Japan); Hirano, Seishiro [Graduate School of Pharmaceutical Sciences, Chiba University, Yayoi, Inage, Chiba 263-8522 (Japan); Research Center for Environmental Risk, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506 (Japan)

    2010-01-15

    It is generally accepted that trivalent arsenicals are more toxic than the corresponding pentavalent arsenicals, since trivalent arsenicals bind the thiol groups of biomolecules, leading to a deterioration in cellular functions. In the present study, we prepared three different arsenic-bound sepharoses and investigated the binding of hepatic cytosolic proteins to pentavalent, trivalent, and glutathione-conjugated trivalent arsenicals. SDS-PAGE showed no proteins bound to pentavalent arsenic specifically. In contrast, we found a number of proteins that have specific and high affinity for trivalent arsenic. Two of those proteins were identified: protein disulfide isomerase-related protein 5 (PDSIRP5) and peroxiredoxin 1/enhancer protein (PRX1/EP). These proteins have vicinal cysteines, as previously reported. In contrast, one of the prominent proteins that did not bind to trivalent arsenic was identified as calreticulin precursor. Although there are 3 cysteines in calreticulin precursor, two of the cysteines are spaced more than 25 amino acids apart. Five synthetic peptides containing 2 vicinal cysteines were prepared to study whether they would inhibit the binding of PDSIRP5, PRX1/EP, and other arsenic-binding proteins to trivalent arsenicals. Only two of the five peptides effectively inhibited binding, suggesting that other amino acids besides the 2 vicinal cysteines may modulate the affinity of cysteine-rich proteins for trivalent arsenicals. We further investigated hepatic cytosolic proteins that bound specifically to glutathione-conjugated trivalent arsenic, which is the most abundant form of arsenical in bile fluid. Four proteins that bound specifically to glutathione-conjugated trivalent arsenic were identified; interestingly, these proteins were different from the trivalent arsenic-binding proteins. These results suggest that although glutathione-conjugation is an important process in the metabolism, excretion, and detoxification of arsenicals, glutathione

  6. Plant ice-binding (antifreeze) proteins

    Science.gov (United States)

    Proteins that determine the temperature at which ice crystals will form in water-based solutions in cells and tissues, that bind to growing ice crystals, thus affecting their size, and that impact ice re-crystallization have been widely-documented and studied in many plant, bacterial, fungal, insect...

  7. Comparison of the ligand binding properties of two homologous rat apocellular retinol-binding proteins expressed in Escherichia coli.

    Science.gov (United States)

    Levin, M S; Locke, B; Yang, N C; Li, E; Gordon, J I

    1988-11-25

    Cellular retinol-binding protein (CRBP) and cellular retinol-binding protein II (CRBP II) are 132-residue cytosolic proteins which have 56% amino acid sequence identity and bind all-trans-retinol as their endogenous ligand. They belong to a family of cytoplasmic proteins which have evolved to bind distinct hydrophobic ligands. Their patterns of tissue-specific and developmental regulation are distinct. We have compared the ligand binding properties of rat apo-CRBP and apo-CRBP II that have been expressed in Escherichia coli. Several observations indicate that the E. coli-derived apoproteins are structurally similar to the native rat proteins: they co-migrate on isoelectric focusing gels; and when complexed with all-trans-retinol, their absorption and excitation/emission spectra are nearly identical to those of the authentic rat holoproteins. Comparative lifetime and acrylamide quenching studies suggest that there are differences in the conformations of apo-CRBP and apo-CRBP II. The interaction of E. coli-derived apo-CRBP and apo-CRBP II with a variety of retinoids was analyzed using spectroscopic techniques. Both apoproteins formed high affinity complexes with all-trans-retinol (K'd approximately 10 nM). In direct binding assays, all-trans-retinal bound to both apoproteins (K'd approximately 50 nM for CRBP; K'd approximately 90 nM for CRBP II). However, all-trans-retinal could displace all-trans-retinol bound to CRBP II but not to CRBP. These observations suggests that there is a specific yet distinct interaction between these two proteins and all-trans-retinal. Apo-CRBP and apo-CRBP II did not demonstrate significant binding to either retinoic acid or methyl retinoate, an uncharged derivative of all-trans-retinoic acid. This indicates that the carboxymethyl group of methyl retinoate cannot be sterically accommodated in their binding pockets and that failure to bind retinoic acid probably is not simply due to the negative charge of its C-15 carboxylate group

  8. Accurate and sensitive quantification of protein-DNA binding affinity.

    Science.gov (United States)

    Rastogi, Chaitanya; Rube, H Tomas; Kribelbauer, Judith F; Crocker, Justin; Loker, Ryan E; Martini, Gabriella D; Laptenko, Oleg; Freed-Pastor, William A; Prives, Carol; Stern, David L; Mann, Richard S; Bussemaker, Harmen J

    2018-04-17

    Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes. Copyright © 2018 the Author(s). Published by PNAS.

  9. SECRET domain of variola virus CrmB protein can be a member of poxviral type II chemokine-binding proteins family

    Directory of Open Access Journals (Sweden)

    Shchelkunov Sergei N

    2010-10-01

    Full Text Available Abstract Background Variola virus (VARV the causative agent of smallpox, eradicated in 1980, have wide spectrum of immunomodulatory proteins to evade host immunity. Recently additional biological activity was discovered for VARV CrmB protein, known to bind and inhibit tumour necrosis factor (TNF through its N-terminal domain homologous to cellular TNF receptors. Besides binding TNF, this protein was also shown to bind with high affinity several chemokines which recruit B- and T-lymphocytes and dendritic cells to sites of viral entry and replication. Ability to bind chemokines was shown to be associated with unique C-terminal domain of CrmB protein. This domain named SECRET (Smallpox virus-Encoded Chemokine Receptor is unrelated to the host proteins and lacks significant homology with other known viral chemokine-binding proteins or any other known protein. Findings De novo modelling of VARV-CrmB SECRET domain spatial structure revealed its apparent structural homology with cowpox virus CC-chemokine binding protein (vCCI and vaccinia virus A41 protein, despite low sequence identity between these three proteins. Potential ligand-binding surface of modelled VARV-CrmB SECRET domain was also predicted to bear prominent electronegative charge which is characteristic to known orthopoxviral chemokine-binding proteins. Conclusions Our results suggest that SECRET should be included into the family of poxviral type II chemokine-binding proteins and that it might have been evolved from the vCCI-like predecessor protein.

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

    Directory of Open Access Journals (Sweden)

    Yoon Sup Choi

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

  11. Noncovalent binding of 4-nitroquinoline-N-oxide to proteins

    International Nuclear Information System (INIS)

    Yamamoto, Osamu

    1979-01-01

    Binding of 4NQO to various kinds of enzymes or proteins was studied. Each one of proteins was mixed with 4NQO in 0.4 mM NaHCO 3 solution and eluted through Ultrogel AcA 22 column. Radioactivity of 14 C-labeled 4NQO found in protein fraction was measured. 4NQO bound hardly to polyglutamic acid and polyaspertic acid, somewhat to serum albumin, insulin, trypsin, RNA polymerase and DNA polymerase, and markedly to ureas which is an SH enzyme. Lactate dehydrogenase, one of SH enzymes, aggregated with 4NQO. The binding of SH enzyme with the N-oxide would be attributable to a noncovalent binding such as >N-O---H-S-, because 4NQO-urease binding yield markedly decreased in the presence of sodium dodecyl sulfate or cysteine, and also 4NQO-bound urease released 4NQO by the addition of sodium dodecyl sulfate. (author)

  12. Noncovalent binding of 4-nitroquinoline-N-oxide to proteins

    Energy Technology Data Exchange (ETDEWEB)

    Yamamoto, O [Hiroshima Univ. (Japan). Research Inst. for Nuclear Medicine and Biology

    1979-12-01

    Binding of 4NQO to various kinds of enzymes or proteins was studied. Each one of proteins was mixed with 4NQO in 0.4 mM NaHCO/sub 3/ solution and eluted through Ultrogel AcA 22 column. Radioactivity of /sup 14/C-labeled 4NQO found in protein fraction was measured. 4NQO bound hardly to polyglutamic acid and polyaspertic acid, somewhat to serum albumin, insulin, trypsin, RNA polymerase and DNA polymerase, and markedly to ureas which is an SH enzyme. Lactate dehydrogenase, one of SH enzymes, aggregated with 4NQO. The binding of SH enzyme with the N-oxide would be attributable to a noncovalent binding such as >N-O---H-S-, because 4NQO-urease binding yield markedly decreased in the presence of sodium dodecyl sulfate or cysteine, and also 4NQO-bound urease released 4NQO by the addition of sodium dodecyl sulfate.

  13. Labelling of pneumococcal penicillin-binding proteins with [3H]propionyl-ampicillin. A rapid method for monitoring penicillin-binding activity

    International Nuclear Information System (INIS)

    Hakenbeck, R.; Kohiyama, M.

    1982-01-01

    Penicillin-binding proteins (PBPs) are membrane components ubiquitous to all bacteria examined so far. Some of them are present in only a few copies per cell. The conventional method of visualizing these proteins consists in binding of radioactive penicillin to the fractions containing PBPs followed by SDS-PAGE and finally fluorography. Although this procedure is laborious, it is necessary for the determination of the identity as well as for the quantification of each PBP. On the other hand, when penicillin-binding conditions are to be examined or binding activity has to be followed through fractionation and purification of PBPs, no fast monitoring device for these proteins has been available. The authors developed a rapid and easy assay for penicillin-binding activity with a filter-binding technique using [ 3 H]propionyl ampicillin ( 3 H-PA) of high specific activity. As little 2μg of crude membranes obtained from the highly penicillin-sensitive, β-lactamase-negative organism Streptococcus pneumoniae, are sufficient to detect binding activity. In this paper they describe optimum conditions for the assay of PBPs and show that this binding activity correlates with the presence of native penicillin-binding proteins. (Auth.)

  14. Recent improvements to Binding MOAD: a resource for protein-ligand binding affinities and structures.

    Science.gov (United States)

    Ahmed, Aqeel; Smith, Richard D; Clark, Jordan J; Dunbar, James B; Carlson, Heather A

    2015-01-01

    For over 10 years, Binding MOAD (Mother of All Databases; http://www.BindingMOAD.org) has been one of the largest resources for high-quality protein-ligand complexes and associated binding affinity data. Binding MOAD has grown at the rate of 1994 complexes per year, on average. Currently, it contains 23,269 complexes and 8156 binding affinities. Our annual updates curate the data using a semi-automated literature search of the references cited within the PDB file, and we have recently upgraded our website and added new features and functionalities to better serve Binding MOAD users. In order to eliminate the legacy application server of the old platform and to accommodate new changes, the website has been completely rewritten in the LAMP (Linux, Apache, MySQL and PHP) environment. The improved user interface incorporates current third-party plugins for better visualization of protein and ligand molecules, and it provides features like sorting, filtering and filtered downloads. In addition to the field-based searching, Binding MOAD now can be searched by structural queries based on the ligand. In order to remove redundancy, Binding MOAD records are clustered in different families based on 90% sequence identity. The new Binding MOAD, with the upgraded platform, features and functionalities, is now equipped to better serve its users. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

    Science.gov (United States)

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

    2001-11-01

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

  16. Definition of IgG- and albumin-binding regions of streptococcal protein G.

    Science.gov (United States)

    Akerström, B; Nielsen, E; Björck, L

    1987-10-05

    Protein G, the immunoglobin G-binding surface protein of group C and G streptococci, also binds serum albumin. The albumin-binding site on protein G is distinct from the immunoglobulin G-binding site. By mild acid hydrolysis of the papain-liberated protein G fragment (35 kDa), a 28-kDa fragment was produced which retained full immunoglobulin G-binding activity (determined by Scatchard plotting) but had lost all albumin-binding capacity. A protein G (65 kDa), isolated after cloning and expression of the protein G gene in Escherichia coli, had comparable affinity to immunoglobulin G (5-10 X 10(10)M-1), but much higher affinity to albumin than the 35- and 28-kDa protein G fragments (31, 2.6, and 0 X 10(9)M-1, respectively). The amino-terminal amino acid sequences of the 65-, 35-, and 28-kDa fragments allowed us to exactly locate the three fragments in an overall sequence map of protein G, based on the partial gene sequences published by Guss et al. (Guss, B., Eliasson, M., Olsson, A., Uhlen, M., Frej, A.-K., Jörnvall, H., Flock, J.-I., and Lindberg, M. (1986) EMBO J. 5, 1567-1575) and Fahnestock et al. (Fahnestock, S. R., Alexander, P., Nagle, J., and Filpula, D. (1986) J. Bacteriol. 167, 870-880). In this map could then be deduced the location of three homologous albumin-binding regions and three homologous immunoglobulin G-binding regions.

  17. Interspecies In Vitro Evaluation of Stereoselective Protein Binding for 3,4-Methylenedioxymethamphetamine

    Directory of Open Access Journals (Sweden)

    Wan Raihana Wan Aasim

    2017-01-01

    Full Text Available Abuse of 3,4-methylenedioxymethamphetamine (MDMA is becoming more common worldwide. To date, there is no information available on stereoselectivity of MDMA protein binding in humans, rats, and mice. Since stereoselectivity plays an important role in MDMA’s pharmacokinetics and pharmacodynamics, in this study we investigated its stereoselectivity in protein binding. The stereoselective protein binding of rac-MDMA was investigated using two different concentrations (20 and 200 ng/mL in human plasma and mouse and rat sera using an ultrafiltration technique. No significant stereoselectivity in protein binding was observed in both human plasma and rat serum; however, a significant stereoselective binding (p<0.05 was observed in mouse serum. Since the protein binding of MDMA in mouse serum is considerably lower than in humans and rats, caution should be exercised when using mice for in vitro studies involving MDMA.

  18. Binding of anthracene to cellular macromolecules in the presence of light. [DNA, HSA

    Energy Technology Data Exchange (ETDEWEB)

    Sinha, B K; Chignell, C F [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (USA)

    1983-01-01

    Ultraviolet radiation (> 295 nm) induced covalent binding of anthracene to DNA which increased with time and was not affected by oxygen. Irradiation in the presence of anthracene induced nicking of Col E/sub 1/ circular DNA and decreased the thermal denaturation temperature of calf thymus DNA. These effects were oxygen dependent, and were decreased by GMP. Irradiation of anthracene and human serum albumin resulted in covalent binding of the hydrocarbon to the protein accompanied by crosslinking of the protein. Protein crosslinking decreased under anaerobic conditions. Irradiation of anthracene bound to liposomes induced lipid peroxidation which was not affected by superoxide dismutase or catalase.

  19. Exploring the composition of protein-ligand binding sites on a large scale.

    Directory of Open Access Journals (Sweden)

    Nickolay A Khazanov

    Full Text Available The residue composition of a ligand binding site determines the interactions available for diffusion-mediated ligand binding, and understanding general composition of these sites is of great importance if we are to gain insight into the functional diversity of the proteome. Many structure-based drug design methods utilize such heuristic information for improving prediction or characterization of ligand-binding sites in proteins of unknown function. The Binding MOAD database if one of the largest curated sets of protein-ligand complexes, and provides a source of diverse, high-quality data for establishing general trends of residue composition from currently available protein structures. We present an analysis of 3,295 non-redundant proteins with 9,114 non-redundant binding sites to identify residues over-represented in binding regions versus the rest of the protein surface. The Binding MOAD database delineates biologically-relevant "valid" ligands from "invalid" small-molecule ligands bound to the protein. Invalids are present in the crystallization medium and serve no known biological function. Contacts are found to differ between these classes of ligands, indicating that residue composition of biologically relevant binding sites is distinct not only from the rest of the protein surface, but also from surface regions capable of opportunistic binding of non-functional small molecules. To confirm these trends, we perform a rigorous analysis of the variation of residue propensity with respect to the size of the dataset and the content bias inherent in structure sets obtained from a large protein structure database. The optimal size of the dataset for establishing general trends of residue propensities, as well as strategies for assessing the significance of such trends, are suggested for future studies of binding-site composition.

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

    DEFF Research Database (Denmark)

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

    2006-01-01

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

  1. Comparative studies of human and chicken retinol-binding proteins and prealbumins.

    Science.gov (United States)

    Kopelman, M; Mokady, S; Cogan, U

    1976-08-09

    Microheterogeneity of retinol-binding proteins of human plasma and urine, and of chicken plasma was studied by polyacrylamide gel electrophoresis. All three protein systems were found microheterogenous. Incorporation of retinol into the protein preparations on the one hand, and depletion of these proteins from retinol on the other hand, enabled us to clarify the extent to which the presence or absence of the ligand affects the apparent heterogeneity. Upon electrophoresis, each of the native proteins displayed two pairs of protein zones. It appeared that within each pair the fast moving band corresponded to aporetinol-binding protein which upon binding of retinol was converted to a holoprotein with a slightly lower mobility. However, it did not seem that proteins of one pair were converted to proteins of the second pair upon binding of retinol, substantiating ghe microheterogenous character of this protein system. A rapid, two step procedure for isolation of prealbumins from plasma is described. The method which consists of DEAE-cellulose chromatography follwed by preparative electrophoresis was utilized to separate human and chicken prealbumins. Routine dodecyl sulphate electrophoresis resulted in partial dissociation of human prealbumin but in no dissociation of the chicken protein. More drastic treatments prior to electrophoresis were needed to effect complete disruption of both proteins into subunits.

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

    Science.gov (United States)

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

    2017-01-01

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

  3. RNA-Binding Proteins in Plant Immunity

    Directory of Open Access Journals (Sweden)

    Virginia Woloshen

    2011-01-01

    Full Text Available Plant defence responses against pathogen infection are crucial to plant survival. The high degree of regulation of plant immunity occurs both transcriptionally and posttranscriptionally. Once transcribed, target gene RNA must be processed prior to translation. This includes polyadenylation, 5′capping, editing, splicing, and mRNA export. RNA-binding proteins (RBPs have been implicated at each level of RNA processing. Previous research has primarily focused on structural RNA-binding proteins of yeast and mammals; however, more recent work has characterized a number of plant RBPs and revealed their roles in plant immune responses. This paper provides an update on the known functions of RBPs in plant immune response regulation. Future in-depth analysis of RBPs and other related players will unveil the sophisticated regulatory mechanisms of RNA processing during plant immune responses.

  4. Leptospiral outer membrane protein microarray, a novel approach to identification of host ligand-binding proteins.

    Science.gov (United States)

    Pinne, Marija; Matsunaga, James; Haake, David A

    2012-11-01

    Leptospirosis is a zoonosis with worldwide distribution caused by pathogenic spirochetes belonging to the genus Leptospira. The leptospiral life cycle involves transmission via freshwater and colonization of the renal tubules of their reservoir hosts. Infection requires adherence to cell surfaces and extracellular matrix components of host tissues. These host-pathogen interactions involve outer membrane proteins (OMPs) expressed on the bacterial surface. In this study, we developed an Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 OMP microarray containing all predicted lipoproteins and transmembrane OMPs. A total of 401 leptospiral genes or their fragments were transcribed and translated in vitro and printed on nitrocellulose-coated glass slides. We investigated the potential of this protein microarray to screen for interactions between leptospiral OMPs and fibronectin (Fn). This approach resulted in the identification of the recently described fibronectin-binding protein, LIC10258 (MFn8, Lsa66), and 14 novel Fn-binding proteins, denoted Microarray Fn-binding proteins (MFns). We confirmed Fn binding of purified recombinant LIC11612 (MFn1), LIC10714 (MFn2), LIC11051 (MFn6), LIC11436 (MFn7), LIC10258 (MFn8, Lsa66), and LIC10537 (MFn9) by far-Western blot assays. Moreover, we obtained specific antibodies to MFn1, MFn7, MFn8 (Lsa66), and MFn9 and demonstrated that MFn1, MFn7, and MFn9 are expressed and surface exposed under in vitro growth conditions. Further, we demonstrated that MFn1, MFn4 (LIC12631, Sph2), and MFn7 enable leptospires to bind fibronectin when expressed in the saprophyte, Leptospira biflexa. Protein microarrays are valuable tools for high-throughput identification of novel host ligand-binding proteins that have the potential to play key roles in the virulence mechanisms of pathogens.

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

    Science.gov (United States)

    Bosdriesz, Evert; Magnúsdóttir, Stefanía; Bruggeman, Frank J; Teusink, Bas; Molenaar, Douwe

    2015-06-01

    Microorganisms rely on binding-protein assisted, active transport systems to scavenge for scarce nutrients. Several advantages of using binding proteins in such uptake systems have been proposed. However, a systematic, rigorous and quantitative analysis of the function of binding proteins is lacking. By combining knowledge of selection pressure and physiochemical constraints, we derive kinetic, thermodynamic, and stoichiometric properties of binding-protein dependent transport systems that enable a maximal import activity per amount of transporter. Under the hypothesis that this maximal specific activity of the transport complex is the selection objective, binding protein concentrations should exceed the concentration of both the scarce nutrient and the transporter. This increases the encounter rate of transporter with loaded binding protein at low substrate concentrations, thereby enhancing the affinity and specific uptake rate. These predictions are experimentally testable, and a number of observations confirm them. © 2015 FEBS.

  6. Structural Basis for a Ribofuranosyl Binding Protein: Insights into the Furanose Specific Transport

    Energy Technology Data Exchange (ETDEWEB)

    Bagaria, A.; Swaminathan, S.; Kumaran, D.; Burley, S. K.

    2011-04-01

    The ATP-binding cassette transporters (ABC-transporters) are members of one of the largest protein superfamilies, with representatives in all extant phyla. These integral membrane proteins utilize the energy of ATP hydrolysis to carry out certain biological processes, including translocation of various substrates across membranes and non-transport related processes such as translation of RNA and DNA repair. Typically, such transport systems in bacteria consist of an ATP binding component, a transmembrane permease, and a periplasmic receptor or binding protein. Soluble proteins found in the periplasm of gram-negative bacteria serve as the primary receptors for transport of many compounds, such as sugars, small peptides, and some ions. Ligand binding activates these periplasmic components, permitting recognition by the membrane spanning domain, which supports for transport and, in some cases, chemotaxis. Transport and chemotaxis processes appear to be independent of one another, and a few mutants of bifunctional periplasmic components reveal the absence of one or the other function. Previously published high-resolution X-ray structures of various periplasmic ligand binding proteins include Arabinose binding protein (ABP), Allose binding protein (ALBP), Glucose-galactose binding protein (GBP) and Ribose binding protein (RBP). Each of these proteins consists of two structurally similar domains connected by a three-stranded hinge region, with ligand buried between the domains. Upon ligand binding and release, various conformational changes have been observed. For RBP, open (apo) and closed (ligand bound) conformations have been reported and so for MBP. The closed/active form of the protein interacts with the integral membrane component of the system in both transport and chemotaxis. Herein, we report 1.9{angstrom} resolution X-ray structure of the R{sub f}BP periplasmic component of an ABC-type sugar transport system from Hahella chejuensis (UniProt Id Q2S7D2) bound to

  7. Effects of protein phosphorylation on color stability of ground meat.

    Science.gov (United States)

    Li, Meng; Li, Xin; Xin, Jianzeng; Li, Zheng; Li, Guixia; Zhang, Yan; Du, Manting; Shen, Qingwu W; Zhang, Dequan

    2017-03-15

    The influence of protein phosphorylation on meat color stability was investigated in this study. Phosphatase and protein kinase inhibitors were added to minced ovine Longissimus thoracis et lumborum (LTL) muscle to manipulate the global phosphorylation of sarcoplasmic proteins. The data obtained show that the rate and extent of pH decline, along with lactate accumulation in postmortem muscle, were related to protein phosphorylation. Analysis of meat color and the relative content of myoglobin redox forms revealed that meat color stability was inversely related to the phosphorylation of sarcoplasmic proteins. Thus, this study suggests that protein phosphorylation may be involved in meat color development by regulating glycolysis and the redox stability of myoglobin. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. PELE:  Protein Energy Landscape Exploration. A Novel Monte Carlo Based Technique.

    Science.gov (United States)

    Borrelli, Kenneth W; Vitalis, Andreas; Alcantara, Raul; Guallar, Victor

    2005-11-01

    Combining protein structure prediction algorithms and Metropolis Monte Carlo techniques, we provide a novel method to explore all-atom energy landscapes. The core of the technique is based on a steered localized perturbation followed by side-chain sampling as well as minimization cycles. The algorithm and its application to ligand diffusion are presented here. Ligand exit pathways are successfully modeled for different systems containing ligands of various sizes:  carbon monoxide in myoglobin, camphor in cytochrome P450cam, and palmitic acid in the intestinal fatty-acid-binding protein. These initial applications reveal the potential of this new technique in mapping millisecond-time-scale processes. The computational cost associated with the exploration is significantly less than that of conventional MD simulations.

  9. Temperature- and pH-dependent effect of lactate on in vitro redox stability of red meat myoglobins.

    Science.gov (United States)

    Nair, M N; Suman, S P; Li, S; Ramanathan, R; Mancini, R A

    2014-01-01

    Our objective was to evaluate the influence of lactate on in vitro redox stability and thermostability of beef, horse, pork, and sheep myoglobins. Lactate (200 mM) had no effect (P>0.05) on redox stability at physiological (pH7.4, 37°C) and meat (pH 5.6, 4°C) conditions. However, lactate increased (Pmeat conditions was species-specific (Pmeat condition suggests that the color stability of lactate-enhanced fresh meat is not due to direct interactions between the ingredient and the heme protein. © 2013.

  10. The binding cavity of mouse major urinary protein is optimised for a variety of ligand binding modes

    Energy Technology Data Exchange (ETDEWEB)

    Pertinhez, Thelma A.; Ferrari, Elena; Casali, Emanuela [Department of Experimental Medicine, University of Parma, Via Volturno, 39, 43100 Parma (Italy); Patel, Jital A. [Department of Chemistry, University of Oxford, Inorganic Chemistry Laboratory, South Parks Road, Oxford OX1 3QR (United Kingdom); Spisni, Alberto, E-mail: alberto.spisni@unipr.it [Department of Experimental Medicine, University of Parma, Via Volturno, 39, 43100 Parma (Italy); Smith, Lorna J., E-mail: lorna.smith@chem.ox.ac.uk [Department of Chemistry, University of Oxford, Inorganic Chemistry Laboratory, South Parks Road, Oxford OX1 3QR (United Kingdom)

    2009-12-25

    {sup 15}N and {sup 1}HN chemical shift data and {sup 15}N relaxation studies have been used to characterise the binding of N-phenyl-naphthylamine (NPN) to mouse major urinary protein (MUP). NPN binds in the {beta}-barrel cavity of MUP, hydrogen bonding to Tyr120 and making extensive non-bonded contacts with hydrophobic side chains. In contrast to the natural pheromone 2-sec-butyl-4,5-dihydrothiazole, NPN binding gives no change to the overall mobility of the protein backbone of MUP. Comparison with 11 different ligands that bind to MUP shows a range of binding modes involving 16 different residues in the {beta}-barrel cavity. These finding justify why MUP is able to adapt to allow for many successful binding partners.

  11. Molecular characterization of Clonorchis sinensis secretory myoglobin: Delineating its role in anti-oxidative survival

    Science.gov (United States)

    2014-01-01

    Background Clonorchiasis is a globally important, neglected food-borne disease caused by Clonorchis sinensis (C. sinensis), and it is highly related to cholangiocarcinoma and hepatocellular carcinoma. Increased molecular evidence has strongly suggested that the adult worm of C. sinensis continuously releases excretory-secretory proteins (ESPs), which play important roles in the parasite-host interactions, to establish successful infection and ensure its own survival. Myoglobin, a hemoprotein, is present in high concentrations in trematodes and ESPs. To further understand the biological function of CsMb and its putative roles in the interactions of C. sinensis with its host, we explored the molecular characterization of CsMb in this paper. Methods We expressed CsMb and its mutants in E. coli BL21 and identified its molecular characteristics using bioinformatics analysis and experimental approaches. Reverse transcription PCR analysis was used to measure myoglobin transcripts of C. sinensis with different culture conditions. The peroxidase activity of CsMb was confirmed by spectrophotometry. We co-cultured RAW264.7 cells with recombinant CsMb (rCsMb), and we then measured the production of hydrogen peroxide (H2O2) and nitric oxide (NO) in addition to the mRNA levels of inducible nitric oxide synthase (iNOS), Cu-Zn superoxide dismutase (SOD1) and Mn superoxide dismutase (SOD2) in activated RAW264.7 cells. Results In the in vitro culture of adult worms, the transcripts of CsMb increased with the increase of oxygen content. Oxidative stress conditions induced by H2O2 increased the levels of CsMb transcripts in a dose-dependent manner. Furthermore, CsMb catalyzed oxidation reactions in the presence of H2O2, and amino acid 34 of CsMb played an essential role in its reaction with H2O2. In addition, CsMb significantly reduced H2O2 and NO levels in LPS-activated macrophages, and CsMb downregulated iNOS and SOD expression in activated macrophages. Conclusion The present study

  12. Rational redesign of neutral endopeptidase binding to merlin and moesin proteins

    Science.gov (United States)

    Niv, Masha Y; Iida, Katsuyuki; Zheng, Rong; Horiguchi, Akio; Shen, Ruoqian; Nanus, David M

    2009-01-01

    Neutral endopeptidase (NEP) is a 90- to 110-kDa cell-surface peptidase that is normally expressed by numerous tissues but whose expression is lost or reduced in a variety of malignancies. The anti-tumorigenic function of NEP is mediated not only by its catalytic activity but also through direct protein–protein interactions of its cytosolic region with several binding partners, including Lyn kinase, PTEN, and ezrin/radixin/moesin (ERM) proteins. We have previously shown that mutation of the K19K20K21 basic cluster in NEPs' cytosolic region to residues QNI disrupts binding to the ERM proteins. Here we show that the ERM-related protein merlin (NF2) does not bind NEP or its cytosolic region. Using experimental data, threading, and sequence analysis, we predicted the involvement of moesin residues E159Q160 in binding to the NEP cytosolic domain. Mutation of these residues to NL (to mimic the corresponding N159L160 residues in the nonbinder merlin) disrupted moesin binding to NEP. Mutation of residues N159L160Y161K162M163 in merlin to the corresponding moesin residues resulted in NEP binding to merlin. This engineered NEP peptide–merlin interaction was diminished by the QNI mutation in NEP, supporting the role of the NEP basic cluster in binding. We thus identified the region of interaction between NEP and moesin, and engineered merlin into a NEP-binding protein. These data form the basis for further exploration of the details of NEP-ERM binding and function. PMID:19388049

  13. Metal binding proteins, recombinant host cells and methods

    Science.gov (United States)

    Summers, Anne O.; Caguiat, Jonathan J.

    2004-06-15

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

  14. A Comparison Study for DNA Motif Modeling on Protein Binding Microarray

    KAUST Repository

    Wong, Ka-Chun; Li, Yue; Peng, Chengbin; Wong, Hau-San

    2015-01-01

    Transcription Factor Binding Sites (TFBSs) are relatively short (5-15 bp) and degenerate. Identifying them is a computationally challenging task. In particular, Protein Binding Microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner; for instance, a typical PBM experiment can measure binding signal intensities of a protein to all possible DNA k-mers (k=810). Since proteins can often bind to DNA with different binding intensities, one of the major challenges is to build motif models which can fully capture the quantitative binding affinity data. To learn DNA motif models from the non-convex objective function landscape, several optimization methods are compared and applied to the PBM motif model building problem. In particular, representative methods from different optimization paradigms have been chosen for modeling performance comparison on hundreds of PBM datasets. The results suggest that the multimodal optimization methods are very effective for capturing the binding preference information from PBM data. In particular, we observe a general performance improvement using di-nucleotide modeling over mono-nucleotide modeling. In addition, the models learned by the best-performing method are applied to two independent applications: PBM probe rotation testing and ChIP-Seq peak sequence prediction, demonstrating its biological applicability.

  15. A Comparison Study for DNA Motif Modeling on Protein Binding Microarray

    KAUST Repository

    Wong, Ka-Chun

    2015-06-11

    Transcription Factor Binding Sites (TFBSs) are relatively short (5-15 bp) and degenerate. Identifying them is a computationally challenging task. In particular, Protein Binding Microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner; for instance, a typical PBM experiment can measure binding signal intensities of a protein to all possible DNA k-mers (k=810). Since proteins can often bind to DNA with different binding intensities, one of the major challenges is to build motif models which can fully capture the quantitative binding affinity data. To learn DNA motif models from the non-convex objective function landscape, several optimization methods are compared and applied to the PBM motif model building problem. In particular, representative methods from different optimization paradigms have been chosen for modeling performance comparison on hundreds of PBM datasets. The results suggest that the multimodal optimization methods are very effective for capturing the binding preference information from PBM data. In particular, we observe a general performance improvement using di-nucleotide modeling over mono-nucleotide modeling. In addition, the models learned by the best-performing method are applied to two independent applications: PBM probe rotation testing and ChIP-Seq peak sequence prediction, demonstrating its biological applicability.

  16. CORRELATION BETWEEN ANGIOTENSIN-CONVERTING ENZYME INHIBITORS LIPOPHILICITY AND PROTEIN BINDING DATA

    Directory of Open Access Journals (Sweden)

    Jasna Trbojević-Stanković

    2012-01-01

    Full Text Available Angiotensin-converting enzyme (ACE inhibitors represent a significant group of drugs primarily used in the treatment of hypertension and congestive heart failure. In this research, seven ACE inhibitors (enalapril, quinapril, fosinopril, lisinopril, cilazapril, ramipril, benazepril were studied to evaluate the relationship between their protein binding and calculated (logP values or ultra-high performance liquid chromatographytandem mass spectrometry (UHPLC-MS and reversed-phase thin-layer chromatography (RP-TLC lipophilicity data (ϕ0, CHI or C0 parameters, respectively. Their protein binding data varied from negligible (lisinopril to 99% (fosinopril, while calculated logPKOWWINvalues ranged from -0.94 (lisinopril to 6.61 (fosinopril. The good correlations were established between protein binding values and logPKOWWIN data (R2=0.7520 as well as between protein binding and chromatographic hydrophobicity data, ϕ0, CHI or C0parameters (R2 were 0.6160, 0.6242 and 0.6547, respectively. The possible application of hydrophobicity data in drugs protein binding evaluation can be of great importance in drug bioavailability.

  17. Differential binding of calmodulin-related proteins to their targets revealed through high-density Arabidopsis protein microarrays

    Science.gov (United States)

    Popescu, Sorina C.; Popescu, George V.; Bachan, Shawn; Zhang, Zimei; Seay, Montrell; Gerstein, Mark; Snyder, Michael; Dinesh-Kumar, S. P.

    2007-01-01

    Calmodulins (CaMs) are the most ubiquitous calcium sensors in eukaryotes. A number of CaM-binding proteins have been identified through classical methods, and many proteins have been predicted to bind CaMs based on their structural homology with known targets. However, multicellular organisms typically contain many CaM-like (CML) proteins, and a global identification of their targets and specificity of interaction is lacking. In an effort to develop a platform for large-scale analysis of proteins in plants we have developed a protein microarray and used it to study the global analysis of CaM/CML interactions. An Arabidopsis thaliana expression collection containing 1,133 ORFs was generated and used to produce proteins with an optimized medium-throughput plant-based expression system. Protein microarrays were prepared and screened with several CaMs/CMLs. A large number of previously known and novel CaM/CML targets were identified, including transcription factors, receptor and intracellular protein kinases, F-box proteins, RNA-binding proteins, and proteins of unknown function. Multiple CaM/CML proteins bound many binding partners, but the majority of targets were specific to one or a few CaMs/CMLs indicating that different CaM family members function through different targets. Based on our analyses, the emergent CaM/CML interactome is more extensive than previously predicted. Our results suggest that calcium functions through distinct CaM/CML proteins to regulate a wide range of targets and cellular activities. PMID:17360592

  18. Characterization of the retinoblastoma binding proteins RBP1 and RBP2

    DEFF Research Database (Denmark)

    Fattaey, A R; Helin, K; Dembski, M S

    1993-01-01

    The retinoblastoma gene product, pRB, regulates cell proliferation by binding to and inhibiting the activity of key growth promoting proteins. Several cellular proteins have been shown to bind directly to pRB and the genes encoding a number of them have been isolated. The protein product of one...

  19. HMMBinder: DNA-Binding Protein Prediction Using HMM Profile Based Features.

    Science.gov (United States)

    Zaman, Rianon; Chowdhury, Shahana Yasmin; Rashid, Mahmood A; Sharma, Alok; Dehzangi, Abdollah; Shatabda, Swakkhar

    2017-01-01

    DNA-binding proteins often play important role in various processes within the cell. Over the last decade, a wide range of classification algorithms and feature extraction techniques have been used to solve this problem. In this paper, we propose a novel DNA-binding protein prediction method called HMMBinder. HMMBinder uses monogram and bigram features extracted from the HMM profiles of the protein sequences. To the best of our knowledge, this is the first application of HMM profile based features for the DNA-binding protein prediction problem. We applied Support Vector Machines (SVM) as a classification technique in HMMBinder. Our method was tested on standard benchmark datasets. We experimentally show that our method outperforms the state-of-the-art methods found in the literature.

  20. HMMBinder: DNA-Binding Protein Prediction Using HMM Profile Based Features

    Directory of Open Access Journals (Sweden)

    Rianon Zaman

    2017-01-01

    Full Text Available DNA-binding proteins often play important role in various processes within the cell. Over the last decade, a wide range of classification algorithms and feature extraction techniques have been used to solve this problem. In this paper, we propose a novel DNA-binding protein prediction method called HMMBinder. HMMBinder uses monogram and bigram features extracted from the HMM profiles of the protein sequences. To the best of our knowledge, this is the first application of HMM profile based features for the DNA-binding protein prediction problem. We applied Support Vector Machines (SVM as a classification technique in HMMBinder. Our method was tested on standard benchmark datasets. We experimentally show that our method outperforms the state-of-the-art methods found in the literature.

  1. Structure and ligand-binding properties of the biogenic amine-binding protein from the saliva of a blood-feeding insect vector of Trypanosoma cruzi

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Xueqing; Chang, Bianca W. [NIH/NIAID, 12735 Twinbrook Parkway, Rockville, MD 20852 (United States); Mans, Ben J. [NIH/NIAID, 12735 Twinbrook Parkway, Rockville, MD 20852 (United States); Agricultural Research Council, Onderstepoort 0110 (South Africa); Ribeiro, Jose M. C.; Andersen, John F., E-mail: jandersen@niaid.nih.gov [NIH/NIAID, 12735 Twinbrook Parkway, Rockville, MD 20852 (United States)

    2013-01-01

    Biogenic amine-binding proteins mediate the anti-inflammatory and antihemostatic activities of blood-feeding insect saliva. The structure of the amine-binding protein from R. prolixus reveals the interaction of biogenic amine ligands with the protein. Proteins that bind small-molecule mediators of inflammation and hemostasis are essential for blood-feeding by arthropod vectors of infectious disease. In ticks and triatomine insects, the lipocalin protein family is greatly expanded and members have been shown to bind biogenic amines, eicosanoids and ADP. These compounds are potent mediators of platelet activation, inflammation and vascular tone. In this paper, the structure of the amine-binding protein (ABP) from Rhodnius prolixus, a vector of the trypanosome that causes Chagas disease, is described. ABP binds the biogenic amines serotonin and norepinephrine with high affinity. A complex with tryptamine shows the presence of a binding site for a single ligand molecule in the central cavity of the β-barrel structure. The cavity contains significant additional volume, suggesting that this protein may have evolved from the related nitrophorin proteins, which bind a much larger heme ligand in the central cavity.

  2. Structure and ligand-binding properties of the biogenic amine-binding protein from the saliva of a blood-feeding insect vector of Trypanosoma cruzi

    International Nuclear Information System (INIS)

    Xu, Xueqing; Chang, Bianca W.; Mans, Ben J.; Ribeiro, Jose M. C.; Andersen, John F.

    2013-01-01

    Biogenic amine-binding proteins mediate the anti-inflammatory and antihemostatic activities of blood-feeding insect saliva. The structure of the amine-binding protein from R. prolixus reveals the interaction of biogenic amine ligands with the protein. Proteins that bind small-molecule mediators of inflammation and hemostasis are essential for blood-feeding by arthropod vectors of infectious disease. In ticks and triatomine insects, the lipocalin protein family is greatly expanded and members have been shown to bind biogenic amines, eicosanoids and ADP. These compounds are potent mediators of platelet activation, inflammation and vascular tone. In this paper, the structure of the amine-binding protein (ABP) from Rhodnius prolixus, a vector of the trypanosome that causes Chagas disease, is described. ABP binds the biogenic amines serotonin and norepinephrine with high affinity. A complex with tryptamine shows the presence of a binding site for a single ligand molecule in the central cavity of the β-barrel structure. The cavity contains significant additional volume, suggesting that this protein may have evolved from the related nitrophorin proteins, which bind a much larger heme ligand in the central cavity

  3. Binding Ligand Prediction for Proteins Using Partial Matching of Local Surface Patches

    Directory of Open Access Journals (Sweden)

    Lee Sael

    2010-12-01

    Full Text Available Functional elucidation of uncharacterized protein structures is an important task in bioinformatics. We report our new approach for structure-based function prediction which captures local surface features of ligand binding pockets. Function of proteins, specifically, binding ligands of proteins, can be predicted by finding similar local surface regions of known proteins. To enable partial comparison of binding sites in proteins, a weighted bipartite matching algorithm is used to match pairs of surface patches. The surface patches are encoded with the 3D Zernike descriptors. Unlike the existing methods which compare global characteristics of the protein fold or the global pocket shape, the local surface patch method can find functional similarity between non-homologous proteins and binding pockets for flexible ligand molecules. The proposed method improves prediction results over global pocket shape-based method which was previously developed by our group.

  4. Binding ligand prediction for proteins using partial matching of local surface patches.

    Science.gov (United States)

    Sael, Lee; Kihara, Daisuke

    2010-01-01

    Functional elucidation of uncharacterized protein structures is an important task in bioinformatics. We report our new approach for structure-based function prediction which captures local surface features of ligand binding pockets. Function of proteins, specifically, binding ligands of proteins, can be predicted by finding similar local surface regions of known proteins. To enable partial comparison of binding sites in proteins, a weighted bipartite matching algorithm is used to match pairs of surface patches. The surface patches are encoded with the 3D Zernike descriptors. Unlike the existing methods which compare global characteristics of the protein fold or the global pocket shape, the local surface patch method can find functional similarity between non-homologous proteins and binding pockets for flexible ligand molecules. The proposed method improves prediction results over global pocket shape-based method which was previously developed by our group.

  5. Nutritional profiling of Eurasian woodcock meat: chemical composition and myoglobin characterization.

    Science.gov (United States)

    Landi, Nicola; Ragucci, Sara; Di Giuseppe, Antonella Ma; Russo, Rosita; Poerio, Elia; Severino, Valeria; Di Maro, Antimo

    2018-04-10

    Meat from birds is a rich source of proteins for the human diet. In this framework, Eurasian woodcock (Scolopax rusticola L.), a medium-small wading bird hunted as game in many Eurasian countries, is considered one of the best meats for culinary purposes. Since the nutritional composition of Eurasian woodcock meat has not yet been reported, we decided to determine the nutritional profile of S. rusticola meat. Macronutrient components (proteins, lipids and fatty acids) were determined, as well as free and total amino acids, and compared with those of the common pheasant. Eurasian woodcock meat contains high levels of proteins and essential amino acids. The levels of unsaturated fatty acids represent a great contribution to the total lipid amount. Among polyunsaturated fatty acids, linoleic acid (C18:2, n-6) is the major essential fatty acid. Finally, we report the characterization of myoglobin (Mb) from Eurasian woodcock. The data revealed that meat from this bird could be a good source of quality raw proteins because of its amino acid composition, and it had a low lipid content. On the other hand, Mb characterization might be of benefit to the meat industry, by providing useful information for the determination of species-specific differences in meat from birds. © 2018 Society of Chemical Industry. © 2018 Society of Chemical Industry.

  6. Effects of ionizing radiation on nitric oxide myoglobin

    International Nuclear Information System (INIS)

    Kamarei, A.R.; Karel, M.

    1983-01-01

    Bovine nitric oxide myoglobin (NOMb) was irradiated with 40-4000 krad of γ-radiation, and the effects on the haem studied using absorption spectroscopy and electron spin resonance (e.s.r.) spectroscopy. The results show the following behaviour: (a) The bright red colour of NOMb changes to brown upon irradiation. This is similar to changes observed in radiation sterilized, nitrite-containing meats. (b) NOMb becomes progressively denitrosylated, with met-myoglobin (metMb) as the immediate product. (c) Upon increasing doses of radiation (up to 800 krad) at O 0 C parallel to NOMb denitrosylation, metMb is gradually converted, by water radiolytic products, to other products, believed to be ferromyoglobin and ferrimyoglobin peroxide. A minor quantity of 'choleglobin-type' pigments may also be formed at the highest doses. (d) Freezing of NOMb has a substantial protective effect against radiation. (e) Native bovine NOMb behaves as a pentaco-ordinate (hfs of 3 peaks with equal intensity); the bond between iron and Nsub(epsilon) is thus dramatically stretched and weakened. (f) Using a thermal energy analyser, no NO could be detected over irradiated NOMb solution, indicating rapid reaction of NO liberated from NOMb by radiation, with radiolytic products of water. (author)

  7. Identification of fibrinogen-binding proteins of Aspergillus fumigatus using proteomic approach.

    Science.gov (United States)

    Upadhyay, Santosh Kumar; Gautam, Poonam; Pandit, Hrishikesh; Singh, Yogendra; Basir, Seemi Farhat; Madan, Taruna

    2012-03-01

    Aspergillus fumigatus, the main etiological agent for various forms of human aspergillosis, gets access to the respiratory system of human host by inhalation of airborne conidia. These conidia possibly adhere to extracellular matrix (ECM) proteins. Among the ECM proteins involved in adherence, fibrinogen is thought to be crucial. Here, we studied whether A. fumigatus three-week culture filtrate (3wcf) proteins promote binding of A. fumigatus to ECM proteins and promote fungal growth. We observed that incubation of ECM with 3wcf proteins led to dose- and time-dependent increase in adherence of conidia to the ECM. In order to identify the catalogue of fibrinogen-binding A. fumigatus proteins, we carried out fibrinogen affinity blotting using two-dimensional gel electrophoresed 3wcf proteins. A total of 15 fibrinogen-binding protein spots corresponding to 7 unique proteins were identified in 3wcf using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF-TOF). Among these, 4 proteins, namely, beta-glucosidase, alpha-mannosidase, pectate lyase A and oryzin precursor were predicted to have cell wall or extracellular localization, whereas amidase family protein and two hypothetical proteins did not display the signal sequence. This study reports seven novel fibrinogen-binding proteins of A. fumigatus, some of which could be further explored for targeting the adhesion phenomenon as antifungal strategy.

  8. Elastin binds to a multifunctional 67-kilodalton peripheral membrane protein

    International Nuclear Information System (INIS)

    Mecham, R.P.; Hinek, A.; Entwistle, R.; Wrenn, D.S.; Griffin, G.L.; Senior, R.M.

    1989-01-01

    Elastin binding proteins from plasma membranes of elastin-producing cells were isolated by affinity chromatography on immobilized elastin peptides. Three proteins of 67, 61, and 55 kDa were released from the elastin resin by guanidine/detergent, soluble elastin peptides, synthetic peptide VGVAPG, or galactoside sugars, but not by synthetic RGD-containing peptide or sugars not related to galactose. All three proteins incorporated radiolabel upon extracellular iodination and contained [ 3 H]leucine following metabolic labeling, confirming that each is a synthetic product of the cell. The 67-kDa protein could be released from the cell surface with lactose-containing buffers, whereas solubilization of the 61- and 55-kDa components required the presence of detergent. Although all three proteins were retained on elastin affinity columns, the 61- and 55-kDa components were retained only in the presence of 67-kDa protein, suggesting that the 67-kDa protein binds elastin and the 61- and 55-kDa proteins bind to the 67-kDa protein. The authors propose that the 67-, 61-, and 55-kDa proteins constitute an elastin-receptor complex that forms a transmembrane link between the extracellular matrix and the intracellular compartment

  9. Value of heart-type fatty acid-binding protein (H-FABP) for ...

    African Journals Online (AJOL)

    Key Words: heart-type fatty acid-binding protein, acute coronary syndrome, biomarker. ... is essential to prevent major complications and death. Routinely used biomarkers such ..... fatty acid binding proteins: their function and physiological sig-.

  10. 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.

  11. Binding of MCM-interacting proteins to ATP-binding site in MCM6

    Directory of Open Access Journals (Sweden)

    Hosoi A

    2016-03-01

    Full Text Available Atsutoshi Hosoi, Taku Sakairi, Yukio Ishimi Graduate School of Science and Engineering, Ibaraki University, Mito, Ibaraki, Japan Abstract: The function of MCM2–7 complex that is a DNA helicase in DNA replication may be regulated by various MCM-interacting proteins, including CDC45, RPA, TIM, TIPIN, Claspin, MCM10, and MCM-BP. It has been shown by immunoprecipitation that human MCM6 interacts with all these proteins in coexpressed insect cells. To determine the region in MCM6 to interact with these proteins, we prepared various truncated forms of MCM6 and examined the interaction of these MCM6 fragments with the MCM-interacting proteins. All these proteins bound to C-terminal half of MCM6, and CDC45, RPA2, TIM, TIPIN, MCM-BP, and MCM10 bound to the fragments containing ATP-binding motifs. CDC45 and RPA2 bound to the smallest fragment containing Walker motif A. Only MCM-BP is bound to the N-terminal half of MCM6. Site-directed mutagenesis study suggests that hydrophobic interaction is involved in the interaction of MCM6 with CDC45 and TIM. These results suggest a possibility that MCM-interacting proteins regulate MCM2–7 function by modulating the ATP-binding ability of the MCM2–7. Keywords: DNA helicase, DNA replication, checkpoint, MCM2–7 proteins

  12. The hydroxyl-functionalized magnetic particles for purification of glycan-binding proteins.

    Science.gov (United States)

    Sun, Xiuxuan; Yang, Ganglong; Sun, Shisheng; Quan, Rui; Dai, Weiwei; Li, Bin; Chen, Chao; Li, Zheng

    2009-12-01

    Glycan-protein interactions play important biological roles in biological processes. Although there are some methods such as glycan arrays that may elucidate recognition events between carbohydrates and protein as well as screen the important glycan-binding proteins, there is a lack of simple effectively separate method to purify them from complex samples. In proteomics studies, fractionation of samples can help to reduce their complexity and to enrich specific classes of proteins for subsequent downstream analyses. Herein, a rapid simple method for purification of glycan-binding proteins from proteomic samples was developed using hydroxyl-coated magnetic particles coupled with underivatized carbohydrate. Firstly, the epoxy-coated magnetic particles were further hydroxyl functionalized with 4-hydroxybenzhydrazide, then the carbohydrates were efficiently immobilized on hydroxyl functionalized surface of magnetic particles by formation of glycosidic bond with the hemiacetal group at the reducing end of the suitable carbohydrates via condensation. All conditions of this method were optimized. The magnetic particle-carbohydrate conjugates were used to purify the glycan-binding proteins from human serum. The fractionated glycan-binding protein population was displayed by SDS-PAGE. The result showed that the amount of 1 mg magnetic particles coupled with mannose in acetate buffer (pH 5.4) was 10 micromol. The fractionated glycan-binding protein population in human serum could be eluted from the magnetic particle-mannose conjugates by 0.1% SDS. The methodology could work together with the glycan microarrays for screening and purification of the important GBPs from complex protein samples.

  13. Oxygen binding to nitric oxide marked hemoglobin

    International Nuclear Information System (INIS)

    Louro, S.R.W.; Ribeiro, P.C.; Bemski, G.

    1979-04-01

    Electron spin resonance spectra of organic phosphate free human hemoglobin marked with nitric oxide at the sixth coordination position of one of the four hemes allow to observe the transition from the tense (T) to the relaxed (R) conformation, as a function of parcial oxygen pressure. The spectra are composites of contributions from α sub(T), α sub(R) and β chains spectra, showing the presence of only two conformations: T and R. In the absence of organic phosphates NO binds to α and β chains with the same probability, but in the presence of phosphates NO combines preferentially with α chains. The dissociation of NO proceeds at least an order of magnitude faster in T than in R configuration. (author) [pt

  14. Ribosomal DNA-binding proteins in the nucleolus of Physarum polycephalum

    International Nuclear Information System (INIS)

    Graham-Lorence, S.E.

    1987-01-01

    In Physarum polycephalum, the nucleoli are extra chromosomal structures containing 200 to 400 copies of a linear 60 kilobase palindromic rDNA molecule. These rDNA molecules are organized into minichromosomes which apparently are held within a nucleolar protein matrix. To obtained evidence for attachment of the rDNA to such a matrix, both intact and lithium diiodosalicylate/NaCl-extracted nucleoli were digested for various lengths of time with micrococcal nuclease, so that portions of the rDNA molecules not attached within the nucleolar structure would be released. Nucleolar DNA-binding proteins were determined by blotting electrophoretically separated proteins from SDS-polyacrylamide gels onto nitrocellulose paper and probing them with radiolabeled DNA. In addition to the histones and lexosome proteins, eight DNA-binding proteins were identified having molecular weights of 25, 38, 47, 53, 55, 67, and 70 kD, with the 47, 53, 67, and 70 kD proteins requiring Ca 2+ for binding

  15. Statistical-mechanical lattice models for protein-DNA binding in chromatin

    International Nuclear Information System (INIS)

    Teif, Vladimir B; Rippe, Karsten

    2010-01-01

    Statistical-mechanical lattice models for protein-DNA binding are well established as a method to describe complex ligand binding equilibria measured in vitro with purified DNA and protein components. Recently, a new field of applications has opened up for this approach since it has become possible to experimentally quantify genome-wide protein occupancies in relation to the DNA sequence. In particular, the organization of the eukaryotic genome by histone proteins into a nucleoprotein complex termed chromatin has been recognized as a key parameter that controls the access of transcription factors to the DNA sequence. New approaches have to be developed to derive statistical-mechanical lattice descriptions of chromatin-associated protein-DNA interactions. Here, we present the theoretical framework for lattice models of histone-DNA interactions in chromatin and investigate the (competitive) DNA binding of other chromosomal proteins and transcription factors. The results have a number of applications for quantitative models for the regulation of gene expression.

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

    Directory of Open Access Journals (Sweden)

    Daniel M Dupont

    Full Text Available Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126 with therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA. We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A controlling uPA activities. One of the aptamers (upanap-126 binds to the area around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12 binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro-uPA complex. The results suggest and highlight that the size and shape of an aptamer as well as the domain organization of a multi-domain protein such as uPA, may provide the basis for extensive sterical interference with protein ligand interactions considered distant from the aptamer binding site.

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

    Science.gov (United States)

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

    2014-03-01

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

  18. Self-Assembly of Protein Monolayers Engineered for Improved Monoclonal Immunoglobulin G Binding

    Directory of Open Access Journals (Sweden)

    Jeremy H. Lakey

    2011-08-01

    Full Text Available Bacterial outer membrane proteins, along with a filling lipid molecule can be modified to form stable self-assembled monolayers on gold. The transmembrane domain of Escherichia coli outer membrane protein A has been engineered to create a scaffold protein to which functional motifs can be fused. In earlier work we described the assembly and structure of an antibody-binding array where the Z domain of Staphylococcus aureus protein A was fused to the scaffold protein. Whilst the binding of rabbit polyclonal immunoglobulin G (IgG to the array is very strong, mouse monoclonal IgG dissociates from the array easily. This is a problem since many immunodiagnostic tests rely upon the use of mouse monoclonal antibodies. Here we describe a strategy to develop an antibody-binding array that will bind mouse monoclonal IgG with lowered dissociation from the array. A novel protein consisting of the scaffold protein fused to two pairs of Z domains separated by a long flexible linker was manufactured. Using surface plasmon resonance the self-assembly of the new protein on gold and the improved binding of mouse monoclonal IgG were demonstrated.

  19. Loss of Selenium-Binding Protein 1 Decreases Sensitivity to Clastogens and Intracellular Selenium Content in HeLa Cells.

    Science.gov (United States)

    Zhao, Changhui; Zeng, Huawei; Wu, Ryan T Y; Cheng, Wen-Hsing

    2016-01-01

    Selenium-binding protein 1 (SBP1) is not a selenoprotein but structurally binds selenium. Loss of SBP1 during carcinogenesis usually predicts poor prognosis. Because genome instability is a hallmark of cancer, we hypothesize that SBP1 sequesters cellular selenium and sensitizes cancer cells to DNA-damaging agents. To test this hypothesis, we knocked down SBP1 expression in HeLa cervical cancer cells by employing a short hairpin RNA (shRNA) approach. Reduced sensitivity to hydrogen peroxide, paraquat and camptothecin, reactive oxygen species content, and intracellular retention of selenium after selenomethionine treatment were observed in SBP1 shRNA HeLa cells. Results from Western analyses showed that treatment of HeLa cells with selenomethionine resulted in increased SBP1 protein expression in a dose-dependent manner. Knockdown of SBP1 rendered HeLa cells increased expression of glutathione peroxidase-1 but not glutathione peroxidase-4 protein levels and accelerated migration from a wound. Altogether, SBP1 retains supplemental selenium and sensitizes HeLa cancer cells to clastogens, suggesting a new cancer treatment strategy by sequestering selenium through SBP1.

  20. Vitamin D binding protein: a multifunctional protein of clinical importance.

    Science.gov (United States)

    Speeckaert, Marijn M; Speeckaert, Reinhart; van Geel, Nanja; Delanghe, Joris R

    2014-01-01

    Since the discovery of group-specific component and its polymorphism by Hirschfeld in 1959, research has put spotlight on this multifunctional transport protein (vitamin D binding protein, DBP). Besides the transport of vitamin D metabolites, DBP is a plasma glycoprotein with many important functions, including sequestration of actin, modulation of immune and inflammatory responses, binding of fatty acids, and control of bone development. A considerable DBP polymorphism has been described with a specific allele distribution in different geographic area. Multiple studies have shed light on the interesting relationship between polymorphisms of the DBP gene and the susceptibility to diseases. In this review, we give an overview of the multifunctional character of DBP and describe the clinical importance of DBP and its polymorphisms. Finally, we discuss the possibilities to use DBP as a novel therapeutic agent.

  1. Strong Ligand-Protein Interactions Derived from Diffuse Ligand Interactions with Loose Binding Sites.

    Science.gov (United States)

    Marsh, Lorraine

    2015-01-01

    Many systems in biology rely on binding of ligands to target proteins in a single high-affinity conformation with a favorable ΔG. Alternatively, interactions of ligands with protein regions that allow diffuse binding, distributed over multiple sites and conformations, can exhibit favorable ΔG because of their higher entropy. Diffuse binding may be biologically important for multidrug transporters and carrier proteins. A fine-grained computational method for numerical integration of total binding ΔG arising from diffuse regional interaction of a ligand in multiple conformations using a Markov Chain Monte Carlo (MCMC) approach is presented. This method yields a metric that quantifies the influence on overall ligand affinity of ligand binding to multiple, distinct sites within a protein binding region. This metric is essentially a measure of dispersion in equilibrium ligand binding and depends on both the number of potential sites of interaction and the distribution of their individual predicted affinities. Analysis of test cases indicates that, for some ligand/protein pairs involving transporters and carrier proteins, diffuse binding contributes greatly to total affinity, whereas in other cases the influence is modest. This approach may be useful for studying situations where "nonspecific" interactions contribute to biological function.

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

    Science.gov (United States)

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

  3. A calmodulin-like protein (LCALA) is a new Leishmania amazonensis candidate for telomere end-binding protein.

    Science.gov (United States)

    Morea, Edna G O; Viviescas, Maria Alejandra; Fernandes, Carlos A H; Matioli, Fabio F; Lira, Cristina B B; Fernandez, Maribel F; Moraes, Barbara S; da Silva, Marcelo S; Storti, Camila B; Fontes, Marcos R M; Cano, Maria Isabel N

    2017-11-01

    Leishmania spp. telomeres are composed of 5'-TTAGGG-3' repeats associated with proteins. We have previously identified LaRbp38 and LaRPA-1 as proteins that bind the G-rich telomeric strand. At that time, we had also partially characterized a protein: DNA complex, named LaGT1, but we could not identify its protein component. Using protein-DNA interaction and competition assays, we confirmed that LaGT1 is highly specific to the G-rich telomeric single-stranded DNA. Three protein bands, with LaGT1 activity, were isolated from affinity-purified protein extracts in-gel digested, and sequenced de novo using mass spectrometry analysis. In silico analysis of the digested peptide identified them as a putative calmodulin with sequences identical to the T. cruzi calmodulin. In the Leishmania genome, the calmodulin ortholog is present in three identical copies. We cloned and sequenced one of the gene copies, named it LCalA, and obtained the recombinant protein. Multiple sequence alignment and molecular modeling showed that LCalA shares homology to most eukaryotes calmodulin. In addition, we demonstrated that LCalA is nuclear, partially co-localizes with telomeres and binds in vivo the G-rich telomeric strand. Recombinant LCalA can bind specifically and with relative affinity to the G-rich telomeric single-strand and to a 3'G-overhang, and DNA binding is calcium dependent. We have described a novel candidate component of Leishmania telomeres, LCalA, a nuclear calmodulin that binds the G-rich telomeric strand with high specificity and relative affinity, in a calcium-dependent manner. LCalA is the first reported calmodulin that binds in vivo telomeric DNA. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Drosophila TDP-43 RNA-Binding Protein Facilitates Association of Sister Chromatid Cohesion Proteins with Genes, Enhancers and Polycomb Response Elements.

    Directory of Open Access Journals (Sweden)

    Amanda Swain

    2016-09-01

    Full Text Available The cohesin protein complex mediates sister chromatid cohesion and participates in transcriptional control of genes that regulate growth and development. Substantial reduction of cohesin activity alters transcription of many genes without disrupting chromosome segregation. Drosophila Nipped-B protein loads cohesin onto chromosomes, and together Nipped-B and cohesin occupy essentially all active transcriptional enhancers and a large fraction of active genes. It is unknown why some active genes bind high levels of cohesin and some do not. Here we show that the TBPH and Lark RNA-binding proteins influence association of Nipped-B and cohesin with genes and gene regulatory sequences. In vitro, TBPH and Lark proteins specifically bind RNAs produced by genes occupied by Nipped-B and cohesin. By genomic chromatin immunoprecipitation these RNA-binding proteins also bind to chromosomes at cohesin-binding genes, enhancers, and Polycomb response elements (PREs. RNAi depletion reveals that TBPH facilitates association of Nipped-B and cohesin with genes and regulatory sequences. Lark reduces binding of Nipped-B and cohesin at many promoters and aids their association with several large enhancers. Conversely, Nipped-B facilitates TBPH and Lark association with genes and regulatory sequences, and interacts with TBPH and Lark in affinity chromatography and immunoprecipitation experiments. Blocking transcription does not ablate binding of Nipped-B and the RNA-binding proteins to chromosomes, indicating transcription is not required to maintain binding once established. These findings demonstrate that RNA-binding proteins help govern association of sister chromatid cohesion proteins with genes and enhancers.

  5. Milk proteins interact with goat Binder of SPerm (BSP) proteins and decrease their binding to sperm.

    Science.gov (United States)

    de Menezes, Erika Bezerra; van Tilburg, Mauricio; Plante, Geneviève; de Oliveira, Rodrigo V; Moura, Arlindo A; Manjunath, Puttaswamy

    2016-11-01

    Seminal plasma Binder of SPerm (BSP) proteins bind to sperm at ejaculation and promote capacitation. When in excess, however, BSP proteins damage the sperm membrane. It has been suggested that milk components of semen extenders associate with BSP proteins, potentially protecting sperm. Thus, this study was conducted to investigate if milk proteins interact with BSP proteins and reduce BSP binding to goat sperm. Using gel filtration chromatography, milk was incubated with goat seminal plasma proteins and loaded onto columns with and without calcium. Milk was also fractionated into parts containing mostly whey proteins or mostly caseins, incubated with seminal plasma proteins and subjected to gel filtration. Eluted fractions were evaluated by immunoblot using anti-goat BSP antibodies, confirming milk protein-BSP protein interactions. As determined by ELISA, milk proteins coated on polystyrene wells bound to increasing of goat BSP proteins. Far-western dot blots confirmed that BSP proteins bound to caseins and β-lactoglobulin in a concentration-dependent manner. Then, cauda epididymal sperm from five goats was incubated with seminal plasma; seminal plasma followed by milk; and milk followed by seminal plasma. Sperm membrane proteins were extracted and evaluated by immunoblotting. The pattern of BSP binding to sperm membrane proteins was reduced by 59.3 % when epididymal sperm were incubated with seminal plasma and then with skimmed milk (p  0.05). In conclusion, goat BSP proteins have an affinity for caseins and whey proteins. Milk reduces BSP binding to goat sperm, depending whether or not sperm had been previously exposed to seminal plasma. Such events may explain the protective effect of milk during goat sperm preservation.

  6. New human erythrocyte protein with binding sites for both spectrin and calmodulin

    International Nuclear Information System (INIS)

    Gardner, K.; Bennett, V.

    1986-01-01

    A new cytoskeletal protein that binds calmodulin has been purified to greater than 95% homogeneity from human erythrocyte cytoskeletons. The protein is a heterodimer with subunits of 103,000 and 97,000 and M/sub r/ = 197,000 calculated from its Stokes radius of 6.9 nm and sedimentation coefficient of 6.8. A binding affinity of this protein for calmodulin has been estimated to be 230 nM by displacement of two different concentrations of 125 I-azidocalmodulin with increasing concentrations of unmodified calmodulin followed by Dixon plot analysis. This protein is present in red cells at approximately 30,000 copies per cell and contains a very tight binding site(s) on cytoskeletons. The protein can be only partially solubilized from isolated cytoskeletons in buffers containing high salt, but can be totally solubilized from red cell ghost membranes by extraction in low ionic strength buffers. Affinity purified IgG against this calmodulin-binding protein identifies crossreacting polypeptide(s) in brain, kidney, testes and retina. Visualization of the calmodulin-binding protein by negative staining, rotary shadowing and unidirectional shadowing indicate that it is a flattened circular molecule with molecular height of 5.4 nm and a diameter of 12.4 nm. Preliminary cosedimentation studies with purified spectrin and F-actin indicate that the site of interaction of this calmodulin-binding protein with the cytoskeleton resides on spectrin

  7. Atomic structure of nitrate-binding protein crucial for photosynthetic productivity

    Energy Technology Data Exchange (ETDEWEB)

    Koropatkin, Nicole M.; Pakrasi, Himadri B.; Smith, Thomas J.

    2006-06-27

    Cyanobacteria, blue-green algae, are the most abundant autotrophs in aquatic environments and form the base of all aquatic food chains by fixing carbon and nitrogen into cellular biomass. The single most important nutrient for photosynthesis and growth is nitrate, which is severely limiting in many aquatic environments particularly the open ocean (1, 2). It is therefore not surprising that NrtA, the solute-binding component of the high-affinity nitrate ABC transporter, is the single-most abundant protein in the plasma membrane of these bacteria (3). Here we describe the first structure of a nitratespecific receptor, NrtA from Synechocystis sp. PCC 6803, complexed with nitrate and determined to a resolution of 1.5Å. NrtA is significantly larger than other oxyanionbinding proteins, representing a new class of transport proteins. From sequence alignments, the only other solute-binding protein in this class is CmpA, a bicarbonatebinding protein. Therefore, these organisms created a novel solute-binding protein for two of the most important nutrients; inorganic nitrogen and carbon. The electrostatic charge distribution of NrtA appears to force the protein off of the membrane while the flexible tether facilitates the delivery of nitrate to the membrane pore. The structure not only details the determinants for nitrate selectivity in NrtA, but also the bicarbonate specificity in CmpA. Nitrate and bicarbonate transport are regulated by the cytoplasmic proteins NrtC and CmpC, respectively. Interestingly, the residues lining the ligand binding pockets suggest that they both bind nitrate. This implies that the nitrogen and carbon uptake pathways are synchronized by intracellular nitrate and nitrite.3 The nitrate ABC transporter of cyanobacteria is composed of four polypeptides (Figure 1): a high-affinity periplasmic solute-binding lipoprotein (NrtA), an integral membrane permease (NrtB), a cytoplasmic ATPase (NrtD), and a unique ATPase/solute-binding fusion protein (Nrt

  8. Phytochrome regulates GTP-binding protein activity in the envelope of pea nuclei

    Science.gov (United States)

    Clark, G. B.; Memon, A. R.; Thompson, G. A. Jr; Roux, S. J.

    1993-01-01

    Three GTP-binding proteins with apparent molecular masses of 27, 28 and 30 kDa have been detected in isolated nuclei of etiolated pea plumules. After LDS-PAGE and transfer to nitrocellulose these proteins bind [32P]GTP in the presence of excess ATP, suggesting that they are monomeric G proteins. When nuclei are disrupted, three proteins co-purify with the nuclear envelope fraction and are highly enriched in this fraction. The level of [32P]GTP-binding for all three protein bands is significantly increased when harvested pea plumules are irradiated by red light, and this effect is reversed by far-red light. The results indicate that GTP-binding activity associated with the nuclear envelope of plant cells is photoreversibly regulated by the pigment phytochrome.

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

    International Nuclear Information System (INIS)

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

    1982-01-01

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

  10. What Is the True Color of Fresh Meat? A Biophysical Undergraduate Laboratory Experiment Investigating the Effects of Ligand Binding on Myoglobin Using Optical, EPR, and NMR Spectroscopy

    Science.gov (United States)

    Linenberger, Kimberly; Bretz, Stacey Lowery; Crowder, Michael W.; McCarrick, Robert; Lorigan, Gary A.; Tierney, David L.

    2011-01-01

    With an increased focus on integrated upper-level laboratories, we present an experiment integrating concepts from inorganic, biological, and physical chemistry content areas. Students investigate the effects of ligand strength on the spectroscopic properties of the heme center in myoglobin using UV-vis, [superscript 1]H NMR, and EPR…

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

    Science.gov (United States)

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

    2016-01-01

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

  12. Near-infrared spectroscopy for monitoring muscle oxygenation

    DEFF Research Database (Denmark)

    Boushel, Robert Christopher; Piantadosi, C A

    2000-01-01

    Near-infrared spectroscopy (NIRS) is a non-invasive method for monitoring oxygen availability and utilization by the tissues. In intact skeletal muscle, NIRS allows semi-quantitative measurements of haemoglobin plus myoglobin oxygenation (tissue O2 stores) and the haemoglobin volume. Specialized...... algorithms allow assessment of the oxidation-reduction (redox) state of the copper moiety (CuA) of mitochondrial cytochrome c oxidase and, with the use of specific tracers, accurate assessment of regional blood flow. NIRS has demonstrated utility for monitoring changes in muscle oxygenation and blood flow...... during submaximal and maximal exercise and under pathophysiological conditions including cardiovascular disease and sepsis. During work, the extent to which skeletal muscles deoxygenate varies according to the type of muscle, type of exercise and blood flow response. In some instances, a strong...

  13. Proteomic based approach for characterizing 4-hydroxy-2-nonenal induced oxidation of buffalo (Bubalus bubalis) and goat (Capra hircus) meat myoglobins

    OpenAIRE

    Maheswarappa, Naveena B.; Rani, K. Usha; Kumar, Y. Praveen; Kulkarni, Vinayak V.; Rapole, Srikanth

    2016-01-01

    Background Myoglobin (Mb) is a sarcoplasmic heme protein primarily responsible for meat color and its chemistry is species specific. 4-hydroxy-2-nonenal (HNE) is a cytotoxic lipid derived aldehyde detected in meat and was reported to covalently adduct with nucleophilic histidine residues of Mb and predispose it to greater oxidation. However, no literature is available on characterization of lipid oxidation induced oxidation of Indian water buffalo (Bubalus bubalis) and goat (Capra hircus) myo...

  14. Nuclear magnetic resonance studies of amino acids and proteins. Side-chain mobility of methionine in the crystalline amonio acid and in crystallne sperm whale (Physeter catodon) myoglobin

    International Nuclear Information System (INIS)

    Keniry, M.A.; Rothgeb, T.M.; Smith, R.L.; Gutowsky, H.S.; Oldfield, E.

    1983-01-01

    Deuterium ( 2 H) nuclear magnetic resonance (NMR) spectra and spin-lattice relaxation times (T 1 ) were obtained of L-[epsilon- 2 H 3 ]methionine, L-[epsilon- 2 H 3 ]methionine in a D,L lattice, and [S-methyl- 2 H 3 ]methionine in the crystalline solid state, as a function of temperature, in addition to obtaining 2 H T 1 and line-width results as a function of temperature on [epsilon- 2 H 3 ]methionine-labeled sperm whale (Physeter catodon) myoglobins by using the method of magnetic ordering. Also recorded were 13 C cross-polarization ''magic-angle'' sample-spinning NMR spectra of [epsilon- 13 C]methionine-labeled crystalline cyanoferrimyoglobin (at 37.7 MHz, corresponding to a magnetic field strength of 3.52 T) and of the same protein in aqueous solution

  15. Calcium phosphate-gold nanoparticles nanocomposite for protein adsorption and mediator-free H{sub 2}O{sub 2} biosensor construction

    Energy Technology Data Exchange (ETDEWEB)

    Xu Qin [College of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225002 (China); Jiangsu Key Laboratory of Environmental Engineering and Monitoring, Yangzhou 225002 (China); Lu Guiju; Bian XiaoJun; Jin Gendi [College of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225002 (China); Wang Wei [School of Chemical and Biological Engineering, Yancheng Institute of Technology, Yancheng, 224051 (China); Hu Xiaoya, E-mail: xyhu@yzu.edu.cn [College of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225002 (China); Jiangsu Key Laboratory of Environmental Engineering and Monitoring, Yangzhou 225002 (China); Wang Yang; Yang Zhanjun [College of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225002 (China)

    2012-04-01

    This work reports a new method for the preparation and application of a kind of biocompatible calcium phosphate-gold nanoparticles (Ca{sub 3}(PO{sub 4}){sub 2}-AuNPs) nanocomposite. UV-vis spectroscopy and transmittance electron microscopy (TEM) have been used to monitor the formation process of the nanocomposite and to examine the interaction between calcium phosphate and gold nanoparticles (AuNPs). The nanocomposite has multiple sites and improved conductivity which make it suitable for the binding of proteins to construct electrochemical sensors. Myoglobin (Mb) adsorbed on the nanocomposite retained its native structure which was proved by Fourier transform infrared spectroscopy (FTIR). Direct electron transfer between the adsorbed Mb and the electrode was observed. Further results demonstrated that the adsorbed Mb has good electrocatalytic activity towards the reduction of H{sub 2}O{sub 2} in the absence of any mediator. Highlights: Black-Right-Pointing-Pointer Using gelatin modified gold nanoparticles to prepare needle-like calcium phosphate. Black-Right-Pointing-Pointer Calcium phosphate provides multiple sites for protein adsorption. Black-Right-Pointing-Pointer Gold nanoparticles act as electron tunneling. Black-Right-Pointing-Pointer Myoglobin adsorbed on the material showed direct electrochemistry and good catalysis.

  16. Juvenile hormone-binding proteins of Melanoplus bivittatus identified by EFDA photoaffinity labeling

    International Nuclear Information System (INIS)

    Winder, B.S.

    1988-01-01

    Proteins that bind juvenile hormone in the hemolymph and fat body of the grasshopper, Melanoplus bivittatus were identified by photoaffinity labeling with radiolabeled epoxyfarnesyl diazoacetate ( 3 H-EFDA), and were characterized by electrophoretic analysis. A protocol was developed which allowed detection of 3 H-EFDA that was covalently linked to proteins upon exposure to ultraviolet light at 254 nm. Quantification of protein-linked 3 H-EFDA by liquid scintillation spectrometry took advantage of the differential solubility of unlinked 3 H-EFDA in toluene alone, and of the protein-linked 3 H-EFDA in toluene plus the detergent, Triton X-100. Competition between EFDA and juvenile hormone (JH) for binding to JH-specific binding sites was measured by hydroxyapatite protein binding assays in the presence of radiolabeled JH or EFDA and competing non-radiolabeled hormone. The protein-linked EFDA was detected on fluorograms of SDS or nondenaturing polyacrylamide gels (PAGE), and by liquid scintillation spectrometry of membranes to which the proteins had been electrophoretically transferred. Proteins which specifically bound JH were identified by photolabeling proteins in the presence and absence of nonlabeled JH-III

  17. Proteome scale identification, classification and structural analysis of iron-binding proteins in bread wheat.

    Science.gov (United States)

    Verma, Shailender Kumar; Sharma, Ankita; Sandhu, Padmani; Choudhary, Neha; Sharma, Shailaja; Acharya, Vishal; Akhter, Yusuf

    2017-05-01

    Bread wheat is one of the major staple foods of worldwide population and iron plays a significant role in growth and development of the plant. In this report, we are presenting the genome wide identification of iron-binding proteins in bread wheat. The wheat genome derived putative proteome was screened for identification of iron-binding sequence motifs. Out of 602 putative iron-binding proteins, 130 were able to produce reliable structural models by homology techniques and further analyzed for the presence of iron-binding structural motifs. The computationally identified proteins appear to bind to ferrous and ferric ions and showed diverse coordination geometries. Glu, His, Asp and Cys amino acid residues were found to be mostly involved in iron binding. We have classified these proteins on the basis of their localization in the different cellular compartments. The identified proteins were further classified into their protein folds, families and functional classes ranging from structure maintenance of cellular components, regulation of gene expression, post translational modification, membrane proteins, enzymes, signaling and storage proteins. This comprehensive report regarding structural iron binding proteome provides useful insights into the diversity of iron binding proteins of wheat plants and further utilized to study their roles in plant growth, development and physiology. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Radiometric immunosorbent assay for the detection of anti-hormone-binding protein antibodies

    International Nuclear Information System (INIS)

    Pierce, E.A.; Dame, M.C.; DeLuca, H.F.

    1986-01-01

    A radiometric immunosorbent assay (RISA) for the detection of monoclonal antibodies to hormone-binding proteins has been developed. The assay involves incubating hybridoma supernatants in microtiter wells that have been coated with goat anti-mouse IgG antibodies. Any mouse IgG in the test supernatant is thus specifically retained in the wells. Radioactive ligand-binding protein complexes are then incubated in the wells. The presence of anti-binding protein antibodies in the supernatant is indicated by specific retention of radioactive ligand-binding protein complexes in the wells. Crude antigen preparations, such as tissue homogenates, can be used to detect antibodies. The assay is capable of detecting antibody at concentrations 20 ng/ml (approx. 100 pM IgG). The RISA has been used successfully to screen for monoclonal antibodies to the intracellular receptor for 1,25-dihydroxyvitamin D 3 and should be useful for the detection of antibodies to ligand-binding proteins in general

  19. A Chitin-binding Protein Purified from Moringa oleifera Seeds Presents Anticandidal Activity by Increasing Cell Membrane Permeability and Reactive Oxygen Species Production

    Directory of Open Access Journals (Sweden)

    João X.S. Neto

    2017-06-01

    Full Text Available Candida species are opportunistic pathogens that infect immunocompromised and/or immunosuppressed patients, particularly in hospital facilities, that besides representing a significant threat to health increase the risk of mortality. Apart from echinocandins and triazoles, which are well tolerated, most of the antifungal drugs used for candidiasis treatment can cause side effects and lead to the development of resistant strains. A promising alternative to the conventional treatments is the use of plant proteins. M. oleifera Lam. is a plant with valuable medicinal properties, including antimicrobial activity. This work aimed to purify a chitin-binding protein from M. oleifera seeds and to evaluate its antifungal properties against Candida species. The purified protein, named Mo-CBP2, represented about 0.2% of the total seed protein and appeared as a single band on native PAGE. By mass spectrometry, Mo-CBP2 presented 13,309 Da. However, by SDS-PAGE, Mo-CBP2 migrated as a single band with an apparent molecular mass of 23,400 Da. Tricine-SDS-PAGE of Mo-CBP2 under reduced conditions revealed two protein bands with apparent molecular masses of 7,900 and 4,600 Da. Altogether, these results suggest that Mo-CBP2 exists in different oligomeric forms. Moreover, Mo-CBP2 is a basic glycoprotein (pI 10.9 with 4.1% (m/m sugar and it did not display hemagglutinating and hemolytic activities upon rabbit and human erythrocytes. A comparative analysis of the sequence of triptic peptides from Mo-CBP2 in solution, after LC-ESI-MS/MS, revealed similarity with other M. oleifera proteins, as the 2S albumin Mo-CBP3 and flocculating proteins, and 2S albumins from different species. Mo-CBP2 possesses in vitro antifungal activity against Candida albicans, C. parapsilosis, C. krusei, and C. tropicalis, with MIC50 and MIC90 values ranging between 9.45–37.90 and 155.84–260.29 μM, respectively. In addition, Mo-CBP2 (18.90 μM increased the cell membrane permeabilization

  20. Cysteine-rich intestinal protein binds zinc during transmucosal zinc transport

    International Nuclear Information System (INIS)

    Hempe, J.M.; Cousins, R.J.

    1991-01-01

    The mechanism of zinc absorption has not been delineated, but kinetic studies show that both passive and carrier-mediated processes are involved. The authors have identified a low molecular mass zinc-binding protein in the soluble fraction of rat intestinal mucosa that could function as an intracellular zinc carrier. The protein was not detected in liver or pancreas, suggesting a role specific to the intestine. The protein binds zinc during transmucosal zinc transport and shows signs of saturation at higher luminal zinc concentrations, characteristics consistent with a role in carrier-mediated zinc absorption. Microsequence analysis of the protein purified by gel-filtration HPCL and SDS/PAGE showed complete identity within the first 41 N-terminal amino acids with the deduced protein sequence of cysteine-rich intestinal protein. These investigators showed that the gene for this protein is developmentally regulated in neonates during the suckling period, conserved in many vertebrate species, and predominantly expressed in the small intestine. Cysteine-rich intestinal protein contains a recently identified conserved sequence of histidine and cysteine residues, the LIM motif, which our results suggest confers metal-binding properties that are important for zinc transport and/or functions of this micronutrient

  1. Specificity and sensitivity of binding proteins in the radioimmunoassay of cortisol

    International Nuclear Information System (INIS)

    Gijzen, A.H.J.

    1977-01-01

    A comparison concerning avidity towards cortisol and 10 other steroids was made between several binding proteins either in solution or bound to cellulose as so called ''solid phase'' reagent. Human blood cortisol binding protein (CBP, transcortin), and two distinctly different cortisol-binding rabbit antisera and the isolated immunoglobulins thereof were compared in their avidity to bind cortisol and several other steroids. The antisera were harvested from rabbits immunized with either cortisol-21-succinyl-albumin (CSA) or cortisol-3-oxim-albumin (COA). The latter antiserum, having the highest titre in cortisol titration, showed the greatest specificity and was most useful as a binding reagent in cortisol radioimmunoassay when used as a solid phase reagent. The determination of cortisol in micro samples of blood serum is possible without steroid extraction or serum protein denaturation and with only minor influence of steroid impurities in the sample to be analyzed. Affinity constants for all compared binding reagents and steroids are given

  2. Fusicoccin-Binding Proteins in Arabidopsis thaliana (L.) Heynh. 1

    Science.gov (United States)

    Meyer, Christiane; Feyerabend, Martin; Weiler, Elmar W.

    1989-01-01

    Using the novel radioligand, [3H]-9′-nor-fusicoccin-8′-alcohol, high affinity binding sites for fusicoccin were characterized in preparations from leaves of Arabidopsis thaliana (L.) Heynh. The binding site copartitioned with the plasmalemma marker, vanadate-sensitive K+, Mg2+-ATPase, when microsomal fractions were further purified by aqueous two-phase partitioning in polyethylene glycol-dextran phase systems and sedimented at an equilibrium density of 1.17 grams per cubic centimeter in continuous sucrose density gradients, as did the ATPase marker. The binding of [3H]-9′-nor-fusicoccin-8′-alcohol was saturable and Scatchard analysis revealed a biphasic plot with two apparent dissociation constants (KD), KD1 = 1.5 nanomolar and KD2 = 42 nanomolar, for the radioligand. Binding was optimal at pH 6, thermolabile, and was reduced by 70% when the membrane vesicles were pretreated with trypsin. The data are consistent with the presence of one or several binding proteins for fusicoccin at the plasma membrane of A. thaliana. Binding of the radioligand was unaffected by pretreatment of the sites with various alkylating and reducing agents, but was reduced by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, diethylpyrocarbonate, chloramine T, and periodate. A number of detergents were tested to find optimum conditions for solubilization. Nonanoyl-N-methylglucamide (50 millimolar) solubilized 70% of the radioligand-binding protein complex in undissociated form. Photoaffinity labeling of membrane preparations with a tritiated azido analog of fusicoccin resulted in the labeling of a 34 ± 1 kilodalton polypeptide. Labeling of this polypeptide, presumably the fusicoccin-binding protein, was severely reduced in the presence of unlabeled fusicoccin. PMID:16666603

  3. In vitro binding of selenium by rat liver mitochondrial selenium-binding protein

    International Nuclear Information System (INIS)

    Brian, W.R.; Hoekstra, W.G.

    1986-01-01

    Last year the authors reported that upon freezing and thawing mitochondria from rats injected with [ 75 Se]Na 2 SeO 3 ( 75 Se-selenite), a 75 Se-binding protein (SeBP) was released. They have studied further in vitro labelling of SeBP. This matrix protein was labelled in vitro when lysed mitochondria (containing non-matrix material) were incubated with 75 Se-selenite but not when matrix material alone was incubated with 75 Se-selenite. Thus, there are one or more promoters of in vitro SeBP labelling in the non-matrix fraction. SeBP was also labelled in vitro when 75 Se-selenite was added to matrix alone and dialyzed. Dialysis tubing, and not the dialysis process, promoted labelling by affecting SeBP and not by affecting 75 Se-selenite. Labelling did not occur when matrix alone and 75 Se-selenite were incubated (not dialyzed) in a glass test tube but did occur in a polystyrene test tube. They hypothesize that non-covalent interactions occur between SeBP and dialysis tubing or polystyrene that expose Se binding sites on the protein. A similar mechanism involving mitochondrial non-matrix material may function in vivo. Non-denaturing disc gel electrophoresis of partially purified SeBP labelled in vivo or in vitro suggested that the same protein was labelled in both conditions. Using in vitro binding techniques, SeBP was also found in sheep liver mitochondrial matrix. This supports the theory that SeBP is important in Se metabolism

  4. The Magnaporthe oryzae Alt A 1-like protein MoHrip1 binds to the plant plasma membrane.

    Science.gov (United States)

    Zhang, Yi; Liang, Yingbo; Dong, Yijie; Gao, Yuhan; Yang, Xiufen; Yuan, Jingjing; Qiu, Dewen

    2017-10-07

    MoHrip1, a protein isolated from Magnaporthe oryzae, belongs to the Alt A 1 (AA1) family. mohrip1 mRNA levels showed inducible expression throughout the infection process in rice. To determine the location of MoHrip1 in M. oryzae, a mohrip1-gfp mutant was generated. Fluorescence microscopy observations and western blotting analysis showed that MoHrip1 was both present in the secretome and abundant in the fungal cell wall. To obtain MoHrip1 protein, we carried out high-yield expression of MoHrip1 in Pichia pastoris. Treatment of tobacco plants with MoHrip1 induced the formation of necrosis, accumulation of reactive oxygen species and expression of several defense-related genes, as well as conferred disease resistance. By fusion to green fluorescent protein, we showed that MoHrip1 was able to bind to the tobacco and rice plant plasma membrane, causing rapid morphological changes at the cellular level, such as cell shrinkage and chloroplast disorganization. These findings indicate that MoHrip1 is a microbe-associated molecular pattern that is perceived by the plant immune system. This is the first study on an AA1 family protein that can bind to the plant plasma membrane. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. D-fructose-binding proteins in bull seminal plasma: Isolation and characterization

    Czech Academy of Sciences Publication Activity Database

    Liberda, J.; Kraus, Marek; Ryšlavá, H.; Vlasáková, M.; Jonáková, Věra; Tichá, M.

    2001-01-01

    Roč. 47, č. 4 (2001), s. 113-119 ISSN 0015-5500 R&D Projects: GA ČR GA303/99/0357; GA ČR GV524/96/K162 Institutional research plan: CEZ:AV0Z5052915 Keywords : bull seminal plasma * non-heparin-binding and heparin-binding proteins * D-fructose-binding proteins Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.519, year: 2001

  6. RNAcontext: a new method for learning the sequence and structure binding preferences of RNA-binding proteins.

    Directory of Open Access Journals (Sweden)

    Hilal Kazan

    2010-07-01

    Full Text Available Metazoan genomes encode hundreds of RNA-binding proteins (RBPs. These proteins regulate post-transcriptional gene expression and have critical roles in numerous cellular processes including mRNA splicing, export, stability and translation. Despite their ubiquity and importance, the binding preferences for most RBPs are not well characterized. In vitro and in vivo studies, using affinity selection-based approaches, have successfully identified RNA sequence associated with specific RBPs; however, it is difficult to infer RBP sequence and structural preferences without specifically designed motif finding methods. In this study, we introduce a new motif-finding method, RNAcontext, designed to elucidate RBP-specific sequence and structural preferences with greater accuracy than existing approaches. We evaluated RNAcontext on recently published in vitro and in vivo RNA affinity selected data and demonstrate that RNAcontext identifies known binding preferences for several control proteins including HuR, PTB, and Vts1p and predicts new RNA structure preferences for SF2/ASF, RBM4, FUSIP1 and SLM2. The predicted preferences for SF2/ASF are consistent with its recently reported in vivo binding sites. RNAcontext is an accurate and efficient motif finding method ideally suited for using large-scale RNA-binding affinity datasets to determine the relative binding preferences of RBPs for a wide range of RNA sequences and structures.

  7. Regulation of activity of the yeast TATA-binding protein through intra ...

    Indian Academy of Sciences (India)

    Unknown

    Abbreviations used: BMH, Bismaleimidohexane; TBP, TATA-binding protein; yTBP, yeast TBP. J. Biosci. | Vol. ... Therefore for full-length TBP, intra-molecular interactions can regulate its activity via a similar ..... simulations (Miaskeiwicz and Ornstein 1996). .... box binding protein (TBP): A molecular dynamics computa-.

  8. Multiple antioxidant proteins protect Chlorobaculum tepidum against oxygen and reactive oxygen species

    DEFF Research Database (Denmark)

    Li, Hui; Jubelirer, Sara; Garcia Costas, Amaya M

    2009-01-01

    include cytochrome bd quinol oxidase, NADH oxidase, rubredoxin oxygen oxidoreductase, several thiol peroxidases, alkyl hydroperoxide reductase, superoxide dismutase, methionine sulfoxide reductase, and rubrerythrin. To test the physiological functions of some of these proteins, ten genes were...

  9. SH2 Domains Serve as Lipid-Binding Modules for pTyr-Signaling Proteins.

    Science.gov (United States)

    Park, Mi-Jeong; Sheng, Ren; Silkov, Antonina; Jung, Da-Jung; Wang, Zhi-Gang; Xin, Yao; Kim, Hyunjin; Thiagarajan-Rosenkranz, Pallavi; Song, Seohyeon; Yoon, Youngdae; Nam, Wonhee; Kim, Ilshin; Kim, Eui; Lee, Dong-Gyu; Chen, Yong; Singaram, Indira; Wang, Li; Jang, Myoung Ho; Hwang, Cheol-Sang; Honig, Barry; Ryu, Sungho; Lorieau, Justin; Kim, You-Me; Cho, Wonhwa

    2016-04-07

    The Src-homology 2 (SH2) domain is a protein interaction domain that directs myriad phosphotyrosine (pY)-signaling pathways. Genome-wide screening of human SH2 domains reveals that ∼90% of SH2 domains bind plasma membrane lipids and many have high phosphoinositide specificity. They bind lipids using surface cationic patches separate from pY-binding pockets, thus binding lipids and the pY motif independently. The patches form grooves for specific lipid headgroup recognition or flat surfaces for non-specific membrane binding and both types of interaction are important for cellular function and regulation of SH2 domain-containing proteins. Cellular studies with ZAP70 showed that multiple lipids bind its C-terminal SH2 domain in a spatiotemporally specific manner and thereby exert exquisite spatiotemporal control over its protein binding and signaling activities in T cells. Collectively, this study reveals how lipids control SH2 domain-mediated cellular protein-protein interaction networks and suggest a new strategy for therapeutic modulation of pY-signaling pathways. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides

    Directory of Open Access Journals (Sweden)

    Weonu Choe

    2016-12-01

    Full Text Available The rapidly increasing application of antibodies has inspired the development of several novel methods to isolate and target antibodies using smart biomaterials that mimic the binding of Fc-receptors to antibodies. The Fc-binding domain of antibodies is the primary binding site for e.g., effector proteins and secondary antibodies, whereas antigens bind to the Fab region. Protein A, G, and L, surface proteins expressed by pathogenic bacteria, are well known to bind immunoglobulin and have been widely exploited in antibody purification strategies. Several difficulties are encountered when bacterial proteins are used in antibody research and application. One of the major obstacles hampering the use of bacterial proteins is sample contamination with trace amounts of these proteins, which can invoke an immune response in the host. Many research groups actively develop synthetic ligands that are able to selectively and strongly bind to antibodies. Among the reported ligands, peptides that bind to the Fc-domain of antibodies are attractive tools in antibody research. Besides their use as high affinity ligands in antibody purification chromatography, Fc-binding peptides are applied e.g., to localize antibodies on nanomaterials and to increase the half-life of proteins in serum. In this review, recent developments of Fc-binding peptides are presented and their binding characteristics and diverse applications are discussed.

  11. Site-directed antibody immobilization using a protein A-gold binding domain fusion protein for enhanced SPR immunosensing.

    Science.gov (United States)

    de Juan-Franco, Elena; Caruz, Antonio; Pedrajas, J R; Lechuga, Laura M

    2013-04-07

    We have implemented a novel strategy for the oriented immobilization of antibodies onto a gold surface based on the use of a fusion protein, the protein A-gold binding domain (PAG). PAG consists of a gold binding peptide (GBP) coupled to the immunoglobulin-binding domains of staphylococcal protein A. This fusion protein provides an easy and fast oriented immobilization of antibodies preserving its native structure, while leaving the antigen binding sites (Fab) freely exposed. Using this immobilization strategy, we have demonstrated the performance of the immunosensing of the human Growth Hormone by SPR. A limit of detection of 90 ng mL(-1) was obtained with an inter-chip variability lower than 7%. The comparison of this method with other strategies for the direct immobilization of antibodies over gold surfaces has showed the enhanced sensitivity provided by the PAG approach.

  12. Cellulose binding domain fusion proteins

    Science.gov (United States)

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  13. Cadmium-binding proteins in midgut gland of freshwater crayfish Procambarus clarkii

    Energy Technology Data Exchange (ETDEWEB)

    Del Ramo, J.; Pastor, A.; Torreblanca, A.; Medina, J.; Diza-Mayans, J.

    1989-02-01

    Metallothioneins, metal binding proteins, were originally isolated and characterized by Margoshes and Vallee. These proteins have a high affinity for various heavy metals, particularly cadmium and mercury and have extensively been studied in mammals. Metal binding proteins have been observed in a variety of marine invertebrates; however, there is very little information available on metal binding proteins in freshwater invertebrates, and particularly in freshwater crustaceans. Cadmium is an ubiquitous non essential element which possesses high toxicity to aquatic organisms. Cadmium binding proteins observed in invertebrates have similar characteristics to mammalian metallothioneins. In 1978, the American red crayfish appeared in Albufera Lake and the surrounding rice fields (Valencia, Spain). Albufera Lake and the surrounding rice fields waters are subjected to very heavy loads of sewage and toxic industrial residues (including heavy metals) from the many urban and wastewaters in this area. In previous reports the authors studied the toxicity and accumulation of cadmium on Procambarus clarkii of Albufera Lake. This crayfish shows a high resistance to cadmium and a great accumulation rate of this metal in several tissues, including midgut gland. Since Procambarus clarkii shows a high resistance to cadmium, the presence of cadmium binding proteins (Cd-BP) in midgut gland of these crayfish would be expected. This report describes results on the characterization of Cd-BPs obtained from cadmium exposed crayfish Procambarus clarkii, demonstrating their presence in this freshwater crayfish.

  14. Application of three-dimensional reduced graphene oxide-gold composite modified electrode for direct electrochemistry and electrocatalysis of myoglobin

    International Nuclear Information System (INIS)

    Shi, Fan; Xi, Jingwen; Hou, Fei; Han, Lin; Li, Guangjiu; Gong, Shixing; Chen, Chanxing; Sun, Wei

    2016-01-01

    In this paper a three-dimensional (3D) reduced graphene oxide (RGO) and gold (Au) composite was synthesized by electrodeposition and used for the electrode modification with carbon ionic liquid electrode (CILE) as the substrate electrode. Myoglobin (Mb) was further immobilized on the surface of 3D RGO–Au/CILE to obtain an electrochemical sensing platform. Direct electrochemistry of Mb on the modified electrode was investigated with a pair of well-defined redox waves appeared on cyclic voltammogram, indicating the realization of direct electron transfer of Mb with the modified electrode. The results can be ascribed to the presence of highly conductive 3D RGO–Au composite on the electrode surface that accelerate the electron transfer rate between the electroactive center of Mb and the electrode. The Mb modified electrode showed excellent electrocatalytic activity to the reduction of trichloroacetic acid in the concentration range from 0.2 to 36.0 mmol/L with the detection limit of 0.06 mmol/L (3σ). - Graphical abstract: Direct electrochemistry of myoglobin was realized on a three-dimensional reduced graphene oxide and gold nanocomposite modified carbon ionic liquid electrode. - Highlights: • A three-dimensional reduced graphene oxide and gold composite was synthesized by electrodeposition. • Myoglobin was immobilized on the modified electrode to obtain an electrochemical sensor. • Direct electrochemistry of myoglobin was realized on the modified electrode. • The myoglobin modified electrode showed excellent electrocatalytic reduction to trichloroacetic acid.

  15. Multiple DNA binding proteins contribute to timing of chromosome replication in E. coli

    DEFF Research Database (Denmark)

    Riber, Leise; Frimodt-Møller, Jakob; Charbon, Godefroid

    2016-01-01

    Chromosome replication in Escherichia coli is initiated from a single origin, oriC. Initiation involves a number of DNA binding proteins, but only DnaA is essential and specific for the initiation process. DnaA is an AAA+ protein that binds both ATP and ADP with similar high affinities. Dna...... replication is initiated, or the time window in which all origins present in a single cell are initiated, i.e. initiation synchrony, or both. Overall, these DNA binding proteins modulate the initiation frequency from oriC by: (i) binding directly to oriC to affect DnaA binding, (ii) altering the DNA topology...... in or around oriC, (iii) altering the nucleotide bound status of DnaA by interacting with non-coding chromosomal sequences, distant from oriC, that are important for DnaA activity. Thus, although DnaA is the key protein for initiation of replication, other DNA-binding proteins act not only on ori...

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  17. A urokinase receptor-associated protein with specific collagen binding properties

    DEFF Research Database (Denmark)

    Behrendt, N; Jensen, O N; Engelholm, L H

    2000-01-01

    membrane-bound lectin with hitherto unknown function. The human cDNA was cloned and sequenced. The protein, designated uPARAP, is a member of the macrophage mannose receptor protein family and contains a putative collagen-binding (fibronectin type II) domain in addition to 8 C-type carbohydrate recognition...... domains. It proved capable of binding strongly to a single type of collagen, collagen V. This collagen binding reaction at the exact site of plasminogen activation on the cell may lead to adhesive functions as well as a contribution to cellular degradation of collagen matrices....

  18. Dynamic SPR monitoring of yeast nuclear protein binding to a cis-regulatory element

    International Nuclear Information System (INIS)

    Mao, Grace; Brody, James P.

    2007-01-01

    Gene expression is controlled by protein complexes binding to short specific sequences of DNA, called cis-regulatory elements. Expression of most eukaryotic genes is controlled by dozens of these elements. Comprehensive identification and monitoring of these elements is a major goal of genomics. In pursuit of this goal, we are developing a surface plasmon resonance (SPR) based assay to identify and monitor cis-regulatory elements. To test whether we could reliably monitor protein binding to a regulatory element, we immobilized a 16 bp region of Saccharomyces cerevisiae chromosome 5 onto a gold surface. This 16 bp region of DNA is known to bind several proteins and thought to control expression of the gene RNR1, which varies through the cell cycle. We synchronized yeast cell cultures, and then sampled these cultures at a regular interval. These samples were processed to purify nuclear lysate, which was then exposed to the sensor. We found that nuclear protein binds this particular element of DNA at a significantly higher rate (as compared to unsynchronized cells) during G1 phase. Other time points show levels of DNA-nuclear protein binding similar to the unsynchronized control. We also measured the apparent association complex of the binding to be 0.014 s -1 . We conclude that (1) SPR-based assays can monitor DNA-nuclear protein binding and that (2) for this particular cis-regulatory element, maximum DNA-nuclear protein binding occurs during G1 phase

  19. Species Differences in the Carbohydrate Binding Preferences of Surfactant Protein D

    DEFF Research Database (Denmark)

    Crouch, Erika C.; Smith, Kelly; McDonald, Barbara

    2006-01-01

    Interactions of surfactant protein D (SP-D) with micro-organisms and organic antigens involve binding to the trimeric neck plus carbohydrate recognition domain (neck+CRD). In these studies, we compared the ligand binding of homologous human, rat, and mouse trimeric neck+CRD fusion proteins, each ...

  20. Lactoferrin binding protein B - a bi-functional bacterial receptor protein.

    Directory of Open Access Journals (Sweden)

    Nicholas K H Ostan

    2017-03-01

    Full Text Available Lactoferrin binding protein B (LbpB is a bi-lobed outer membrane-bound lipoprotein that comprises part of the lactoferrin (Lf receptor complex in Neisseria meningitidis and other Gram-negative pathogens. Recent studies have demonstrated that LbpB plays a role in protecting the bacteria from cationic antimicrobial peptides due to large regions rich in anionic residues in the C-terminal lobe. Relative to its homolog, transferrin-binding protein B (TbpB, there currently is little evidence for its role in iron acquisition and relatively little structural and biophysical information on its interaction with Lf. In this study, a combination of crosslinking and deuterium exchange coupled to mass spectrometry, information-driven computational docking, bio-layer interferometry, and site-directed mutagenesis was used to probe LbpB:hLf complexes. The formation of a 1:1 complex of iron-loaded Lf and LbpB involves an interaction between the Lf C-lobe and LbpB N-lobe, comparable to TbpB, consistent with a potential role in iron acquisition. The Lf N-lobe is also capable of binding to negatively charged regions of the LbpB C-lobe and possibly other sites such that a variety of higher order complexes are formed. Our results are consistent with LbpB serving dual roles focused primarily on iron acquisition when exposed to limited levels of iron-loaded Lf on the mucosal surface and effectively binding apo Lf when exposed to high levels at sites of inflammation.

  1. The spliceosome-associated protein Mfap1 binds to VCP in Drosophila.

    Directory of Open Access Journals (Sweden)

    Sandra Rode

    Full Text Available Posttranscriptional regulation of gene expression contributes to many developmental transitions. Previously, we found that the AAA chaperone Valosin-Containing Protein (VCP regulates ecdysone-dependent dendrite pruning of Drosophila class IV dendritic arborization (c4da neurons via an effect on RNA metabolism. In a search for RNA binding proteins associated with VCP, we identified the spliceosome-associated protein Mfap1, a component of the tri-snRNP complex. Mfap1 is a nucleolar protein in neurons and its levels are regulated by VCP. Mfap1 binds to VCP and TDP-43, a disease-associated RNA-binding protein. via distinct regions in its N- and C-terminal halfs. Similar to vcp mutations, Mfap1 overexpression causes c4da neuron dendrite pruning defects and mislocalization of TDP-43 in these cells, but genetic analyses show that Mfap1 is not a crucial VCP target during dendrite pruning. Finally, rescue experiments with a lethal mfap1 mutant show that the VCP binding region is not essential for Mfap1 function, but may act to increase its stability or activity.

  2. Small GTP-binding proteins in human endothelial cells

    NARCIS (Netherlands)

    de Leeuw, H. P.; Koster, P. M.; Calafat, J.; Janssen, H.; van Zonneveld, A. J.; van Mourik, J. A.; Voorberg, J.

    1998-01-01

    Small GTP-binding proteins of the Ras superfamily control an extensive number of intracellular events by alternating between GDP- and GTP-bound conformation. The presence of members of this protein family was examined in human umbilical vein endothelial cells employing RT-PCR. Sequence analysis of

  3. Predicting Flavin and Nicotinamide Adenine Dinucleotide-Binding Sites in Proteins Using the Fragment Transformation Method

    Directory of Open Access Journals (Sweden)

    Chih-Hao Lu

    2015-01-01

    Full Text Available We developed a computational method to identify NAD- and FAD-binding sites in proteins. First, we extracted from the Protein Data Bank structures of proteins that bind to at least one of these ligands. NAD-/FAD-binding residue templates were then constructed by identifying binding residues through the ligand-binding database BioLiP. The fragment transformation method was used to identify structures within query proteins that resembled the ligand-binding templates. By comparing residue types and their relative spatial positions, potential binding sites were identified and a ligand-binding potential for each residue was calculated. Setting the false positive rate at 5%, our method predicted NAD- and FAD-binding sites at true positive rates of 67.1% and 68.4%, respectively. Our method provides excellent results for identifying FAD- and NAD-binding sites in proteins, and the most important is that the requirement of conservation of residue types and local structures in the FAD- and NAD-binding sites can be verified.

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

    Science.gov (United States)

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

    1993-01-01

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

  5. Extreme sequence divergence but conserved ligand-binding specificity in Streptococcus pyogenes M protein.

    Directory of Open Access Journals (Sweden)

    2006-05-01

    Full Text Available Many pathogenic microorganisms evade host immunity through extensive sequence variability in a protein region targeted by protective antibodies. In spite of the sequence variability, a variable region commonly retains an important ligand-binding function, reflected in the presence of a highly conserved sequence motif. Here, we analyze the limits of sequence divergence in a ligand-binding region by characterizing the hypervariable region (HVR of Streptococcus pyogenes M protein. Our studies were focused on HVRs that bind the human complement regulator C4b-binding protein (C4BP, a ligand that confers phagocytosis resistance. A previous comparison of C4BP-binding HVRs identified residue identities that could be part of a binding motif, but the extended analysis reported here shows that no residue identities remain when additional C4BP-binding HVRs are included. Characterization of the HVR in the M22 protein indicated that two relatively conserved Leu residues are essential for C4BP binding, but these residues are probably core residues in a coiled-coil, implying that they do not directly contribute to binding. In contrast, substitution of either of two relatively conserved Glu residues, predicted to be solvent-exposed, had no effect on C4BP binding, although each of these changes had a major effect on the antigenic properties of the HVR. Together, these findings show that HVRs of M proteins have an extraordinary capacity for sequence divergence and antigenic variability while retaining a specific ligand-binding function.

  6. UV-induced cross-linking of abscisic acid to binding proteins

    International Nuclear Information System (INIS)

    Cornelussen, M.H.M.; Karssen, C.M.; Loon, L.C. van

    1995-01-01

    Conditions for UV-induced cross-linking of abscisic acid (ABA) through its enone chromophore to binding proteins were evaluated. The effects of a UV-light band between 260 and 530 nm on both unconjugated and protein-conjugated ABA, as well as on anti-ABA antibodies as models of ABA-binding proteins were determined. UV irradiation caused both isomerization and photolysis of ABA, but increasing the lower irradiation boundary to 345 nm strongly reduced photolysis and largely prevented isomerization. When conjugated to alkaline phosphatase (AP), ABA remained stable when using either a 320 or a 345 nm filter. At these wavelengths both binding of ABA to antibodies as well as AP enzymatic activity were maintained. UV-induced cross-linking of monoclonal anti-ABA antibodies to immobilized ABA was analysed by immunoassays. Optimal cross-linking was achieved after a 5 min irradiation period at 0°, using a long pass, cut-on filter to quench wavelengths below 290 nm. This cross-linking faithfully reflected cognate binding activity. (author)

  7. Molecular cloning of the gene for the human placental GTP-binding protein Gp (G25K): Identification of this GTP-binding protein as the human homolog of the yeast cell-division-cycle protein CDC42

    International Nuclear Information System (INIS)

    Shinjo, K.; Koland, J.G.; Hart, M.J.; Narasimhan, V.; Cerione, R.A.; Johnson, D.I.; Evans, T.

    1990-01-01

    The authors have isolated cDNA clones from a human placental library that code for a low molecular weight GTP-binding protein originally designated G p (also called G25K). This identification is based on comparisons with the available peptide sequences for the purified human G p protein and the use of two highly specific anti-peptide antibodies. The predicted amino acid sequence of the protein is very similar to those of various members of the ras superfamily of low molecular weight GTP-binding proteins, including the N-, Ki-, and Ha-ras proteins (30-35% identical), the rho proteins and the rac proteins. The highest degree of sequence identity (80%) is found with the Saccharomyces cerevisiae cell division-cycle protein CDC42. The human placental gene, which they designate CDC42Hs, complements the cdc42-1 mutation in S. cerevisiae, which suggests that this GTP-binding protein is the human homolog of the yeast protein

  8. Calculation of protein-ligand binding affinities.

    Science.gov (United States)

    Gilson, Michael K; Zhou, Huan-Xiang

    2007-01-01

    Accurate methods of computing the affinity of a small molecule with a protein are needed to speed the discovery of new medications and biological probes. This paper reviews physics-based models of binding, beginning with a summary of the changes in potential energy, solvation energy, and configurational entropy that influence affinity, and a theoretical overview to frame the discussion of specific computational approaches. Important advances are reported in modeling protein-ligand energetics, such as the incorporation of electronic polarization and the use of quantum mechanical methods. Recent calculations suggest that changes in configurational entropy strongly oppose binding and must be included if accurate affinities are to be obtained. The linear interaction energy (LIE) and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) methods are analyzed, as are free energy pathway methods, which show promise and may be ready for more extensive testing. Ultimately, major improvements in modeling accuracy will likely require advances on multiple fronts, as well as continued validation against experiment.

  9. Histone acetylation and CREB binding protein are required for neuronal resistance against ischemic injury.

    Directory of Open Access Journals (Sweden)

    Ferah Yildirim

    Full Text Available Epigenetic transcriptional regulation by histone acetylation depends on the balance between histone acetyltransferase (HAT and deacetylase activities (HDAC. Inhibition of HDAC activity provides neuroprotection, indicating that the outcome of cerebral ischemia depends crucially on the acetylation status of histones. In the present study, we characterized the changes in histone acetylation levels in ischemia models of focal cerebral ischemia and identified cAMP-response element binding protein (CREB-binding protein (CBP as a crucial factor in the susceptibility of neurons to ischemic stress. Both neuron-specific RNA interference and neurons derived from CBP heterozygous knockout mice showed increased damage after oxygen-glucose deprivation (OGD in vitro. Furthermore, we demonstrated that ischemic preconditioning by a short (5 min subthreshold occlusion of the middle cerebral artery (MCA, followed 24 h afterwards by a 30 min occlusion of the MCA, increased histone acetylation levels in vivo. Ischemic preconditioning enhanced CBP recruitment and histone acetylation at the promoter of the neuroprotective gene gelsolin leading to increased gelsolin expression in neurons. Inhibition of CBP's HAT activity attenuated neuronal ischemic preconditioning. Taken together, our findings suggest that the levels of CBP and histone acetylation determine stroke outcome and are crucially associated with the induction of an ischemia-resistant state in neurons.

  10. Distribution of PASTA domains in penicillin-binding proteins and serine/threonine kinases of Actinobacteria.

    Science.gov (United States)

    Ogawara, Hiroshi

    2016-09-01

    PASTA domains (penicillin-binding protein and serine/threonine kinase-associated domains) have been identified in penicillin-binding proteins and serine/threonine kinases of Gram-positive Firmicutes and Actinobacteria. They are believed to bind β-lactam antibiotics, and be involved in peptidoglycan metabolism, although their biological function is not definitively clarified. Actinobacteria, especially Streptomyces species, are distinct in that they undergo complex cellular differentiation and produce various antibiotics including β-lactams. This review focuses on the distribution of PASTA domains in penicillin-binding proteins and serine/threonine kinases in Actinobacteria. In Actinobacteria, PASTA domains are detectable exclusively in class A but not in class B penicillin-binding proteins, in sharp contrast to the cases in other bacteria. In penicillin-binding proteins, PASTA domains distribute independently from taxonomy with some distribution bias. Particularly interesting thing is that no Streptomyces species have penicillin-binding protein with PASTA domains. Protein kinases in Actinobacteria possess 0 to 5 PASTA domains in their molecules. Protein kinases in Streptomyces can be classified into three groups: no PASTA domain, 1 PASTA domain and 4 PASTA domain-containing groups. The 4 PASTA domain-containing groups can be further divided into two subgroups. The serine/threonine kinases in different groups may perform different functions. The pocket region in one of these subgroup is more dense and extended, thus it may be involved in binding of ligands like β-lactams more efficiently.

  11. Binding of rare earths to serum proteins and DNA

    International Nuclear Information System (INIS)

    Rosoff, B.; Spencer, H.

    1979-01-01

    In order to investigate further the physiological behavior of rare earths and rare earth chelates, studies of the binding of 46 Sc, 91 Y, and 140 La to serum proteins and to nucleic acids were performed using the methods of equilibrium dialysis and ultrafiltration. The binding of lanthanum and yttrium as the chlorides to α-globulin increased as the free rare earth concentration increased. When scandium and lanthanum were chelated in nitrilotriacetate (NTA) the binding to α-globulin was considerably less and there was no binding to albumin. The binding of 46 Sc chelated to ethylenediamine di(O-hydroxyphenylacetate) (EDDHA) was five times greater than of 46 Sc chloride. When the free scandium concentration was increased, the moles bound per mole of protein increased proportionally and the binding was reversible. Scandium was 100% filterable from a mixture of human serum and from the scandium chelates with high stability constants scandium diethylenetriaminepentaacetate (ScDTPA), scandium ethylenediaminetetraacetate (ScEDTA) and scandium cyclohexane trans-1,2-diaminetetraacetate (ScCDTA) respectively. In contrast, only 2% of the scandium was filterable when scandium nitrilotriacetate, a scandium chelate of low stability constant, was used. (Auth.)

  12. A rapid method for myoglobin radioimmunoanalysis as a diagnostic tool in myocardial infarction

    International Nuclear Information System (INIS)

    Grachev, M.A.; Matveev, L.E.; Pressman, E.K.; Roschke, V.V.

    1982-01-01

    Stone et al. have elaborated a RIA-method for the determination of myoglobin, and found that increase of its concentration in serum is a reliable criterion for the diagnosis of myocardial infarction. However, the test took 24-28 h. Subsequently, the time of the analysis has been reduced to 5-6 h. Recently, a rapid method for the determination of myoglobin has been proposed based upon the use of antiserum immobilized on a powdered carrier. This method takes a little more than 1 h. The procedure according to Roxin et al. is fast due to its non-equilibrium character; after the incubation (30 min) the reaction of the antigen with the immobilized antibody still remains far from equilibrium. It is generally believed that non-equilibrium RIA procedures are less convenient than equilibrium ones for practical clinical applications. According to the RIA procedure proposed here, the time saving compared with the established methods is achieved by using relatively-high concentrations of radioactive myoglobin of moderate specific radioactivity. Under these conditions, the kinetic plateau is reached in 15-20 min. Hence, the total time of the analysis to obtain a standard curve and results for five unknown sera is 55 min. Therefore, the method becomes more useful as a guide in the treatment of myocardial infarction. (Auth.)

  13. Functional assignment to JEV proteins using SVM.

    Science.gov (United States)

    Sahoo, Ganesh Chandra; Dikhit, Manas Ranjan; Das, Pradeep

    2008-01-01

    Identification of different protein functions facilitates a mechanistic understanding of Japanese encephalitis virus (JEV) infection and opens novel means for drug development. Support vector machines (SVM), useful for predicting the functional class of distantly related proteins, is employed to ascribe a possible functional class to Japanese encephalitis virus protein. Our study from SVMProt and available JE virus sequences suggests that structural and nonstructural proteins of JEV genome possibly belong to diverse protein functions, are expected to occur in the life cycle of JE virus. Protein functions common to both structural and non-structural proteins are iron-binding, metal-binding, lipid-binding, copper-binding, transmembrane, outer membrane, channels/Pores - Pore-forming toxins (proteins and peptides) group of proteins. Non-structural proteins perform functions like actin binding, zinc-binding, calcium-binding, hydrolases, Carbon-Oxygen Lyases, P-type ATPase, proteins belonging to major facilitator family (MFS), secreting main terminal branch (MTB) family, phosphotransfer-driven group translocators and ATP-binding cassette (ABC) family group of proteins. Whereas structural proteins besides belonging to same structural group of proteins (capsid, structural, envelope), they also perform functions like nuclear receptor, antibiotic resistance, RNA-binding, DNA-binding, magnesium-binding, isomerase (intra-molecular), oxidoreductase and participate in type II (general) secretory pathway (IISP).

  14. Evolving Transcription Factor Binding Site Models From Protein Binding Microarray Data

    KAUST Repository

    Wong, Ka-Chun; Peng, Chengbin; Li, Yue

    2016-01-01

    Protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner. In this paper, we describe the PBM motif model building problem. We apply several evolutionary computation methods and compare their performance with the interior point method, demonstrating their performance advantages. In addition, given the PBM domain knowledge, we propose and describe a novel method called kmerGA which makes domain-specific assumptions to exploit PBM data properties to build more accurate models than the other models built. The effectiveness and robustness of kmerGA is supported by comprehensive performance benchmarking on more than 200 datasets, time complexity analysis, convergence analysis, parameter analysis, and case studies. To demonstrate its utility further, kmerGA is applied to two real world applications: 1) PBM rotation testing and 2) ChIP-Seq peak sequence prediction. The results support the biological relevance of the models learned by kmerGA, and thus its real world applicability.

  15. Evolving Transcription Factor Binding Site Models From Protein Binding Microarray Data

    KAUST Repository

    Wong, Ka-Chun

    2016-02-02

    Protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner. In this paper, we describe the PBM motif model building problem. We apply several evolutionary computation methods and compare their performance with the interior point method, demonstrating their performance advantages. In addition, given the PBM domain knowledge, we propose and describe a novel method called kmerGA which makes domain-specific assumptions to exploit PBM data properties to build more accurate models than the other models built. The effectiveness and robustness of kmerGA is supported by comprehensive performance benchmarking on more than 200 datasets, time complexity analysis, convergence analysis, parameter analysis, and case studies. To demonstrate its utility further, kmerGA is applied to two real world applications: 1) PBM rotation testing and 2) ChIP-Seq peak sequence prediction. The results support the biological relevance of the models learned by kmerGA, and thus its real world applicability.

  16. Singlet oxygen-mediated protein oxidation

    DEFF Research Database (Denmark)

    Wright, Adam; Bubb, William A; Hawkins, Clare Louise

    2002-01-01

    Singlet oxygen (1O2) is generated by a number of enzymes as well as by UV or visible light in the presence of a sensitizer and has been proposed as a damaging agent in a number of pathologies including cataract, sunburn, and skin cancers. Proteins, and Cys, Met, Trp, Tyr and His side chains...... in particular, are major targets for 1O2 as a result of their abundance and high rate constants for reaction. In this study it is shown that long-lived peroxides are formed on free Tyr, Tyr residues in peptides and proteins, and model compounds on exposure to 1O2 generated by both photochemical and chemical....... These studies demonstrate that long-lived Tyr-derived peroxides are formed on proteins exposed to 1O2 and that these may promote damage to other targets via further radical generation....

  17. Effect of benzimidazol-derivatives on the DNA-protein binding formation after UV-radiation of chromatin

    International Nuclear Information System (INIS)

    Mil', E.M.; Binyukov, V.I.; Zhil'tsova, V.M.; Stolyarova, L.G.; Kuznetsov, Yu.V.

    1991-01-01

    Effect of benzimidazol-derivatives on the DNA-protein binding formation was studied after UV-radiation of chromatin. These derivatives were shown to protect chromatin from UV-induced DNA-protein binding formation. Structural analog contained two aminomethyl residuals sensibilized additional binding formation in chromatin. Results suggested, that benzimidazol interacted with DNA, while aminomethyl groups interacted with protein and sensibilized binding of DNA, whilt aminomethyl groups interacted with protein and sensibilized binding of DNA with histone H1

  18. Zinc fingers, zinc clusters, and zinc twists in DNA-binding protein domains

    International Nuclear Information System (INIS)

    Vallee, B.L.; Auld, D.S.; Coleman, J.E.

    1991-01-01

    The authors recognize three distinct motifs of DNA-binding zinc proteins: (i) zinc fingers, (ii) zinc clusters, and (iii) zinc twists. Until very recently, x-ray crystallographic or NMR three-dimensional structure analyses of DNA-binding zinc proteins have not been available to serve as standards of reference for the zinc binding sites of these families of proteins. Those of the DNA-binding domains of the fungal transcription factor GAL4 and the rat glucocorticoid receptor are the first to have been determined. Both proteins contain two zinc binding sites, and in both, cysteine residues are the sole zinc ligands. In GAL4, two zinc atoms are bound to six cysteine residues which form a zinc cluster akin to that of metallothionein; the distance between the two zinc atoms of GAL4 is ∼3.5 angstrom. In the glucocorticoid receptor, each zinc atom is bound to four cysteine residues; the interatomic zinc-zinc distance is ∼13 angstrom, and in this instance, a zinc twist is represented by a helical DNA recognition site located between the two zinc atoms. Zinc clusters and zinc twists are here recognized as two distinctive motifs in DNA-binding proteins containing multiple zinc atoms. For native zinc fingers, structural data do not exist as yet; consequently, the interatomic distances between zinc atoms are not known. As further structural data become available, the structural and functional significance of these different motifs in their binding to DNA and other proteins participating in the transmission of the genetic message will become apparent

  19. A Single Rainbow Trout Cobalamin-binding Protein Stands in for Three Human Binders

    DEFF Research Database (Denmark)

    Greibe, Eva Holm; Fedosov, Sergey; Sorensen, Boe S

    2012-01-01

    affinity for the cobalamin analog cobinamide. Like haptocorrin and transcobalamin, the trout cobalamin-binding protein was present in plasma and recognized ligands with altered nucleotide moiety. Like intrinsic factors, the trout cobalamin-binding protein was present in the stomach and resisted degradation...... by trypsin and chymotrypsin. It also resembled intrinsic factor in the composition of conserved residues in the primary cobalamin-binding site in the C terminus. The trout cobalamin-binding protein was glycosylated and displayed spectral properties comparable with those of haptocorrin and intrinsic factor...

  20. Fatty Acid Binding Proteins in Prostate Cancer

    National Research Council Canada - National Science Library

    Jett, Marti

    2000-01-01

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

  1. Immuno-histochemical localization of cholesterol binding proteins in ...

    African Journals Online (AJOL)

    This manuscript aims to investigate immunocytochemical localization of cholesterol binding proteins (CBPs) in semi-thin sections of midgut of Schistocerca gregaria (Forskal). For this purpose ... Further, same protein was also localized in other tissues like fat body, testis, and ovary of male and female insects of S. gregaria.

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

    NARCIS (Netherlands)

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

    2016-01-01

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

  3. A microscopic insight from conformational thermodynamics to functional ligand binding in proteins.

    Science.gov (United States)

    Sikdar, Samapan; Chakrabarti, J; Ghosh, Mahua

    2014-12-01

    We show that the thermodynamics of metal ion-induced conformational changes aid to understand the functions of protein complexes. This is illustrated in the case of a metalloprotein, alpha-lactalbumin (aLA), a divalent metal ion binding protein. We use the histograms of dihedral angles of the protein, generated from all-atom molecular dynamics simulations, to calculate conformational thermodynamics. The thermodynamically destabilized and disordered residues in different conformational states of a protein are proposed to serve as binding sites for ligands. This is tested for β-1,4-galactosyltransferase (β4GalT) binding to the Ca(2+)-aLA complex, in which the binding residues are known. Among the binding residues, the C-terminal residues like aspartate (D) 116, glutamine (Q) 117, tryptophan (W) 118 and leucine (L) 119 are destabilized and disordered and can dock β4GalT onto Ca(2+)-aLA. No such thermodynamically favourable binding residues can be identified in the case of the Mg(2+)-aLA complex. We apply similar analysis to oleic acid binding and predict that the Ca(2+)-aLA complex can bind to oleic acid through the basic histidine (H) 32 of the A2 helix and the hydrophobic residues, namely, isoleucine (I) 59, W60 and I95, of the interfacial cleft. However, the number of destabilized and disordered residues in Mg(2+)-aLA are few, and hence, the oleic acid binding to Mg(2+)-bound aLA is less stable than that to the Ca(2+)-aLA complex. Our analysis can be generalized to understand the functionality of other ligand bound proteins.

  4. Characterization of the dextran-binding domain in the glucan-binding protein C of Streptococcus mutans.

    Science.gov (United States)

    Takashima, Y; Fujita, K; Ardin, A C; Nagayama, K; Nomura, R; Nakano, K; Matsumoto-Nakano, M

    2015-10-01

    Streptococcus mutans produces multiple glucan-binding proteins (Gbps), among which GbpC encoded by the gbpC gene is known to be a cell-surface-associated protein involved in dextran-induced aggregation. The purpose of the present study was to characterize the dextran-binding domain of GbpC using bioinformatics analysis and molecular techniques. Bioinformatics analysis specified five possible regions containing molecular binding sites termed GB1 through GB5. Next, truncated recombinant GbpC (rGbpC) encoding each region was produced using a protein expression vector and five deletion mutant strains were generated, termed CDGB1 through CDGB5 respectively. The dextran-binding rates of truncated rGbpC that included the GB1, GB3, GB4 and GB5 regions in the upstream sequences were higher than that of the construct containing GB2 in the downstream region. In addition, the rates of dextran-binding for strains CDGB4 and CD1, which was entire gbpC deletion mutant, were significantly lower than for the other strains, while those of all other deletion mutants were quite similar to that of the parental strain MT8148. Biofilm structures formed by CDGB4 and CD1 were not as pronounced as that of MT8148, while those formed by other strains had greater density as compared to that of CD1. Our results suggest that the dextran-binding domain may be located in the GB4 region in the interior of the gbpC gene. Bioinformatics analysis is useful for determination of functional domains in many bacterial species. © 2015 The Society for Applied Microbiology.

  5. Genetic analysis of RPA single-stranded DNA binding protein in Haloferax volcanii

    OpenAIRE

    Stroud, A. L.

    2012-01-01

    Replication protein A (RPA) is a single-stranded DNA-binding protein that is present in all three domains of life. The roles of RPA include stabilising and protecting single- stranded DNA from nuclease degradation during DNA replication and repair. To achieve this, RPA uses an oligosaccharide-binding fold (OB fold) to bind single- stranded DNA. Haloferax volcanii encodes three RPAs – RPA1, RPA2 and RPA3, of which rpa1 and rpa3 are in operons with genes encoding associated proteins (APs). ...

  6. Effect of Ca2+ on the promiscuous target-protein binding of calmodulin.

    Directory of Open Access Journals (Sweden)

    Annie M Westerlund

    2018-04-01

    Full Text Available Calmodulin (CaM is a calcium sensing protein that regulates the function of a large number of proteins, thus playing a crucial part in many cell signaling pathways. CaM has the ability to bind more than 300 different target peptides in a Ca2+-dependent manner, mainly through the exposure of hydrophobic residues. How CaM can bind a large number of targets while retaining some selectivity is a fascinating open question. Here, we explore the mechanism of CaM selective promiscuity for selected target proteins. Analyzing enhanced sampling molecular dynamics simulations of Ca2+-bound and Ca2+-free CaM via spectral clustering has allowed us to identify distinct conformational states, characterized by interhelical angles, secondary structure determinants and the solvent exposure of specific residues. We searched for indicators of conformational selection by mapping solvent exposure of residues in these conformational states to contacts in structures of CaM/target peptide complexes. We thereby identified CaM states involved in various binding classes arranged along a depth binding gradient. Binding Ca2+ modifies the accessible hydrophobic surface of the two lobes and allows for deeper binding. Apo CaM indeed shows shallow binding involving predominantly polar and charged residues. Furthermore, binding to the C-terminal lobe of CaM appears selective and involves specific conformational states that can facilitate deep binding to target proteins, while binding to the N-terminal lobe appears to happen through a more flexible mechanism. Thus the long-ranged electrostatic interactions of the charged residues of the N-terminal lobe of CaM may initiate binding, while the short-ranged interactions of hydrophobic residues in the C-terminal lobe of CaM may account for selectivity. This work furthers our understanding of the mechanism of CaM binding and selectivity to different target proteins and paves the way towards a comprehensive model of CaM selectivity.

  7. Membrane proteins bind lipids selectively to modulate their structure and function.

    Science.gov (United States)

    Laganowsky, Arthur; Reading, Eamonn; Allison, Timothy M; Ulmschneider, Martin B; Degiacomi, Matteo T; Baldwin, Andrew J; Robinson, Carol V

    2014-06-05

    Previous studies have established that the folding, structure and function of membrane proteins are influenced by their lipid environments and that lipids can bind to specific sites, for example, in potassium channels. Fundamental questions remain however regarding the extent of membrane protein selectivity towards lipids. Here we report a mass spectrometry approach designed to determine the selectivity of lipid binding to membrane protein complexes. We investigate the mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis and aquaporin Z (AqpZ) and the ammonia channel (AmtB) from Escherichia coli, using ion mobility mass spectrometry (IM-MS), which reports gas-phase collision cross-sections. We demonstrate that folded conformations of membrane protein complexes can exist in the gas phase. By resolving lipid-bound states, we then rank bound lipids on the basis of their ability to resist gas phase unfolding and thereby stabilize membrane protein structure. Lipids bind non-selectively and with high avidity to MscL, all imparting comparable stability; however, the highest-ranking lipid is phosphatidylinositol phosphate, in line with its proposed functional role in mechanosensation. AqpZ is also stabilized by many lipids, with cardiolipin imparting the most significant resistance to unfolding. Subsequently, through functional assays we show that cardiolipin modulates AqpZ function. Similar experiments identify AmtB as being highly selective for phosphatidylglycerol, prompting us to obtain an X-ray structure in this lipid membrane-like environment. The 2.3 Å resolution structure, when compared with others obtained without lipid bound, reveals distinct conformational changes that re-position AmtB residues to interact with the lipid bilayer. Our results demonstrate that resistance to unfolding correlates with specific lipid-binding events, enabling a distinction to be made between lipids that merely bind from those that modulate membrane

  8. Fn3 proteins engineered to recognize tumor biomarker mesothelin internalize upon binding.

    Directory of Open Access Journals (Sweden)

    Allison R Sirois

    Full Text Available Mesothelin is a cell surface protein that is overexpressed in numerous cancers, including breast, ovarian, lung, liver, and pancreatic tumors. Aberrant expression of mesothelin has been shown to promote tumor progression and metastasis through interaction with established tumor biomarker CA125. Therefore, molecules that specifically bind to mesothelin have potential therapeutic and diagnostic applications. However, no mesothelin-targeting molecules are currently approved for routine clinical use. While antibodies that target mesothelin are in development, some clinical applications may require a targeting molecule with an alternative protein fold. For example, non-antibody proteins are more suitable for molecular imaging and may facilitate diverse chemical conjugation strategies to create drug delivery complexes. In this work, we engineered variants of the fibronectin type III domain (Fn3 non-antibody protein scaffold to bind to mesothelin with high affinity, using directed evolution and yeast surface display. Lead engineered Fn3 variants were solubly produced and purified from bacterial culture at high yield. Upon specific binding to mesothelin on human cancer cell lines, the engineered Fn3 proteins internalized and co-localized to early endosomes. To our knowledge, this is the first report of non-antibody proteins engineered to bind mesothelin. The results validate that non-antibody proteins can be engineered to bind to tumor biomarker mesothelin, and encourage the continued development of engineered variants for applications such as targeted diagnostics and therapeutics.

  9. Crystallographic structure and substrate-binding interactions of the molybdate-binding protein of the phytopathogen Xanthomonas axonopodis pv. citri.

    Science.gov (United States)

    Balan, Andrea; Santacruz-Pérez, Carolina; Moutran, Alexandre; Ferreira, Luís Carlos Souza; Neshich, Goran; Gonçalves Barbosa, João Alexandre Ribeiro

    2008-02-01

    In Xanthomonas axonopodis pv. citri (Xac or X. citri), the modA gene codes for a periplasmic protein (ModA) that is capable of binding molybdate and tungstate as part of the ABC-type transporter required for the uptake of micronutrients. In this study, we report the crystallographic structure of the Xac ModA protein with bound molybdate. The Xac ModA structure is similar to orthologs with known three-dimensional structures and consists of two nearly symmetrical domains separated by a hinge region where the oxyanion-binding site lies. Phylogenetic analysis of different ModA orthologs based on sequence alignments revealed three groups of molybdate-binding proteins: bacterial phytopathogens, enterobacteria and soil bacteria. Even though the ModA orthologs are segregated into different groups, the ligand-binding hydrogen bonds are mostly conserved, except for Archaeglobus fulgidus ModA. A detailed discussion of hydrophobic interactions in the active site is presented and two new residues, Ala38 and Ser151, are shown to be part of the ligand-binding pocket.

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

    DEFF Research Database (Denmark)

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

    1995-01-01

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

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    are the oldest evolutionary derivatives followed by IF and HC (the latter being present only in reptiles and most but not all mammals). Our findings suggest that the only cobalamin-binding protein in zebrafish is an intermediate between the three human cobalamin binders. These findings support the hypothesis...

  12. Generation of Oxygen Free Radicals by Proflavine: Implication in Protein Degradation

    Directory of Open Access Journals (Sweden)

    Mansour K.M. Gatasheh

    2017-07-01

    Full Text Available Proflavine, an acridine dye, is a known DNA intercalating agent. In the present study, we show that proflavine alone on photoillumination can generate reactive oxygen species (ROS. These proflavine-derived ROS cause damage to proteins, and this effect is enhanced when the divalent metal ion Cu (II is included in the reaction. Bathocuproine, a specific Cu (I sequestering agent, when present in the reaction mixture containing Cu (II, was found to inhibit the protein degradation, showing that Cu (I is an essential intermediate in the reaction. The effect of several scavengers of ROS such as superoxide dismutase, sodium azide, potassium iodide, and thiourea were examined on the protein damaging reaction. Potassium iodide was found to be the most effective in inhibiting protein damage followed by sodium azide and thiourea. Our results indicate the involvement of superoxide, singlet oxygen, triplet oxygen, and hydroxyl radicals in proflavine-induced damage to proteins.

  13. The primary structure of fatty-acid-binding protein from nurse shark liver. Structural and evolutionary relationship to the mammalian fatty-acid-binding protein family.

    Science.gov (United States)

    Medzihradszky, K F; Gibson, B W; Kaur, S; Yu, Z H; Medzihradszky, D; Burlingame, A L; Bass, N M

    1992-02-01

    The primary structure of a fatty-acid-binding protein (FABP) isolated from the liver of the nurse shark (Ginglymostoma cirratum) was determined by high-performance tandem mass spectrometry (employing multichannel array detection) and Edman degradation. Shark liver FABP consists of 132 amino acids with an acetylated N-terminal valine. The chemical molecular mass of the intact protein determined by electrospray ionization mass spectrometry (Mr = 15124 +/- 2.5) was in good agreement with that calculated from the amino acid sequence (Mr = 15121.3). The amino acid sequence of shark liver FABP displays significantly greater similarity to the FABP expressed in mammalian heart, peripheral nerve myelin and adipose tissue (61-53% sequence similarity) than to the FABP expressed in mammalian liver (22% similarity). Phylogenetic trees derived from the comparison of the shark liver FABP amino acid sequence with the members of the mammalian fatty-acid/retinoid-binding protein gene family indicate the initial divergence of an ancestral gene into two major subfamilies: one comprising the genes for mammalian liver FABP and gastrotropin, the other comprising the genes for mammalian cellular retinol-binding proteins I and II, cellular retinoic-acid-binding protein myelin P2 protein, adipocyte FABP, heart FABP and shark liver FABP, the latter having diverged from the ancestral gene that ultimately gave rise to the present day mammalian heart-FABP, adipocyte FABP and myelin P2 protein sequences. The sequence for intestinal FABP from the rat could be assigned to either subfamily, depending on the approach used for phylogenetic tree construction, but clearly diverged at a relatively early evolutionary time point. Indeed, sequences proximately ancestral or closely related to mammalian intestinal FABP, liver FABP, gastrotropin and the retinoid-binding group of proteins appear to have arisen prior to the divergence of shark liver FABP and should therefore also be present in elasmobranchs

  14. Predictions of RNA-binding ability and aggregation propensity of proteins

    OpenAIRE

    Agostini, Federico, 1985-

    2014-01-01

    RNA-binding proteins (RBPs) control the fate of a multitude of coding and non-coding transcripts. Formation of ribonucleoprotein (RNP) complexes fine-tunes regulation of post-transcriptional events and influences gene expression. Recently, it has been observed that non-canonical proteins with RNA-binding ability are enriched in structurally disordered and low-complexity regions that are generally involved in functional and dysfunctional associations. Therefore, it is possible that interaction...

  15. Cartilage Acidic Protein 2 a hyperthermostable, high affinity calcium-binding protein.

    Science.gov (United States)

    Anjos, Liliana; Gomes, Ana S; Melo, Eduardo P; Canário, Adelino V; Power, Deborah M

    2013-03-01

    Cartilage Acidic Protein 2 (CRTAC2) is a novel protein present from prokaryotes to vertebrates with abundant expression in the teleost fish pituitary gland and an isoform of CRTAC1, a chondrocyte marker in humans. The two proteins are non-integrins containing N-terminal integrin-like Ca(2+)-binding motifs and their structure and function remain to be assigned. Structural studies of recombinant sea bream (sb)CRTAC2 revealed it is composed of 8.8% α-helix, 33.4% β-sheet and 57.8% unordered protein. sbCRTAC2 bound Ca(2+) with high affinity (K(d)=1.46nM) and favourable Gibbs free energy (∆G=-12.4kcal/mol). The stoichiometry for Ca(2+) bound to sbCRTAC2 at saturation indicated six Ca(2+) ligand-binding sites exist per protein molecule. No conformational change in sbCRTAC2 occurred in the presence of Ca(2+). Fluorescence emission revealed that the tertiary structure of the protein is hyperthermostable between 25°C and 95°C and the fully unfolded state is only induced by chemical denaturing (4M GndCl). sbCRTAC has a widespread tissue distribution and is present as high molecular weight aggregates, although strong reducing conditions promote formation of the monomer. sbCRTAC2 promotes epithelial cell outgrowth in vitro suggesting it may share functional homology with mammalian CRTAC1, recently implicated in cell-cell and cell-matrix interactions. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. Elastic neutron scattering of dry and rehydrated trehalose coated carboxy-myoglobin

    International Nuclear Information System (INIS)

    Librizzi, Fabio; Vitrano, Eugenio; Paciaroni, Alessandro; Cordone, Lorenzo

    2008-01-01

    We report here a comparison between the hydrogen atoms mean square displacements measured by elastic neutron scattering on trehalose coated carboxy-myoglobin, at ILL on the backscattering spectrometers IN13 and IN16. An inconsistency is observed when comparing the mean square displacements measured on the two spectrometer, on samples of identical composition, since they resulted of larger amplitude on IN13 (either in condition of drought or after overnight rehydration under 75% D 2 O atmosphere), notwithstanding the lower time window accessible on this instrument with respect to IN16. Such inconsistency disappears when the data obtained on this last spectrometer are analyzed in two separate ranges of the exchanged wave vector q. The analysis of the data collected on IN13 in terms of the two-well model [W. Doster, S. Cusak, W. Petry, Nature 337 (1989) 754] gives relevant information on the enthalpy and entropy values involved in the interconversion among substates in dry and rehydrated trehalose coated protein samples

  17. Elastic neutron scattering of dry and rehydrated trehalose coated carboxy-myoglobin

    Energy Technology Data Exchange (ETDEWEB)

    Librizzi, Fabio; Vitrano, Eugenio [Dipartimento di Scienze Fisiche e Astronomiche and CNISM, Universita di Palermo, Via Archirafi 36, I-90123 Palermo (Italy); Paciaroni, Alessandro [Dipartimento di Fisica dell' Universita di Perugia, INFM-CRS SOFT and CEMIN - Centro di Eccellenza per i Materiali Innovativi e Nanostrutturati, Via A. Pascoli, I-06123 Perugia (Italy); Cordone, Lorenzo [Dipartimento di Scienze Fisiche e Astronomiche and CNISM, Universita di Palermo, Via Archirafi 36, I-90123 Palermo (Italy)], E-mail: cordone@fisica.unipa.it

    2008-04-18

    We report here a comparison between the hydrogen atoms mean square displacements measured by elastic neutron scattering on trehalose coated carboxy-myoglobin, at ILL on the backscattering spectrometers IN13 and IN16. An inconsistency is observed when comparing the mean square displacements measured on the two spectrometer, on samples of identical composition, since they resulted of larger amplitude on IN13 (either in condition of drought or after overnight rehydration under 75% D{sub 2}O atmosphere), notwithstanding the lower time window accessible on this instrument with respect to IN16. Such inconsistency disappears when the data obtained on this last spectrometer are analyzed in two separate ranges of the exchanged wave vector q. The analysis of the data collected on IN13 in terms of the two-well model [W. Doster, S. Cusak, W. Petry, Nature 337 (1989) 754] gives relevant information on the enthalpy and entropy values involved in the interconversion among substates in dry and rehydrated trehalose coated protein samples.

  18. UPF201 Archaeal Specific Family Members Reveals Structural Similarity to RNA-Binding Proteins but Low Likelihood for RNA-Binding Function

    Energy Technology Data Exchange (ETDEWEB)

    Rao, K.N.; Swaminathan, S.; Burley, S. K.

    2008-12-11

    We have determined X-ray crystal structures of four members of an archaeal specific family of proteins of unknown function (UPF0201; Pfam classification: DUF54) to advance our understanding of the genetic repertoire of archaea. Despite low pairwise amino acid sequence identities (10-40%) and the absence of conserved sequence motifs, the three-dimensional structures of these proteins are remarkably similar to one another. Their common polypeptide chain fold, encompassing a five-stranded antiparallel {beta}-sheet and five {alpha}-helices, proved to be quite unexpectedly similar to that of the RRM-type RNA-binding domain of the ribosomal L5 protein, which is responsible for binding the 5S- rRNA. Structure-based sequence alignments enabled construction of a phylogenetic tree relating UPF0201 family members to L5 ribosomal proteins and other structurally similar RNA binding proteins, thereby expanding our understanding of the evolutionary purview of the RRM superfamily. Analyses of the surfaces of these newly determined UPF0201 structures suggest that they probably do not function as RNA binding proteins, and that this domain specific family of proteins has acquired a novel function in archaebacteria, which awaits experimental elucidation.

  19. RNA-binding proteins involved in post-transcriptional regulation in bacteria

    Directory of Open Access Journals (Sweden)

    Elke eVan Assche

    2015-03-01

    Full Text Available Post-transcriptional regulation is a very important mechanism to control gene expression in changing environments. In the past decade, a lot of interest has been directed towards the role of small RNAs in bacterial post-transcriptional regulation. However, small RNAs are not the only molecules controlling gene expression at this level, RNA-binding proteins play an important role as well. CsrA and Hfq are the two best studied bacterial proteins of this type, but recently, additional proteins involved in post-transcriptional control have been identified. This review focuses on the general working mechanisms of post-transcriptionally active RNA-binding proteins, which include (i adaptation of the susceptibility of mRNAs and sRNAs to RNases, (ii modulating the accessibility of the ribosome binding site of mRNAs, (iii recruiting and assisting in the interaction of mRNAs with other molecules and (iv regulating transcription terminator / antiterminator formation, and gives an overview of both the well-studied and the newly identified proteins that are involved in post-transcriptional regulatory processes. Additionally, the post-transcriptional mechanisms by which the expression or the activity of these proteins is regulated, are described. For many of the newly identified proteins, however, mechanistic questions remain. Most likely, more post-transcriptionally active proteins will be identified in the future.

  20. Interaction of the anaphase-promoting complex/cyclosome and proteasome protein complexes with multiubiquitin chain-binding proteins

    DEFF Research Database (Denmark)

    Seeger, Michael; Hartmann-Petersen, Rasmus; Wilkinson, Caroline R M

    2003-01-01

    Fission yeast Rhp23 and Pus1 represent two families of multiubiquitin chain-binding proteins that associate with the proteasome. We show that both proteins bind to different regions of the proteasome subunit Mts4. The binding site for Pus1 was mapped to a cluster of repetitive sequences also found...... in the proteasome subunit SpRpn2 and the anaphase-promoting complex/cyclosome (APC/C) subunit Cut4. The putative role of Pus1 as a factor involved in allocation of ubiquitinylated substrates for the proteasome is discussed....

  1. Role of Electrostatics in Protein-RNA Binding: The Global vs the Local Energy Landscape.

    Science.gov (United States)

    Ghaemi, Zhaleh; Guzman, Irisbel; Gnutt, David; Luthey-Schulten, Zaida; Gruebele, Martin

    2017-09-14

    U1A protein-stem loop 2 RNA association is a basic step in the assembly of the spliceosomal U1 small nuclear ribonucleoprotein. Long-range electrostatic interactions due to the positive charge of U1A are thought to provide high binding affinity for the negatively charged RNA. Short range interactions, such as hydrogen bonds and contacts between RNA bases and protein side chains, favor a specific binding site. Here, we propose that electrostatic interactions are as important as local contacts in biasing the protein-RNA energy landscape toward a specific binding site. We show by using molecular dynamics simulations that deletion of two long-range electrostatic interactions (K22Q and K50Q) leads to mutant-specific alternative RNA bound states. One of these states preserves short-range interactions with aromatic residues in the original binding site, while the other one does not. We test the computational prediction with experimental temperature-jump kinetics using a tryptophan probe in the U1A-RNA binding site. The two mutants show the distinct predicted kinetic behaviors. Thus, the stem loop 2 RNA has multiple binding sites on a rough RNA-protein binding landscape. We speculate that the rough protein-RNA binding landscape, when biased to different local minima by electrostatics, could be one way that protein-RNA interactions evolve toward new binding sites and novel function.

  2. Concentration of serum thyroid hormone binding proteins after 131I treatment of hyperthyroidism

    International Nuclear Information System (INIS)

    Harrop, J.S.; Hopton, M.R.; Lazarus, J.H.

    1981-01-01

    Serum concentrations of the thyroid hormone binding proteins, thyroxine binding globulin, prealbumin, and albumin were determined in 30 thyrotoxic patients before and after 131 I treatment. Each patient was placed into one of three groups according to response to treatment. The serum concentration of all three proteins rose significantly in 10 patients who became euthyroid, and a greater increase was seen in 10 patients who developed hypothyroidism. There was no significant change in thyroid hormone binding protein concentrations in 10 subjects who remained hyperthyroid. Changes in the concentration of thyroid hormone binding proteins should be borne in mind when total thyroid hormone concentrations are used to monitor the progress of patients receiving treatment for hyperthyroidism. (author)

  3. Probing the structural basis of oxygen binding in a cofactor-independent dioxygenase.

    Science.gov (United States)

    Li, Kunhua; Fielding, Elisha N; Condurso, Heather L; Bruner, Steven D

    2017-07-01

    The enzyme DpgC is included in the small family of cofactor-independent dioxygenases. The chemistry of DpgC is uncommon as the protein binds and utilizes dioxygen without the aid of a metal or organic cofactor. Previous structural and biochemical studies identified the substrate-binding mode and the components of the active site that are important in the catalytic mechanism. In addition, the results delineated a putative binding pocket and migration pathway for the co-substrate dioxygen. Here, structural biology is utilized, along with site-directed mutagenesis, to probe the assigned dioxygen-binding pocket. The key residues implicated in dioxygen trafficking were studied to probe the process of binding, activation and chemistry. The results support the proposed chemistry and provide insight into the general mechanism of dioxygen binding and activation.

  4. A mutation in the mitochondrial protein UQCRB promotes angiogenesis through the generation of mitochondrial reactive oxygen species

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Junghwa [Chemical Genomics National Research Lab., Department of Biotechnology, Translational Research Center for Protein Function Control, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Jung, Hye Jin [Department of Pharmaceutical Engineering, Sun Moon University, Asansi, Chungnam 330-150 (Korea, Republic of); Jeong, Seung Hun; Kim, Hyoung Kyu; Han, Jin [National Research Laboratory for Mitochondrial Signaling, Department of Physiology, College of Medicine, Department of Health Sciences and Technology, Cardiovascular and Metabolic Disease Center, Inje University, Busan (Korea, Republic of); Kwon, Ho Jeong, E-mail: kwonhj@yonsei.ac.kr [Chemical Genomics National Research Lab., Department of Biotechnology, Translational Research Center for Protein Function Control, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Department of Internal Medicine, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of)

    2014-12-12

    Highlights: • We constructed mitochondrial protein UQCRB mutant stable cell lines on the basis of a human case report. • These mutant cell lines exhibit pro-angiogenic activity with enhanced VEGF expression. • Proliferation of mutant cell lines was regulated by UQCRB inhibitors. • UQCRB may have a functional role in angiogenesis. - Abstract: Ubiquinol-cytochrome c reductase binding protein (UQCRB) is one of the subunits of mitochondrial complex III and is a target protein of the natural anti-angiogenic small molecule terpestacin. Previously, the biological role of UQCRB was thought to be limited to the maintenance of complex III. However, the identification and validation of UQCRB as a target protein of terpestacin enabled the role of UQCRB in oxygen sensing and angiogenesis to be elucidated. To explore the biological role of this protein further, UQCRB mutant stable cell lines were generated on the basis of a human case report. We demonstrated that these cell lines exhibited glycolytic and pro-angiogenic activities via mitochondrial reactive oxygen species (mROS)-mediated HIF1 signal transduction. Furthermore, a morphological abnormality in mitochondria was detected in UQCRB mutant stable cell lines. In addition, the proliferative effect of the UQCRB mutants was significantly regulated by the UQCRB inhibitors terpestacin and A1938. Collectively, these results provide a molecular basis for UQCRB-related biological processes and reveal potential key roles of UQCRB in angiogenesis and mitochondria-mediated metabolic disorders.

  5. A mutation in the mitochondrial protein UQCRB promotes angiogenesis through the generation of mitochondrial reactive oxygen species

    International Nuclear Information System (INIS)

    Chang, Junghwa; Jung, Hye Jin; Jeong, Seung Hun; Kim, Hyoung Kyu; Han, Jin; Kwon, Ho Jeong

    2014-01-01

    Highlights: • We constructed mitochondrial protein UQCRB mutant stable cell lines on the basis of a human case report. • These mutant cell lines exhibit pro-angiogenic activity with enhanced VEGF expression. • Proliferation of mutant cell lines was regulated by UQCRB inhibitors. • UQCRB may have a functional role in angiogenesis. - Abstract: Ubiquinol-cytochrome c reductase binding protein (UQCRB) is one of the subunits of mitochondrial complex III and is a target protein of the natural anti-angiogenic small molecule terpestacin. Previously, the biological role of UQCRB was thought to be limited to the maintenance of complex III. However, the identification and validation of UQCRB as a target protein of terpestacin enabled the role of UQCRB in oxygen sensing and angiogenesis to be elucidated. To explore the biological role of this protein further, UQCRB mutant stable cell lines were generated on the basis of a human case report. We demonstrated that these cell lines exhibited glycolytic and pro-angiogenic activities via mitochondrial reactive oxygen species (mROS)-mediated HIF1 signal transduction. Furthermore, a morphological abnormality in mitochondria was detected in UQCRB mutant stable cell lines. In addition, the proliferative effect of the UQCRB mutants was significantly regulated by the UQCRB inhibitors terpestacin and A1938. Collectively, these results provide a molecular basis for UQCRB-related biological processes and reveal potential key roles of UQCRB in angiogenesis and mitochondria-mediated metabolic disorders

  6. Change of conformation and internal dynamics of supercoiled DNA upon binding of Escherichia coli single-strand binding protein

    International Nuclear Information System (INIS)

    Langowski, J.; Benight, A.S.; Fujimoto, B.S.; Schurr, J.M.; Schomburg, U.

    1985-01-01

    The influence of Escherichia coli single-strand binding (SSB) protein on the conformation and internal dynamics of pBR322 and pUC8 supercoiled DNAs has been investigated by using dynamic light scattering at 632.8 and 351.1 nm and time-resolved fluorescence polarization anisotropy of intercalated ethidium. SSB protein binds to both DNAs up to a stoichiometry that is sufficient to almost completely relax the superhelical turns. Upon saturation binding, the translational diffusion coefficients (D 0 ) of both DNAs decrease by approximately 20%. Apparent diffusion coefficients (D/sub app/) obtained from dynamic light scattering display the well-known increase with K 2 (K = scattering vector), leveling off toward a plateau value (D/sub plat/) at high K 2 . For both DNAs, the difference D/sub plat/ - D 0 increases upon relaxation of supercoils by SSB protein, which indicates a corresponding enhancement of the subunit mobilities in internal motions. Fluorescence polarization anisotropy measurements on free and complexed pBR322 DNA indicate a (predominantly) uniform torsional rigidity for the saturated DNA/SSB protein complex that is significantly reduced compared to the free DNA. These observations are all consistent with the notion that binding of SSB protein is accompanied by a gradual loss of supercoils and saturates when the superhelical twist is largely removed

  7. Sampling protein motion and solvent effect during ligand binding

    Science.gov (United States)

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

    2012-01-01

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

  8. Rational design of a conformation-switchable Ca2+- and Tb3+-binding protein without the use of multiple coupled metal-binding sites.

    Science.gov (United States)

    Li, Shunyi; Yang, Wei; Maniccia, Anna W; Barrow, Doyle; Tjong, Harianto; Zhou, Huan-Xiang; Yang, Jenny J

    2008-10-01

    Ca2+, as a messenger of signal transduction, regulates numerous target molecules via Ca2+-induced conformational changes. Investigation into the determinants for Ca2+-induced conformational change is often impeded by cooperativity between multiple metal-binding sites or protein oligomerization in naturally occurring proteins. To dissect the relative contributions of key determinants for Ca2+-dependent conformational changes, we report the design of a single-site Ca2+-binding protein (CD2.trigger) created by altering charged residues at an electrostatically sensitive location on the surface of the host protein rat Cluster of Differentiation 2 (CD2).CD2.trigger binds to Tb3+ and Ca2+ with dissociation constants of 0.3 +/- 0.1 and 90 +/- 25 microM, respectively. This protein is largely unfolded in the absence of metal ions at physiological pH, but Tb3+ or Ca2+ binding results in folding of the native-like conformation. Neutralization of the charged coordination residues, either by mutation or protonation, similarly induces folding of the protein. The control of a major conformational change by a single Ca2+ ion, achieved on a protein designed without reliance on sequence similarity to known Ca2+-dependent proteins and coupled metal-binding sites, represents an important step in the design of trigger proteins.

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

    Science.gov (United States)

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

    2017-12-01

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

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

    International Nuclear Information System (INIS)

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

    2009-01-01

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

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

    Science.gov (United States)

    Hung, Che-Lun; Hua, Guan-Jie

    2013-01-01

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

  12. Mycobacterial laminin-binding histone-like protein mediates collagen-dependent cytoadherence

    Directory of Open Access Journals (Sweden)

    André Alves Dias

    2012-12-01

    Full Text Available When grown in the presence of exogenous collagen I, Mycobacterium bovis BCG was shown to form clumps. Scanning electron microscopy examination of these clumps revealed the presence of collagen fibres cross-linking the bacilli. Since collagen is a major constituent of the eukaryotic extracellular matrices, we assayed BCG cytoadherence in the presence of exogenous collagen I. Collagen increased the interaction of the bacilli with A549 type II pneumocytes or U937 macrophages, suggesting that BCG is able to recruit collagen to facilitate its attachment to host cells. Using an affinity chromatography approach, we have isolated a BCG collagen-binding protein corresponding to the previously described mycobacterial laminin-binding histone-like protein (LBP/Hlp, a highly conserved protein associated with the mycobacterial cell wall. Moreover, Mycobacterium leprae LBP/Hlp, a well-characterized adhesin, was also able to bind collagen I. Finally, using recombinant fragments of M. leprae LBP/Hlp, we mapped the collagen-binding activity within the C-terminal domain of the adhesin. Since this protein was already shown to be involved in the recognition of laminin and heparan sulphate-containing proteoglycans, the present observations reinforce the adhesive activities of LBP/Hlp, which can be therefore considered as a multifaceted mycobacterial adhesin, playing an important role in both leprosy and tuberculosis pathogenesis.

  13. Membrane-associated 41-kDa GTP-binding protein in collagen-induced platelet activation

    International Nuclear Information System (INIS)

    Walker, G.; Bourguignon, L.Y.

    1990-01-01

    Initially we established that the binding of collagen to human blood platelets stimulates both the rapid loss of PIP2 and the generation of inositol-4,5-bisphosphate (IP2) and inositol-1,4,5-triphosphate (IP3). These results indicate that the binding of collagen stimulates inositol phospholipid-specific phospholipase C during platelet activation. The fact that GTP or GTP-gamma-S augments, and pertussis toxin inhibits, collagen-induced IP3 formation suggests that a GTP-binding protein or (or proteins) may be directly involved in the regulation of phospholipase C-mediated phosphoinositide turnover in human platelets. We have used several complementary techniques to isolate and characterize a platelet 41-kDa polypeptide (or polypeptides) that has a number of structural and functional similarities to the regulatory alpha i subunit of the GTP-binding proteins isolated from bovine brain. This 41-kDa polypeptide (or polypeptides) is found to be closely associated with at least four membrane glycoproteins (e.g., gp180, gp110, gp95, and gp75) in a 330-kDa complex that can be dissociated by treatment with high salt plus urea. Most important, we have demonstrated that antilymphoma 41-kDa (alpha i subunit of GTP-binding proteins) antibody cross-reacts with the platelet 41-kDa protein (or proteins) and the alpha i subunit of bovine brain Gi alpha proteins, and blocks GTP/collagen-induced IP3 formation. These data provide strong evidence that the 41-kDa platelet GTP-binding protein (or proteins) is directly involved in collagen-induced signal transduction during platelet activation

  14. Membrane-associated 41-kDa GTP-binding protein in collagen-induced platelet activation

    Energy Technology Data Exchange (ETDEWEB)

    Walker, G.; Bourguignon, L.Y. (Univ. of Miami Medical School, FL (USA))

    1990-08-01

    Initially we established that the binding of collagen to human blood platelets stimulates both the rapid loss of PIP2 and the generation of inositol-4,5-bisphosphate (IP2) and inositol-1,4,5-triphosphate (IP3). These results indicate that the binding of collagen stimulates inositol phospholipid-specific phospholipase C during platelet activation. The fact that GTP or GTP-gamma-S augments, and pertussis toxin inhibits, collagen-induced IP3 formation suggests that a GTP-binding protein or (or proteins) may be directly involved in the regulation of phospholipase C-mediated phosphoinositide turnover in human platelets. We have used several complementary techniques to isolate and characterize a platelet 41-kDa polypeptide (or polypeptides) that has a number of structural and functional similarities to the regulatory alpha i subunit of the GTP-binding proteins isolated from bovine brain. This 41-kDa polypeptide (or polypeptides) is found to be closely associated with at least four membrane glycoproteins (e.g., gp180, gp110, gp95, and gp75) in a 330-kDa complex that can be dissociated by treatment with high salt plus urea. Most important, we have demonstrated that antilymphoma 41-kDa (alpha i subunit of GTP-binding proteins) antibody cross-reacts with the platelet 41-kDa protein (or proteins) and the alpha i subunit of bovine brain Gi alpha proteins, and blocks GTP/collagen-induced IP3 formation. These data provide strong evidence that the 41-kDa platelet GTP-binding protein (or proteins) is directly involved in collagen-induced signal transduction during platelet activation.

  15. Subcellular distribution of calcium-binding proteins and a calcium-ATPase in canine pancreas

    International Nuclear Information System (INIS)

    Nigam, S.K.; Towers, T.

    1990-01-01

    Using a 45Ca blot-overlay assay, we monitored the subcellular fractionation pattern of several Ca binding proteins of apparent molecular masses 94, 61, and 59 kD. These proteins also appeared to stain blue with Stains-All. Additionally, using a monoclonal antiserum raised against canine cardiac sarcoplasmic reticulum Ca-ATPase, we examined the subcellular distribution of a canine pancreatic 110-kD protein recognized by this antiserum. This protein had the same electrophoretic mobility as the cardiac protein against which the antiserum was raised. The three Ca binding proteins and the Ca-ATPase cofractionated into the rough microsomal fraction (RM), previously shown to consist of highly purified RER, in a pattern highly similar to that of the RER marker, ribophorin I. To provide further evidence for an RER localization, native RM were subjected to isopycnic flotation in sucrose gradients. The Ca binding proteins and the Ca-ATPase were found in dense fractions, along with ribophorin I. When RM were stripped of ribosomes with puromycin/high salt, the Ca binding proteins and the Ca-ATPase exhibited a shift to less dense fractions, as did ribophorin I. We conclude that, in pancreas, the Ca binding proteins and Ca-ATPase we detect are localized to the RER (conceivably a subcompartment of the RER) or, possibly, a structure intimately associated with the RER

  16. A web server for analysis, comparison and prediction of protein ligand binding sites.

    Science.gov (United States)

    Singh, Harinder; Srivastava, Hemant Kumar; Raghava, Gajendra P S

    2016-03-25

    One of the major challenges in the field of system biology is to understand the interaction between a wide range of proteins and ligands. In the past, methods have been developed for predicting binding sites in a protein for a limited number of ligands. In order to address this problem, we developed a web server named 'LPIcom' to facilitate users in understanding protein-ligand interaction. Analysis, comparison and prediction modules are available in the "LPIcom' server to predict protein-ligand interacting residues for 824 ligands. Each ligand must have at least 30 protein binding sites in PDB. Analysis module of the server can identify residues preferred in interaction and binding motif for a given ligand; for example residues glycine, lysine and arginine are preferred in ATP binding sites. Comparison module of the server allows comparing protein-binding sites of multiple ligands to understand the similarity between ligands based on their binding site. This module indicates that ATP, ADP and GTP ligands are in the same cluster and thus their binding sites or interacting residues exhibit a high level of similarity. Propensity-based prediction module has been developed for predicting ligand-interacting residues in a protein for more than 800 ligands. In addition, a number of web-based tools have been integrated to facilitate users in creating web logo and two-sample between ligand interacting and non-interacting residues. In summary, this manuscript presents a web-server for analysis of ligand interacting residue. This server is available for public use from URL http://crdd.osdd.net/raghava/lpicom .

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

    Science.gov (United States)

    Liao, Jielou

    2004-03-01

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

  18. Reactions of metal-substituted myoglobins with excess electrons studied by pulse radiolysis and low-temperature gamma-radiolysis

    International Nuclear Information System (INIS)

    Miki, Hideho; Nakajima, Atushi; Ogasawara, Masaaki; Tamura, Mamoru

    1990-01-01

    Reactions of metal-substituted myoglobins with excess electrons in electron-pulse-irradiated aqueous solutions at room temperature and γ-irradiated aqueous matrices at 77 K were studied for the purpose of probing the functional role of heme iron. The rate constants for the reactions of various myoglobins with hydrated electrons were not much different from each other, and were close to those of diffusion-controlled reactions. In contrast, the reduction rates of myoglobins with dithionite depended markedly on the kind of central metals in the myoglobins. The difference was interpreted in terms of Marcus' theory for electron-transfer reactions. Effects of the 6-coordinate structure of the cobalt(III) species on the reaction with dithionite was also discussed. The steady-state optical-absorption measurements of γ-irradiated matrices containing cobaltimyoglobin at 77 K demonstrated the reduction of cobalt(III) species by excess electrons produced by the action of ionizing radiation. It was shown, by electron-spin resonance spectroscopy, that a 6-coordinated cobalt(II) species produced at 77 K transformed to a 5-coordinate one at higher temperatures, as reported previously. However, structural relaxation was not observed by optical spectroscopy either in the solutions or in the low-temperature matrices. It was concluded, therefore, that the intermediate 6-coordinate cobalt(II) species gave an optical absorption spectrum which was indistinguishable from that of the relaxed 5-coordinate cobalt(II) species. (author)

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

    Directory of Open Access Journals (Sweden)

    Ryoko Tsukahara

    2014-01-01

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

  20. GenProBiS: web server for mapping of sequence variants to protein binding sites.

    Science.gov (United States)

    Konc, Janez; Skrlj, Blaz; Erzen, Nika; Kunej, Tanja; Janezic, Dusanka

    2017-07-03

    Discovery of potentially deleterious sequence variants is important and has wide implications for research and generation of new hypotheses in human and veterinary medicine, and drug discovery. The GenProBiS web server maps sequence variants to protein structures from the Protein Data Bank (PDB), and further to protein-protein, protein-nucleic acid, protein-compound, and protein-metal ion binding sites. The concept of a protein-compound binding site is understood in the broadest sense, which includes glycosylation and other post-translational modification sites. Binding sites were defined by local structural comparisons of whole protein structures using the Protein Binding Sites (ProBiS) algorithm and transposition of ligands from the similar binding sites found to the query protein using the ProBiS-ligands approach with new improvements introduced in GenProBiS. Binding site surfaces were generated as three-dimensional grids encompassing the space occupied by predicted ligands. The server allows intuitive visual exploration of comprehensively mapped variants, such as human somatic mis-sense mutations related to cancer and non-synonymous single nucleotide polymorphisms from 21 species, within the predicted binding sites regions for about 80 000 PDB protein structures using fast WebGL graphics. The GenProBiS web server is open and free to all users at http://genprobis.insilab.org. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  1. Intramyocardial oxygen transport by quantitative diffuse reflectance spectroscopy in calves

    Science.gov (United States)

    Lindbergh, Tobias; Larsson, Marcus; Szabó, Zoltán; Casimir-Ahn, Henrik; Strömberg, Tomas

    2010-03-01

    Intramyocardial oxygen transport was assessed during open-chest surgery in calves by diffuse reflectance spectroscopy using a small intramuscular fiber-optic probe. The sum of hemo- and myoglobin tissue fraction and oxygen saturation, the tissue fraction and oxidation of cytochrome aa3, and the tissue fraction of methemoglobin were estimated using a calibrated empirical light transport model. Increasing the oxygen content in the inhaled gas, 21%-50%-100%, in five calves (group A) gave an increasing oxygen saturation of 19+/-4%, 24+/-5%, and 28+/-8% (panimals increased with LVAD pump speed (p<0.001, ANOVA) and with oxygen content in inhaled gas (p<0.001, ANOVA). The cytochrome aa3 oxidation level was above 96% in both group A and group B calves, including the two cases involving cardiac arrest. In conclusion, the estimated tissue fractions and oxygenation/oxidation levels of the myocardial chromophores during respiratory and hemodynamic provocations were in agreement with previously presented results, demonstrating the potential of the method.

  2. Ligand binding turns moth pheromone-binding protein into a pH sensor: effect on the Antheraea polyphemus PBP1 conformation.

    Science.gov (United States)

    Katre, Uma V; Mazumder, Suman; Prusti, Rabi K; Mohanty, Smita

    2009-11-13

    In moths, pheromone-binding proteins (PBPs) are responsible for the transport of the hydrophobic pheromones to the membrane-bound receptors across the aqueous sensillar lymph. We report here that recombinant Antheraea polyphemus PBP1 (ApolPBP1) picks up hydrophobic molecule(s) endogenous to the Escherichia coli expression host that keeps the protein in the "open" (bound) conformation at high pH but switches to the "closed" (free) conformation at low pH. This finding has bearing on the solution structures of undelipidated lepidopteran moth PBPs determined thus far. Picking up a hydrophobic molecule from the host expression system could be a common feature for lipid-binding proteins. Thus, delipidation is critical for bacterially expressed lipid-binding proteins. We have shown for the first time that the delipidated ApolPBP1 exists primarily in the closed form at all pH levels. Thus, current views on the pH-induced conformational switch of PBPs hold true only for the ligand-bound open conformation of the protein. Binding of various ligands to delipidated ApolPBP1 studied by solution NMR revealed that the protein in the closed conformation switches to the open conformation only at or above pH 6.0 with a protein to ligand stoichiometry of approximately 1:1. Mutation of His(70) and His(95) to alanine drives the equilibrium toward the open conformation even at low pH for the ligand-bound protein by eliminating the histidine-dependent pH-induced conformational switch. Thus, the delipidated double mutant can bind ligand even at low pH in contrast to the wild type protein as revealed by fluorescence competitive displacement assay using 1-aminoanthracene and solution NMR.

  3. Fibroblast growth factor regulates insulin-like growth factor-binding protein production by vascular smooth muscle cells.

    Science.gov (United States)

    Ververis, J; Ku, L; Delafontaine, P

    1994-02-01

    Insulin-like growth factor I is an important mitogen for vascular smooth muscle cells, and its effects are regulated by several binding proteins. Western ligand blotting of conditioned medium from rat aortic smooth muscle cells detected a 24 kDa binding protein and a 28 kDa glycosylated variant of this protein, consistent with insulin-like growth factor binding protein-4 by size. Low amounts of a glycosylated 38 to 42 kDa doublet (consistent with binding protein-3) and a 31 kDa non-glycosylated protein also were present. Basic fibroblast growth factor markedly increased secretion of the 24 kDa binding protein and its 28 kDa glycosylated variant. This effect was dose- and time-dependent and was inhibited by co-incubation with cycloheximide. Crosslinking of [125I]-insulin-like growth factor I to cell monolayers revealed no surface-associated binding proteins, either basally or after agonist treatment. Induction of binding protein production by fibroblast growth factor at sites of vascular injury may be important in vascular proliferative responses in vivo.

  4. Cyanobacterial Oxygenic Photosynthesis is Protected by Flavodiiron Proteins

    Directory of Open Access Journals (Sweden)

    Yagut Allahverdiyeva

    2015-03-01

    Full Text Available Flavodiiron proteins (FDPs, also called flavoproteins, Flvs are modular enzymes widely present in Bacteria and Archaea. The evolution of cyanobacteria and oxygenic photosynthesis occurred in concert with the modulation of typical bacterial FDPs. Present cyanobacterial FDPs are composed of three domains, the β-lactamase-like, flavodoxin-like and flavin-reductase like domains. Cyanobacterial FDPs function as hetero- and homodimers and are involved in the regulation of photosynthetic electron transport. Whilst Flv2 and Flv4 proteins are limited to specific cyanobacterial species (β-cyanobacteria and function in photoprotection of Photosystem II, Flv1 and Flv3 proteins, functioning in the “Mehler-like” reaction and safeguarding Photosystem I under fluctuating light conditions, occur in nearly all cyanobacteria and additionally in green algae, mosses and lycophytes. Filamentous cyanobacteria have additional FDPs in heterocyst cells, ensuring a microaerobic environment for the function of the nitrogenase enzyme under the light. Here, the evolution, occurrence and functional mechanisms of various FDPs in oxygenic photosynthetic organisms are discussed.

  5. The mycobacterial DNA-binding protein 1 (MDP1 from Mycobacterium bovis BCG influences various growth characteristics

    Directory of Open Access Journals (Sweden)

    Maurischat Sven

    2008-06-01

    Full Text Available Abstract Background Pathogenic mycobacteria such as M. tuberculosis, M. bovis or M. leprae are characterised by their extremely slow growth rate which plays an important role in mycobacterial virulence and eradication of the bacteria. Various limiting factors influence the generation time of mycobacteria, and the mycobacterial DNA-binding protein 1 (MDP1 has also been implicated in growth regulation. Our strategy to investigate the role of MDP1 in mycobacterial growth consisted in the generation and characterisation of a M. bovis BCG derivative expressing a MDP1-antisense gene. Results The expression rate of the MDP1 protein in the recombinant M. bovis BCG containing the MDP1-antisense plasmid was reduced by about 50% compared to the reference strain M. bovis BCG containing the empty vector. In comparison to this reference strain, the recombinant M. bovis BCG grew faster in broth culture and reached higher cell masses in stationary phase. Likewise its intracellular growth in mouse and human macrophages was ameliorated. Bacterial clumping in broth culture was reduced by the antisense plasmid. The antisense plasmid increased the susceptibility of the bacteria towards Ampicillin. 2-D protein gels of bacteria maintained under oxygen-poor conditions demonstrated a reduction in the number and the intensity of many protein spots in the antisense strain compared to the reference strain. Conclusion The MDP1 protein has a major impact on various growth characteristics of M. bovis BCG. It plays an important role in virulence-related traits such as aggregate formation and intracellular multiplication. Its impact on the protein expression in a low-oxygen atmosphere indicates a role in the adaptation to the hypoxic conditions present in the granuloma.

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

    International Nuclear Information System (INIS)

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

    2007-01-01

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

  7. Near infrared spectroscopy (NIRS) to monitor tissue haemoglobin (and myoglobin) oxygenation

    NARCIS (Netherlands)

    Scheeren, T. W. L.

    2010-01-01

    Introduction: Tissue oxygenation may be monitored noninvasively by near infrared spectroscopy (NIRS) both on the thenar eminescence (muscle) and on the forehead (brain). Thenar measurement have been used to guide therapy in trauma patients ( 1 ) and to determine the prognosis of septic patients ( 2

  8. Photoaffinity labeling of serum vitamin D binding protein by 3-deoxy-3-azido-25-hydroxyvitamin D3

    International Nuclear Information System (INIS)

    Link, R.P.; Kutner, A.; Schnoes, H.K.; DeLuca, H.F.

    1987-01-01

    3-Deoxy-3-azido-25-hydroxyvitamin D3 was covalently incorporated in the 25-hydroxyvitamin D3 binding site of purified human plasma vitamin D binding protein. Competition experiments showed that 3-deoxy-3-azido-25-hydroxyvitamin D3 and 25-hydroxyvitamin D3 bind at the same site on the protein. Tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was synthesized from tritiated 25-hydroxyvitamin D3, retaining the high specific activity of the parent compound. The tritiated azido label bound reversibly to human vitamin D binding protein in the dark and covalently to human vitamin D binding protein after exposure to ultraviolet light. Reversible binding of tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was compared to tritiated 25-hydroxyvitamin D3 binding to human vitamin D binding protein. Scatchard analysis of the data indicated equivalent maximum density binding sites with a KD,app of 0.21 nM for 25-hydroxyvitamin D3 and a KD,app of 1.3 nM for the azido derivative. Covalent binding was observed only after exposure to ultraviolet irradiation, with an average of 3% of the reversibly bound label becoming covalently bound to vitamin D binding protein. The covalent binding was reduced 70-80% when 25-hydroxyvitamin D3 was present, indicating strong covalent binding at the vitamin D binding site of the protein. When tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was incubated with human plasma in the absence and presence of 25-hydroxyvitamin D3, 12% of the azido derivative was reversibly bound to vitamin D binding protein. After ultraviolet irradiation, four plasma proteins covalently bound the azido label, but vitamin D binding protein was the only protein of the four that was unlabeled in the presence of 25-hydroxyvitamin D3

  9. An improved UPLC method for the detection of undeclared horse meat addition by using myoglobin as molecular marker.

    Science.gov (United States)

    Di Giuseppe, Antonella M A; Giarretta, Nicola; Lippert, Martina; Severino, Valeria; Di Maro, Antimo

    2015-02-15

    In 2013, following the scandal of the presence of undeclared horse meat in various processed beef products across the Europe, several researches have been undertaken for the safety of consumer health. In this framework, an improved UPLC separation method has been developed to detect the presence of horse myoglobin in raw meat samples. The separation of both horse and beef myoglobins was achieved in only seven minutes. The methodology was improved by preparing mixtures with different composition percentages of horse and beef meat. By using myoglobin as marker, low amounts (0.50mg/0.50g, w/w; ∼0.1%) of horse meat can be detected and quantified in minced raw meat samples with high reproducibility and sensitivity, thus offering a valid alternative to conventional PCR techniques. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. 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

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

    DEFF Research Database (Denmark)

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

    2005-01-01

    The envelope protein (gp120) of human immunodeficiency virus (HIV) contains highly conserved mannosylated oligosaccharides. These glycoconjugates contribute to resistance to antibody neutralization, and binding to cell surface lectins on macrophages and dendritic cells. Mannose-binding lectin (MBL......) binds to gp120 and plays a role in defence against the virus. In this study it is demonstrated that surfactant protein D (SP-D) binds to gp120 and inhibits HIV infectivity at significantly lower concentrations than MBL. The binding of SP-D was mediated by its calcium-dependent carbohydrate......-binding activity and was dependent on glycosylation of gp120. Native dodecameric SP-D bound to HIV gp120 more strongly than native trimeric SP-D. Since one common polymorphic form of SP-D is predominantly expressed as trimers and associated with lower blood levels, these individuals may have less effective innate...

  12. Identification and characterization of Taenia solium enolase as a plasminogen-binding protein.

    Science.gov (United States)

    Ayón-Núñez, Dolores A; Fragoso, Gladis; Espitia, Clara; García-Varela, Martín; Soberón, Xavier; Rosas, Gabriela; Laclette, Juan P; Bobes, Raúl J

    2018-06-01

    The larval stage of Taenia solium (cysticerci) is the causal agent of human and swine cysticercosis. When ingested by the host, T. solium eggs are activated and hatch in the intestine, releasing oncospheres that migrate to various tissues and evolve into cysticerci. Plasminogen (Plg) receptor proteins have been reported to play a role in migration processes for several pathogens. This work is aimed to identify Plg-binding proteins in T. solium cysticerci and determine whether T. solium recombinant enolase (rTsEnoA) is capable of specifically binding and activating human Plg. To identify Plg-binding proteins, a 2D-SDS-PAGE ligand blotting was performed, and recognized spots were identified by MS/MS. Seven proteins from T. solium cysticerci were found capable of binding Plg: fascicilin-1, fasciclin-2, enolase, MAPK, annexin, actin, and cytosolic malate dehydrogenase. To determine whether rTsEnoA binds human Plg, a ligand blotting was performed and the results were confirmed by ELISA both in the presence and absence of εACA, a competitive Plg inhibitor. Finally, rTsEnoA-bound Plg was activated to plasmin in the presence of tPA. To better understand the evolution of enolase isoforms in T. solium, a phylogenetic inference analysis including 75 enolase amino acid sequences was conducted. The origin of flatworm enolase isoforms, except for Eno4, is independent of their vertebrate counterparts. Therefore, herein we propose to designate tapeworm protein isoforms as A, B, C, and 4. In conclusion, recombinant enolase showed a strong plasminogen binding and activating activity in vitro. T. solium enolase could play a role in parasite invasion along with other plasminogen-binding proteins. Copyright © 2018 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2014-07-01

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

  14. Zinc(II) and the single-stranded DNA binding protein of bacteriophage T4

    International Nuclear Information System (INIS)

    Gauss, P.; Krassa, K.B.; McPheeters, D.S.; Nelson, M.A.; Gold, L.

    1987-01-01

    The DNA binding domain of the gene 32 protein of the bacteriophage T4 contains a single zinc-finger sequence. The gene 32 protein is an extensively studied member of a class of proteins that bind relatively nonspecifically to single-stranded DNA. The authors have sequenced and characterized mutations in gene 32 whose defective proteins are activated by increasing the Zn(II) concentration in the growth medium. The results identify a role for the gene 32 protein in activation of T4 late transcription. Several eukaryotic proteins with zinc fingers participate in activation of transcription, and the gene 32 protein of T4 should provide a simple, well-characterized system in which genetics can be utilized to study the role of a zinc finger in nucleic acid binding and gene expression

  15. Novel RNA-binding properties of the MTG chromatin regulatory proteins

    NARCIS (Netherlands)

    S. Rossetti (Stefano); L. van Unen (Leontine); N. Sacchi; A.T. Hoogeveen (Andre)

    2008-01-01

    textabstractBackground: The myeloid translocation gene (MTG) proteins are non-DNA-binding transcriptional regulators capable of interacting with chromatin modifying proteins. As a consequence of leukemia-associated chromosomal translocations, two of the MTG proteins, MTG8 and MTG16, are fused to the

  16. Polyamine binding to proteins in oat and Petunia protoplasts

    Science.gov (United States)

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

    1989-01-01

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

  17. Detecting local ligand-binding site similarity in nonhomologous proteins by surface patch comparison.

    Science.gov (United States)

    Sael, Lee; Kihara, Daisuke

    2012-04-01

    Functional elucidation of proteins is one of the essential tasks in biology. Function of a protein, specifically, small ligand molecules that bind to a protein, can be predicted by finding similar local surface regions in binding sites of known proteins. Here, we developed an alignment free local surface comparison method for predicting a ligand molecule which binds to a query protein. The algorithm, named Patch-Surfer, represents a binding pocket as a combination of segmented surface patches, each of which is characterized by its geometrical shape, the electrostatic potential, the hydrophobicity, and the concaveness. Representing a pocket by a set of patches is effective to absorb difference of global pocket shape while capturing local similarity of pockets. The shape and the physicochemical properties of surface patches are represented using the 3D Zernike descriptor, which is a series expansion of mathematical 3D function. Two pockets are compared using a modified weighted bipartite matching algorithm, which matches similar patches from the two pockets. Patch-Surfer was benchmarked on three datasets, which consist in total of 390 proteins that bind to one of 21 ligands. Patch-Surfer showed superior performance to existing methods including a global pocket comparison method, Pocket-Surfer, which we have previously introduced. Particularly, as intended, the accuracy showed large improvement for flexible ligand molecules, which bind to pockets in different conformations. Copyright © 2011 Wiley Periodicals, Inc.

  18. Growth of thin films of low molecular weight proteins by matrix assisted pulsed laser evaporation (MAPLE)

    DEFF Research Database (Denmark)

    Matei, Andreea; Schou, Jørgen; Constantinescu, C.

    2011-01-01

    Thin films of lysozyme and myoglobin grown by matrix assisted pulsed laser evaporation (MAPLE) from a water ice matrix have been investigated. The deposition rate of these two low molecular weight proteins (lysozyme: 14307 amu and myoglobin: 17083 amu) exhibits a maximum of about 1–2 ng/cm2 per....... The results for lysozyme demonstrate that the fragmentation rate of the proteins during the MAPLE process is not influenced by the pH of the water solution prior to freezing....

  19. Preliminary structural characterization of human SOUL, a haem-binding protein

    International Nuclear Information System (INIS)

    Freire, Filipe; Romão, Maria João; Macedo, Anjos L.; Aveiro, Susana S.; Goodfellow, Brian J.; Carvalho, Ana Luísa

    2009-01-01

    This manuscript describes the overexpression, purification and crystallization of human SOUL protein (hSOUL). hSOUL is a 23 kDa haem-binding protein that was first identified as the PP23 protein isolated from human full-term placenta. Human SOUL (hSOUL) is a 23 kDa haem-binding protein that was first identified as the PP 23 protein isolated from human full-term placentas. Here, the overexpression, purification and crystallization of hSOUL are reported. The crystals belonged to space group P6 4 22, with unit-cell parameters a = b = 145, c = 60 Å and one protein molecule in the asymmetric unit. X-ray diffraction data were collected to 3.5 Å resolution at the ESRF. A preliminary model of the three-dimensional structure of hSOUL was obtained by molecular replacement using the structures of murine p22HBP, obtained by solution NMR, as search models

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

    Directory of Open Access Journals (Sweden)

    О. V. Sviatenko

    2014-04-01

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

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

    DEFF Research Database (Denmark)

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

    2014-01-01

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

  2. Acyl-CoA binding protein and epidermal barrier function

    DEFF Research Database (Denmark)

    Bloksgaard, Maria; Neess, Ditte; Færgeman, Nils J

    2014-01-01

    The acyl-CoA binding protein (ACBP) is a 10kDa intracellular protein expressed in all eukaryotic species and mammalian tissues investigated. It binds acyl-CoA esters with high specificity and affinity and is thought to act as an intracellular transporter of acyl-CoA esters between different...... includes tousled and greasy fur, development of alopecia and scaling of the skin with age. Furthermore, epidermal barrier function is compromised causing a ~50% increase in transepidermal water loss relative to that of wild type mice. Lipidomic analyses indicate that this is due to significantly reduced...

  3. Leishmania replication protein A-1 binds in vivo single-stranded telomeric DNA

    International Nuclear Information System (INIS)

    Neto, J.L. Siqueira; Lira, C.B.B.; Giardini, M.A.; Khater, L.; Perez, A.M.; Peroni, L.A.; Reis, J.R.R. dos; Freitas-Junior, L.H.; Ramos, C.H.I.; Cano, M.I.N.

    2007-01-01

    Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in different events of DNA metabolism. In yeast, subunits 1 (RPA-1) and 2 (RPA-2) work also as telomerase recruiters and, in humans, the complex unfolds G-quartet structures formed by the 3' G-rich telomeric strand. In most eukaryotes, RPA-1 and RPA-2 bind DNA using multiple OB fold domains. In trypanosomatids, including Leishmania, RPA-1 has a canonical OB fold and a truncated RFA-1 structural domain. In Leishmania amazonensis, RPA-1 alone can form a complex in vitro with the telomeric G-rich strand. In this work, we show that LaRPA-1 is a nuclear protein that associates in vivo with Leishmania telomeres. We mapped the boundaries of the OB fold DNA-binding domain using deletion mutants. Since Leishmania and other trypanosomatids lack homologues of known telomere end binding proteins, our results raise questions about the function of RPA-1 in parasite telomeres

  4. Beauty is in the eye of the beholder: proteins can recognize binding sites of homologous proteins in more than one way.

    Directory of Open Access Journals (Sweden)

    Juliette Martin

    2010-06-01

    Full Text Available Understanding the mechanisms of protein-protein interaction is a fundamental problem with many practical applications. The fact that different proteins can bind similar partners suggests that convergently evolved binding interfaces are reused in different complexes. A set of protein complexes composed of non-homologous domains interacting with homologous partners at equivalent binding sites was collected in 2006, offering an opportunity to investigate this point. We considered 433 pairs of protein-protein complexes from the ABAC database (AB and AC binary protein complexes sharing a homologous partner A and analyzed the extent of physico-chemical similarity at the atomic and residue level at the protein-protein interface. Homologous partners of the complexes were superimposed using Multiprot, and similar atoms at the interface were quantified using a five class grouping scheme and a distance cut-off. We found that the number of interfacial atoms with similar properties is systematically lower in the non-homologous proteins than in the homologous ones. We assessed the significance of the similarity by bootstrapping the atomic properties at the interfaces. We found that the similarity of binding sites is very significant between homologous proteins, as expected, but generally insignificant between the non-homologous proteins that bind to homologous partners. Furthermore, evolutionarily conserved residues are not colocalized within the binding sites of non-homologous proteins. We could only identify a limited number of cases of structural mimicry at the interface, suggesting that this property is less generic than previously thought. Our results support the hypothesis that different proteins can interact with similar partners using alternate strategies, but do not support convergent evolution.

  5. Protein aggregation in food models: effect of γ-irradiation and lipid oxidation

    International Nuclear Information System (INIS)

    Delincee, H.; Paul, P.

    1981-01-01

    Myoglobin and serum albumin have been irradiated in aqueous solution in the presence of varying amounts of carbohydrates and lipids, and the yield of protein aggregates has been determined by gel filtration. With myoglobin the formation of aggregates evolving from the reaction with oxidizing lipids was observed, which was not found for serum albumin. The production of protein-lipid complexes, in which lipid material was occluded in the high-molecular aggregates by physical forces was demonstrated. Gel filtration and gel electrophoresis, both in the presence of SDS, and thin-layer isoelectric focusing revealed distinct structural differenes between the protein aggregates induced by irradiation and the aggregates formed by interaction with oxidizing lipids

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  7. RNA-Binding Proteins Revisited – The Emerging Arabidopsis mRNA Interactome

    KAUST Repository

    Köster, Tino

    2017-04-13

    RNA–protein interaction is an important checkpoint to tune gene expression at the RNA level. Global identification of proteins binding in vivo to mRNA has been possible through interactome capture – where proteins are fixed to target RNAs by UV crosslinking and purified through affinity capture of polyadenylated RNA. In Arabidopsis over 500 RNA-binding proteins (RBPs) enriched in UV-crosslinked samples have been identified. As in mammals and yeast, the mRNA interactomes came with a few surprises. For example, a plethora of the proteins caught on RNA had not previously been linked to RNA-mediated processes, for example proteins of intermediary metabolism. Thus, the studies provide unprecedented insights into the composition of the mRNA interactome, highlighting the complexity of RNA-mediated processes.

  8. RNA-Binding Proteins Revisited – The Emerging Arabidopsis mRNA Interactome

    KAUST Repository

    Kö ster, Tino; Marondedze, Claudius; Meyer, Katja; Staiger, Dorothee

    2017-01-01

    RNA–protein interaction is an important checkpoint to tune gene expression at the RNA level. Global identification of proteins binding in vivo to mRNA has been possible through interactome capture – where proteins are fixed to target RNAs by UV crosslinking and purified through affinity capture of polyadenylated RNA. In Arabidopsis over 500 RNA-binding proteins (RBPs) enriched in UV-crosslinked samples have been identified. As in mammals and yeast, the mRNA interactomes came with a few surprises. For example, a plethora of the proteins caught on RNA had not previously been linked to RNA-mediated processes, for example proteins of intermediary metabolism. Thus, the studies provide unprecedented insights into the composition of the mRNA interactome, highlighting the complexity of RNA-mediated processes.

  9. Variations in riboflavin binding by human plasma: identification of immunoglobulins as the major proteins responsible

    International Nuclear Information System (INIS)

    Innis, W.S.; McCormick, D.B.; Merrill, A.H. Jr.

    1985-01-01

    Riboflavin binding by plasma proteins from healthy human subjects was examined by equilibrium dialysis using a physiological concentration of [2-14C]riboflavin (0.04 microM). Binding ranged from 0.080 to 0.917 pmole of riboflavin/mg of protein (with a mean +/- SD of 0.274 +/- 0.206), which corresponded to 4.14 to 49.4 pmole/ml of plasma (15.5 +/- 11.0) (N = 34). Males and females yielded similar results. Upon fractionation of plasma by gel filtration, the major riboflavin-binding components eluted with albumin and gamma-globulins. Albumin was purified and found to bind riboflavin only very weakly (Kd = 3.8 to 10.4 mM), although FMN and photochemical degradation products (e.g., lumiflavine and lumichrome) were more tightly bound. Binding in the gamma-globulin fraction was attributed to IgG and IGA because the binding protein(s) and immunoglobulins copurified using various methods were removed by treatment of plasma with protein A-agarose, and were coincident upon immunoelectrophoresis followed by autoradiography to detect [2-14C]riboflavin. Differences among the plasma samples correlated with the binding recovered with the immunoglobulins. Binding was not directly related to the total IgG or IgA levels of subjects. Hence, it appears that the binding is due to a subfraction of these proteins. These findings suggest that riboflavin-binding immunoglobulins are a major cause of variations in riboflavin binding in human circulation, and may therefore affect the utilization of this micronutrient

  10. Structure and Calcium Binding Properties of a Neuronal Calcium-Myristoyl Switch Protein, Visinin-Like Protein 3.

    Science.gov (United States)

    Li, Congmin; Lim, Sunghyuk; Braunewell, Karl H; Ames, James B

    2016-01-01

    Visinin-like protein 3 (VILIP-3) belongs to a family of Ca2+-myristoyl switch proteins that regulate signal transduction in the brain and retina. Here we analyze Ca2+ binding, characterize Ca2+-induced conformational changes, and determine the NMR structure of myristoylated VILIP-3. Three Ca2+ bind cooperatively to VILIP-3 at EF2, EF3 and EF4 (KD = 0.52 μM and Hill slope of 1.8). NMR assignments, mutagenesis and structural analysis indicate that the covalently attached myristoyl group is solvent exposed in Ca2+-bound VILIP-3, whereas Ca2+-free VILIP-3 contains a sequestered myristoyl group that interacts with protein residues (E26, Y64, V68), which are distinct from myristate contacts seen in other Ca2+-myristoyl switch proteins. The myristoyl group in VILIP-3 forms an unusual L-shaped structure that places the C14 methyl group inside a shallow protein groove, in contrast to the much deeper myristoyl binding pockets observed for recoverin, NCS-1 and GCAP1. Thus, the myristoylated VILIP-3 protein structure determined in this study is quite different from those of other known myristoyl switch proteins (recoverin, NCS-1, and GCAP1). We propose that myristoylation serves to fine tune the three-dimensional structures of neuronal calcium sensor proteins as a means of generating functional diversity.

  11. Molecular Properties of Globin Channels and Pores: Role of Cholesterol in Ligand Binding and Movement

    Directory of Open Access Journals (Sweden)

    Gene A Morrill

    2016-09-01

    Full Text Available ABSTRACT: Globins contain one or more cavities that control or affect such functions as ligand movement and ligand binding. Here we report that the extended globin family [cytoglobin (Cygb; neuroglobin (Ngb; myoglobin (Mb; hemoglobin (Hb subunits Hba(α and Hbb(β] contain either a transmembrane (TM helix or pore-lining region as well as internal cavities. Protein motif/domain analyses indicate that Ngb and Hbb each contain 5 cholesterol-binding (CRAC/CARC domains and 1 caveolin binding motif, whereas the Cygb dimer has 6 cholesterol-binding domains but lacks caveolin-binding motifs. Mb and Hba each exhibit 2 cholesterol-binding domains and also lack caveolin-binding motifs. The Hb αβ-tetramer contains 14 cholesterol-binding domains. Computer algorithms indicate that Cygb and Ngb cavities display multiple partitions and C-terminal pore-lining regions, whereas Mb has three major cavities plus a C-terminal pore-lining region. The Hb tetramer exhibits a large internal cavity but the subunits differ in that they contain a C-terminal TM helix (Hba and pore-lining region (Hbb. The cavities include 43 of 190 Cygb residues, 38 of 151 of Ngb residues, 55 of 154 Mb residues and 137 of 688 residues in the Hb tetramer. Each cavity complex includes 6 to 8 residues of the TM helix or pore-lining region and CRAC/CARC domains exist within all cavities. Erythrocyte Hb αβ-tetramers are largely cytosolic but also bind to a membrane anion exchange protein, band 3, which contains a large internal cavity and 12 TM helices (5 being pore-lining regions. The Hba TM helix may be the erythrocyte membrane band 3 attachment site. Band 3 contributes 4 caveolin binding motifs and 10 CRAC/CARC domains. Cholesterol binding may create lipid-disordered phases that alter globin cavities and facilitate ligand movement, permitting ion channel formation and conformational changes that orchestrate anion and ligand (O2, CO2, NO movement within the large internal cavities and

  12. Integrating protein structures and precomputed genealogies in the Magnum database: Examples with cellular retinoid binding proteins

    Directory of Open Access Journals (Sweden)

    Bradley Michael E

    2006-02-01

    Full Text Available Abstract Background When accurate models for the divergent evolution of protein sequences are integrated with complementary biological information, such as folded protein structures, analyses of the combined data often lead to new hypotheses about molecular physiology. This represents an excellent example of how bioinformatics can be used to guide experimental research. However, progress in this direction has been slowed by the lack of a publicly available resource suitable for general use. Results The precomputed Magnum database offers a solution to this problem for ca. 1,800 full-length protein families with at least one crystal structure. The Magnum deliverables include 1 multiple sequence alignments, 2 mapping of alignment sites to crystal structure sites, 3 phylogenetic trees, 4 inferred ancestral sequences at internal tree nodes, and 5 amino acid replacements along tree branches. Comprehensive evaluations revealed that the automated procedures used to construct Magnum produced accurate models of how proteins divergently evolve, or genealogies, and correctly integrated these with the structural data. To demonstrate Magnum's capabilities, we asked for amino acid replacements requiring three nucleotide substitutions, located at internal protein structure sites, and occurring on short phylogenetic tree branches. In the cellular retinoid binding protein family a site that potentially modulates ligand binding affinity was discovered. Recruitment of cellular retinol binding protein to function as a lens crystallin in the diurnal gecko afforded another opportunity to showcase the predictive value of a browsable database containing branch replacement patterns integrated with protein structures. Conclusion We integrated two areas of protein science, evolution and structure, on a large scale and created a precomputed database, known as Magnum, which is the first freely available resource of its kind. Magnum provides evolutionary and structural

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

    Science.gov (United States)

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

    2015-01-01

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

  14. Sequence-based prediction of protein-binding sites in DNA: comparative study of two SVM models.

    Science.gov (United States)

    Park, Byungkyu; Im, Jinyong; Tuvshinjargal, Narankhuu; Lee, Wook; Han, Kyungsook

    2014-11-01

    As many structures of protein-DNA complexes have been known in the past years, several computational methods have been developed to predict DNA-binding sites in proteins. However, its inverse problem (i.e., predicting protein-binding sites in DNA) has received much less attention. One of the reasons is that the differences between the interaction propensities of nucleotides are much smaller than those between amino acids. Another reason is that DNA exhibits less diverse sequence patterns than protein. Therefore, predicting protein-binding DNA nucleotides is much harder than predicting DNA-binding amino acids. We computed the interaction propensity (IP) of nucleotide triplets with amino acids using an extensive dataset of protein-DNA complexes, and developed two support vector machine (SVM) models that predict protein-binding nucleotides from sequence data alone. One SVM model predicts protein-binding nucleotides using DNA sequence data alone, and the other SVM model predicts protein-binding nucleotides using both DNA and protein sequences. In a 10-fold cross-validation with 1519 DNA sequences, the SVM model that uses DNA sequence data only predicted protein-binding nucleotides with an accuracy of 67.0%, an F-measure of 67.1%, and a Matthews correlation coefficient (MCC) of 0.340. With an independent dataset of 181 DNAs that were not used in training, it achieved an accuracy of 66.2%, an F-measure 66.3% and a MCC of 0.324. Another SVM model that uses both DNA and protein sequences achieved an accuracy of 69.6%, an F-measure of 69.6%, and a MCC of 0.383 in a 10-fold cross-validation with 1519 DNA sequences and 859 protein sequences. With an independent dataset of 181 DNAs and 143 proteins, it showed an accuracy of 67.3%, an F-measure of 66.5% and a MCC of 0.329. Both in cross-validation and independent testing, the second SVM model that used both DNA and protein sequence data showed better performance than the first model that used DNA sequence data. To the best of

  15. Pharmacophore screening of the protein data bank for specific binding site chemistry.

    Science.gov (United States)

    Campagna-Slater, Valérie; Arrowsmith, Andrew G; Zhao, Yong; Schapira, Matthieu

    2010-03-22

    A simple computational approach was developed to screen the Protein Data Bank (PDB) for putative pockets possessing a specific binding site chemistry and geometry. The method employs two commonly used 3D screening technologies, namely identification of cavities in protein structures and pharmacophore screening of chemical libraries. For each protein structure, a pocket finding algorithm is used to extract potential binding sites containing the correct types of residues, which are then stored in a large SDF-formatted virtual library; pharmacophore filters describing the desired binding site chemistry and geometry are then applied to screen this virtual library and identify pockets matching the specified structural chemistry. As an example, this approach was used to screen all human protein structures in the PDB and identify sites having chemistry similar to that of known methyl-lysine binding domains that recognize chromatin methylation marks. The selected genes include known readers of the histone code as well as novel binding pockets that may be involved in epigenetic signaling. Putative allosteric sites were identified on the structures of TP53BP1, L3MBTL3, CHEK1, KDM4A, and CREBBP.

  16. Label-free electrochemical detection of singlet oxygen protein damage

    Czech Academy of Sciences Publication Activity Database

    Vargová, Veronika; Gimenez, R.E.; Černocká, Hana; Trujillo, D.C.; Tulli, F.; Zanini, V.I.P.; Paleček, Emil; Borsarelli, C.D.; Ostatná, Veronika

    2016-01-01

    Roč. 187, JAN 2016 (2016), s. 662-669 ISSN 0013-4686 R&D Projects: GA ČR GA13-00956S Institutional support: RVO:68081707 Keywords : singlet oxygen protein damage * surface-attached protein stability * mercury and carbon electrodes Subject RIV: BO - Biophysics Impact factor: 4.798, year: 2016

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-06-01

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

  18. Structure solution of DNA-binding proteins and complexes with ARCIMBOLDO libraries

    Energy Technology Data Exchange (ETDEWEB)

    Pröpper, Kevin [University of Göttingen, (Germany); Instituto de Biologia Molecular de Barcelona (IBMB-CSIC), (Spain); Meindl, Kathrin; Sammito, Massimo [Instituto de Biologia Molecular de Barcelona (IBMB-CSIC), (Spain); Dittrich, Birger; Sheldrick, George M. [University of Göttingen, (Germany); Pohl, Ehmke, E-mail: ehmke.pohl@durham.ac.uk [Durham University, (United Kingdom); Usón, Isabel, E-mail: ehmke.pohl@durham.ac.uk [Instituto de Biologia Molecular de Barcelona (IBMB-CSIC), (Spain); Institucio Catalana de Recerca i Estudis Avancats (ICREA), (Spain); University of Göttingen, (Germany)

    2014-06-01

    The structure solution of DNA-binding protein structures and complexes based on the combination of location of DNA-binding protein motif fragments with density modification in a multi-solution frame is described. Protein–DNA interactions play a major role in all aspects of genetic activity within an organism, such as transcription, packaging, rearrangement, replication and repair. The molecular detail of protein–DNA interactions can be best visualized through crystallography, and structures emphasizing insight into the principles of binding and base-sequence recognition are essential to understanding the subtleties of the underlying mechanisms. An increasing number of high-quality DNA-binding protein structure determinations have been witnessed despite the fact that the crystallographic particularities of nucleic acids tend to pose specific challenges to methods primarily developed for proteins. Crystallographic structure solution of protein–DNA complexes therefore remains a challenging area that is in need of optimized experimental and computational methods. The potential of the structure-solution program ARCIMBOLDO for the solution of protein–DNA complexes has therefore been assessed. The method is based on the combination of locating small, very accurate fragments using the program Phaser and density modification with the program SHELXE. Whereas for typical proteins main-chain α-helices provide the ideal, almost ubiquitous, small fragments to start searches, in the case of DNA complexes the binding motifs and DNA double helix constitute suitable search fragments. The aim of this work is to provide an effective library of search fragments as well as to determine the optimal ARCIMBOLDO strategy for the solution of this class of structures.

  19. Identification of pheromone components and their binding affinity to the odorant binding protein CcapOBP83a-2 of the Mediterranean fruit fly, Ceratitis capitata

    Czech Academy of Sciences Publication Activity Database

    Siciliano, P.; He, X. L.; Woodcock, C.; Pickett, J. A.; Field, L. M.; Birkett, M. A.; Kalinová, Blanka; Gomulski, L. M.; Scolari, F.; Gasperi, G.; Malacrida, A. R.; Zhou, J. J.

    2014-01-01

    Roč. 48, May (2014), s. 51-62 ISSN 0965-1748 Institutional support: RVO:61388963 Keywords : medfly * Ceratitis capitata * olfaction * odorant binding protein * pheromone binding protein * pheromone * binding studies * protein expression * electroantennography * GC-EAG * fluorescence displacement Subject RIV: CE - Biochemistry Impact factor: 3.450, year: 2014

  20. Myoglobin-Catalyzed Olefination of Aldehydes.

    Science.gov (United States)

    Tyagi, Vikas; Fasan, Rudi

    2016-02-12

    The olefination of aldehydes constitutes a most valuable and widely adopted strategy for constructing carbon-carbon double bonds in organic chemistry. While various synthetic methods have been made available for this purpose, no biocatalysts are known to mediate this transformation. Reported herein is that engineered myoglobin variants can catalyze the olefination of aldehydes in the presence of α-diazoesters with high catalytic efficiency (up to 4,900 turnovers) and excellent E diastereoselectivity (92-99.9 % de). This transformation could be applied to the olefination of a variety of substituted benzaldehydes and heteroaromatic aldehydes, also in combination with different alkyl α-diazoacetate reagents. This work provides a first example of biocatalytic aldehyde olefination and extends the spectrum of synthetically valuable chemical transformations accessible using metalloprotein-based catalysts. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. The clinical significance of fatty acid binding proteins

    Directory of Open Access Journals (Sweden)

    Barbara Choromańska

    2011-11-01

    Full Text Available Excessive levels of free fatty acids are toxic to cells. The human body has evolved a defense mechanism in the form of small cytoplasmic proteins called fatty acid binding proteins (FABPs that bind long-chain fatty acids (LCFA, and then refer them to appropriate intracellular disposal sites (oxidation in mitochondria and peroxisomes or storage in the endoplasmic reticulum. So far, nine types of these proteins have been described, and their name refers to the place in which they were first identified or where they can be found in the greatest concentration. The most important FABPs were isolated from the liver (L-FABP, heart (H-FABP, intestine (I-FABP, brain (B-FABP, epidermis (E-FABP and adipocytes (A-FABP. Determination of H-FABP is used in the diagnosis of myocardial infarction, and L-FABP in kidney lesions of different etiologies. It is postulated that FABPs play an important role in the pathogenesis of metabolic diseases. Elevated levels of A-FABP have been found in the pericardial fat tissue and were associated with cardiac dysfunction in obese people. A rise in A-FABP has been observed in patients with type II diabetes. I-FABP is known as a marker of cell damage in the small intestine. Increased concentration of B-FABP has been associated with human brain tumors such as glioblastoma and astrocytoma, as well as with neurodegenerative diseases (Alzheimer’s, Parkinson’s and other disorders of cognitive function. The aim of this work was to present current data on the clinical significance of fatty acid binding proteins.

  2. Species specificity for HBsAg binding protein endonexin II

    NARCIS (Netherlands)

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

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

  3. Contribution of Human Oral Cells to Astringency by Binding Salivary Protein/Tannin Complexes.

    Science.gov (United States)

    Soares, Susana; Ferrer-Galego, Raúl; Brandão, Elsa; Silva, Mafalda; Mateus, Nuno; Freitas, Victor de

    2016-10-10

    The most widely accepted mechanism to explain astringency is the interaction and precipitation of salivary proteins by food tannins, in particular proline-rich proteins. However, other mechanisms have been arising to explain astringency, such as binding of tannins to oral cells. In this work, an experimental method was adapted to study the possible contribution of both salivary proteins and oral cells to astringency induced by grape seed procyanidin fractions. Overall, in the absence of salivary proteins, the extent of procyanidin complexation with oral cells increased with increasing procyanidin degree of polymerization (mDP). Procyanidin fractions rich in monomers were the ones with the lowest ability to bind to oral cells. In the presence of salivary proteins and for procyanidins with mDP 2 the highest concentrations (1.5 and 2.0 mM) resulted in an increased binding of procyanidins to oral cells. This was even more evident for fractions III and IV at 1.0 mM and upper concentrations. Regarding the salivary proteins affected, it was possible to observe a decrease of P-B peptide and aPRP proteins for fractions II and III. This decrease is greater as the procyanidins' mDP increases. In fact, for fraction IV an almost total depletion of all salivary proteins was observed. This decrease is due to the formation of insoluble salivary protein/procyanidin complexes. Altogether, these data suggest that some procyanidins are able to bind to oral cells and that the salivary proteins interact with procyanidins forming salivary protein/procyanidin complexes that are also able to link to oral cells. The procyanidins that remain unbound to oral cells are able to bind to salivary proteins forming a large network of salivary protein/procyanidin complexes. Overall, the results presented herein provide one more step to understand food oral astringency onset.

  4. Peptide microarrays to probe for competition for binding sites in a protein interaction network

    NARCIS (Netherlands)

    Sinzinger, M.D.S.; Ruttekolk, I.R.R.; Gloerich, J.; Wessels, H.; Chung, Y.D.; Adjobo-Hermans, M.J.W.; Brock, R.E.

    2013-01-01

    Cellular protein interaction networks are a result of the binding preferences of a particular protein and the entirety of interactors that mutually compete for binding sites. Therefore, the reconstruction of interaction networks by the accumulation of interaction networks for individual proteins

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

    International Nuclear Information System (INIS)

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

    1979-01-01

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

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

    Science.gov (United States)

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

    2003-10-24

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

  7. Isolation of low-molecular-weight lead-binding protein from human erythrocytes

    International Nuclear Information System (INIS)

    Raghavan, S.R.V.; Gonick, H.C.

    1977-01-01

    In blood, lead is mainly associated with erythrocytes and only a very small amount is found in plasma. Previously it was thought that the lead was bound to the erythrocyte cell membrane but more recently it has been observed that lead is bound primarily to the cell contents, ostensibly hemoglobin. In examining the lead-binding properties of normal human erythrocytes and those of lead-exposed industrial workers, we have found that, whereas lead binds only to hemoglobin in normal erythrocytes, there is also appreciable binding of lead to a low-molecular weight-protein in erythrocytes from lead-exposed workers. The synthesis of this protein may be induced by lead exposure. The 10,000 molecular weight protein may act as a storage site and mechanism for segregating lead in a non-toxic form

  8. Space-related pharma-motifs for fast search of protein binding motifs and polypharmacological targets.

    Science.gov (United States)

    Chiu, Yi-Yuan; Lin, Chun-Yu; Lin, Chih-Ta; Hsu, Kai-Cheng; Chang, Li-Zen; Yang, Jinn-Moon

    2012-01-01

    To discover a compound inhibiting multiple proteins (i.e. polypharmacological targets) is a new paradigm for the complex diseases (e.g. cancers and diabetes). In general, the polypharmacological proteins often share similar local binding environments and motifs. As the exponential growth of the number of protein structures, to find the similar structural binding motifs (pharma-motifs) is an emergency task for drug discovery (e.g. side effects and new uses for old drugs) and protein functions. We have developed a Space-Related Pharmamotifs (called SRPmotif) method to recognize the binding motifs by searching against protein structure database. SRPmotif is able to recognize conserved binding environments containing spatially discontinuous pharma-motifs which are often short conserved peptides with specific physico-chemical properties for protein functions. Among 356 pharma-motifs, 56.5% interacting residues are highly conserved. Experimental results indicate that 81.1% and 92.7% polypharmacological targets of each protein-ligand complex are annotated with same biological process (BP) and molecular function (MF) terms, respectively, based on Gene Ontology (GO). Our experimental results show that the identified pharma-motifs often consist of key residues in functional (active) sites and play the key roles for protein functions. The SRPmotif is available at http://gemdock.life.nctu.edu.tw/SRP/. SRPmotif is able to identify similar pharma-interfaces and pharma-motifs sharing similar binding environments for polypharmacological targets by rapidly searching against the protein structure database. Pharma-motifs describe the conservations of binding environments for drug discovery and protein functions. Additionally, these pharma-motifs provide the clues for discovering new sequence-based motifs to predict protein functions from protein sequence databases. We believe that SRPmotif is useful for elucidating protein functions and drug discovery.

  9. How oxygen attacks [FeFe] hydrogenases from photosynthetic organisms

    Science.gov (United States)

    Stripp, Sven T.; Goldet, Gabrielle; Brandmayr, Caterina; Sanganas, Oliver; Vincent, Kylie A.; Haumann, Michael; Armstrong, Fraser A.; Happe, Thomas

    2009-01-01

    Green algae such as Chlamydomonas reinhardtii synthesize an [FeFe] hydrogenase that is highly active in hydrogen evolution. However, the extreme sensitivity of [FeFe] hydrogenases to oxygen presents a major challenge for exploiting these organisms to achieve sustainable photosynthetic hydrogen production. In this study, the mechanism of oxygen inactivation of the [FeFe] hydrogenase CrHydA1 from C. reinhardtii has been investigated. X-ray absorption spectroscopy shows that reaction with oxygen results in destruction of the [4Fe-4S] domain of the active site H-cluster while leaving the di-iron domain (2FeH) essentially intact. By protein film electrochemistry we were able to determine the order of events leading up to this destruction. Carbon monoxide, a competitive inhibitor of CrHydA1 which binds to an Fe atom of the 2FeH domain and is otherwise not known to attack FeS clusters in proteins, reacts nearly two orders of magnitude faster than oxygen and protects the enzyme against oxygen damage. These results therefore show that destruction of the [4Fe-4S] cluster is initiated by binding and reduction of oxygen at the di-iron domain—a key step that is blocked by carbon monoxide. The relatively slow attack by oxygen compared to carbon monoxide suggests that a very high level of discrimination can be achieved by subtle factors such as electronic effects (specific orbital overlap requirements) and steric constraints at the active site. PMID:19805068

  10. Mechanisms of zinc binding to the solute-binding protein AztC and transfer from the metallochaperone AztD.

    Science.gov (United States)

    Neupane, Durga P; Avalos, Dante; Fullam, Stephanie; Roychowdhury, Hridindu; Yukl, Erik T

    2017-10-20

    Bacteria can acquire the essential metal zinc from extremely zinc-limited environments by using ATP-binding cassette (ABC) transporters. These transporters are critical virulence factors, relying on specific and high-affinity binding of zinc by a periplasmic solute-binding protein (SBP). As such, the mechanisms of zinc binding and release among bacterial SBPs are of considerable interest as antibacterial drug targets. Zinc SBPs are characterized by a flexible loop near the high-affinity zinc-binding site. The function of this structure is not always clear, and its flexibility has thus far prevented structural characterization by X-ray crystallography. Here, we present intact structures for the zinc-specific SBP AztC from the bacterium Paracoccus denitrificans in the zinc-bound and apo-states. A comparison of these structures revealed that zinc loss prompts significant structural rearrangements, mediated by the formation of a sodium-binding site in the apo-structure. We further show that the AztC flexible loop has no impact on zinc-binding affinity, stoichiometry, or protein structure, yet is essential for zinc transfer from the metallochaperone AztD. We also found that 3 His residues in the loop appear to temporarily coordinate zinc and then convey it to the high-affinity binding site. Thus, mutation of any of these residues to Ala abrogated zinc transfer from AztD. Our structural and mechanistic findings conclusively identify a role for the AztC flexible loop in zinc acquisition from the metallochaperone AztD, yielding critical insights into metal binding by AztC from both solution and AztD. These proteins are highly conserved in human pathogens, making this work potentially useful for the development of novel antibiotics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Maltose-binding protein effectively stabilizes the partially closed conformation of the ATP-binding cassette transporter MalFGK2

    KAUST Repository

    Weng, Jingwei; Gu, Shuo; Gao, Xin; Huang, Xuhui; Wang, Wenning

    2017-01-01

    Maltose transporter MalFGK2 is a type-I importer in the ATP-binding cassette (ABC) transporter superfamily. Upon the binding of its periplasmic binding protein, MalE, the ATPase activity of MalFGK2 can be greatly enhanced. Crystal structures of the MalFGK2-MalE-maltose complex in a so-called

  12. Maltose-binding protein effectively stabilizes the partially closed conformation of the ATP-binding cassette transporter MalFGK2

    KAUST Repository

    Weng, Jingwei

    2017-02-23

    Maltose transporter MalFGK2 is a type-I importer in the ATP-binding cassette (ABC) transporter superfamily. Upon the binding of its periplasmic binding protein, MalE, the ATPase activity of MalFGK2 can be greatly enhanced. Crystal structures of the MalFGK2-MalE-maltose complex in a so-called

  13. Conserved RNA-Binding Proteins Required for Dendrite Morphogenesis in Caenorhabditis elegans Sensory Neurons

    Science.gov (United States)

    Antonacci, Simona; Forand, Daniel; Wolf, Margaret; Tyus, Courtney; Barney, Julia; Kellogg, Leah; Simon, Margo A.; Kerr, Genevieve; Wells, Kristen L.; Younes, Serena; Mortimer, Nathan T.; Olesnicky, Eugenia C.; Killian, Darrell J.

    2015-01-01

    The regulation of dendritic branching is critical for sensory reception, cell−cell communication within the nervous system, learning, memory, and behavior. Defects in dendrite morphology are associated with several neurologic disorders; thus, an understanding of the molecular mechanisms that govern dendrite morphogenesis is important. Recent investigations of dendrite morphogenesis have highlighted the importance of gene regulation at the posttranscriptional level. Because RNA-binding proteins mediate many posttranscriptional mechanisms, we decided to investigate the extent to which conserved RNA-binding proteins contribute to dendrite morphogenesis across phyla. Here we identify a core set of RNA-binding proteins that are important for dendrite morphogenesis in the PVD multidendritic sensory neuron in Caenorhabditis elegans. Homologs of each of these genes were previously identified as important in the Drosophila melanogaster dendritic arborization sensory neurons. Our results suggest that RNA processing, mRNA localization, mRNA stability, and translational control are all important mechanisms that contribute to dendrite morphogenesis, and we present a conserved set of RNA-binding proteins that regulate these processes in diverse animal species. Furthermore, homologs of these genes are expressed in the human brain, suggesting that these RNA-binding proteins are candidate regulators of dendrite development in humans. PMID:25673135

  14. Dsc E3 ligase localization to the Golgi requires the ATPase Cdc48 and cofactor Ufd1 for activation of sterol regulatory element-binding protein in fission yeast.

    Science.gov (United States)

    Burr, Risa; Ribbens, Diedre; Raychaudhuri, Sumana; Stewart, Emerson V; Ho, Jason; Espenshade, Peter J

    2017-09-29

    Sterol regulatory element-binding proteins (SREBPs) in the fission yeast Schizosaccharomyces pombe regulate lipid homeostasis and the hypoxic response under conditions of low sterol or oxygen availability. SREBPs are cleaved in the Golgi through the combined action of the Dsc E3 ligase complex, the rhomboid protease Rbd2, and the essential ATPases associated with diverse cellular activities (AAA + ) ATPase Cdc48. The soluble SREBP N-terminal transcription factor domain is then released into the cytosol to enter the nucleus and regulate gene expression. Previously, we reported that Cdc48 binding to Rbd2 is required for Rbd2-mediated SREBP cleavage. Here, using affinity chromatography and mass spectrometry experiments, we identified Cdc48-binding proteins in S. pombe , generating a list of many previously unknown potential Cdc48-binding partners. We show that the established Cdc48 cofactor Ufd1 is required for SREBP cleavage but does not interact with the Cdc48-Rbd2 complex. Cdc48-Ufd1 is instead required at a step prior to Rbd2 function, during Golgi localization of the Dsc E3 ligase complex. Together, these findings demonstrate that two distinct Cdc48 complexes, Cdc48-Ufd1 and Cdc48-Rbd2, are required for SREBP activation and low-oxygen adaptation in S. pombe . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Effect of fullerenol surface chemistry on nanoparticle binding-induced protein misfolding

    Science.gov (United States)

    Radic, Slaven; Nedumpully-Govindan, Praveen; Chen, Ran; Salonen, Emppu; Brown, Jared M.; Ke, Pu Chun; Ding, Feng

    2014-06-01

    Fullerene and its derivatives with different surface chemistry have great potential in biomedical applications. Accordingly, it is important to delineate the impact of these carbon-based nanoparticles on protein structure, dynamics, and subsequently function. Here, we focused on the effect of hydroxylation -- a common strategy for solubilizing and functionalizing fullerene -- on protein-nanoparticle interactions using a model protein, ubiquitin. We applied a set of complementary computational modeling methods, including docking and molecular dynamics simulations with both explicit and implicit solvent, to illustrate the impact of hydroxylated fullerenes on the structure and dynamics of ubiquitin. We found that all derivatives bound to the model protein. Specifically, the more hydrophilic nanoparticles with a higher number of hydroxyl groups bound to the surface of the protein via hydrogen bonds, which stabilized the protein without inducing large conformational changes in the protein structure. In contrast, fullerene derivatives with a smaller number of hydroxyl groups buried their hydrophobic surface inside the protein, thereby causing protein denaturation. Overall, our results revealed a distinct role of surface chemistry on nanoparticle-protein binding and binding-induced protein misfolding.Fullerene and its derivatives with different surface chemistry have great potential in biomedical applications. Accordingly, it is important to delineate the impact of these carbon-based nanoparticles on protein structure, dynamics, and subsequently function. Here, we focused on the effect of hydroxylation -- a common strategy for solubilizing and functionalizing fullerene -- on protein-nanoparticle interactions using a model protein, ubiquitin. We applied a set of complementary computational modeling methods, including docking and molecular dynamics simulations with both explicit and implicit solvent, to illustrate the impact of hydroxylated fullerenes on the structure and

  16. Recombinant fusion protein of albumin-retinol binding protein inactivates stellate cells

    International Nuclear Information System (INIS)

    Choi, Soyoung; Park, Sangeun; Kim, Suhyun; Lim, Chaeseung; Kim, Jungho; Cha, Dae Ryong; Oh, Junseo

    2012-01-01

    Highlights: ► We designed novel recombinant albumin-RBP fusion proteins. ► Expression of fusion proteins inactivates pancreatic stellate cells (PSCs). ► Fusion proteins are successfully internalized into and inactivate PSCs. ► RBP moiety mediates cell specific uptake of fusion protein. -- Abstract: Quiescent pancreatic- (PSCs) and hepatic- (HSCs) stellate cells store vitamin A (retinol) in lipid droplets via retinol binding protein (RBP) receptor and, when activated by profibrogenic stimuli, they transform into myofibroblast-like cells which play a key role in the fibrogenesis. Despite extensive investigations, there is, however, currently no appropriate therapy available for tissue fibrosis. We previously showed that the expression of albumin, composed of three homologous domains (I–III), inhibits stellate cell activation, which requires its high-affinity fatty acid-binding sites asymmetrically distributed in domain I and III. To attain stellate cell-specific uptake, albumin (domain I/III) was coupled to RBP; RBP-albumin domain III (R-III) and albumin domain I -RBP-albumin III (I-R-III). To assess the biological activity of fusion proteins, cultured PSCs were used. Like wild type albumin, expression of R-III or I-R-III in PSCs after passage 2 (activated PSCs) induced phenotypic reversal from activated to fat-storing cells. On the other hand, R-III and I-R-III, but not albumin, secreted from transfected 293 cells were successfully internalized into and inactivated PSCs. FPLC-purified R-III was found to be internalized into PSCs via caveolae-mediated endocytosis, and its efficient cellular uptake was also observed in HSCs and podocytes among several cell lines tested. Moreover, tissue distribution of intravenously injected R-III was closely similar to that of RBP. Therefore, our data suggest that albumin-RBP fusion protein comprises of stellate cell inactivation-inducing moiety and targeting moiety, which may lead to the development of effective anti

  17. Binding proteins of somatomedins and their functions

    International Nuclear Information System (INIS)

    Kostelecka, Z.; Blahovec, J.

    1998-01-01

    In this paper the functions of binding proteins are discussed. One variable that provides insulin-like growth factors (IGFs) control at the extracellular level is the presence of high-affinity, soluble insulin-like growth factor proteins (IGFBPs). IGFBP-1 inhibits IGF effect on human osteosarcoma cells. Increased concentration of IGFBP-3 inhibits the proliferation of breast cancer cell line MCF 7 either directly or by competition for IGF receptors. Maybe IGFBPs work as anti-mitogens and IGFs are potential promotors of cancer growth

  18. Ligand Binding Domain Protein in Tetracycline-Inducible Expression ...

    African Journals Online (AJOL)

    binding domain proteins in E. coli using a tetracycline inducible system. To allow for ... development of molecular ligands with improved therapeutic windows. Keywords: Nuclear receptor ..... functional recombinant cannabinoid receptor CB2 in ...

  19. Oxygen binding properties, capillary densities and heart weights in high altitude camelids.

    Science.gov (United States)

    Jürgens, K D; Pietschmann, M; Yamaguchi, K; Kleinschmidt, T

    1988-01-01

    The oxygen binding properties of the blood of the camelid species vicuna, llama, alpaca and dromedary camel were measured and evaluated with respect to interspecific differences. The highest blood oxygen affinity, not only among camelids but of all mammals investigated so far, was found in the vicuna (P50 = 17.6 Torr compared to 20.3-21.6 Torr in the other species). Low hematocrits (23-34%) and small red blood cells (21-30 microns 3) are common features of all camelids, but the lowest values are found in the Lama species. Capillary densities were determined in heart and soleus muscle of vicuna and llama. Again, the vicuna shows exceptional values (3720 cap/mm2 on average in the heart) for a mammal of this body size. Finally, heart weight as percent of body weight is higher in the vicuna (0.7-0.9%) than in the other camelids studied (0.5-0.7%). The possibility that these parameters, measured in New World tylopodes at sea level, are not likely to change considerably with transfer to high altitude, is discussed. In the vicuna, a unique combination of the following features seems to be responsible for an outstanding physical capability at high altitude: saturation of blood with oxygen in the lung is favored by a high blood oxygen affinity, oxygen supply being facilitated by low diffusion distances in the muscle tissue. Loading, as well as unloading, of oxygen is improved by a relatively high oxygen transfer conductance of the red blood cells, which is due to their small size and which compensates the negative effect of a low hematocrit on the oxygen conductance of blood.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. Activity of cAMP-dependent protein kinases and cAMP-binding proteins of rat kidney cytosol during dehydration

    International Nuclear Information System (INIS)

    Zelenina, M.N.; Solenov, E.I.; Ivanova, L.N.

    1985-01-01

    The activity of cAMP-dependent protein kinases, the binding of cAMP, and the spectrum of cAMP-binding proteins in the cytosol of the renal papilla was studied in intact rats and in rats after 24 h on a water-deprived diet. It was found that the activation of protein kinases by 10 -6 M cAMP is significantly higher in the experimental animals than in the intact animals. In chromatography on DEAE-cellulose, the positions of the peaks of specific reception of cAMP corresponded to the peaks of the regulatory subunits of cAMP-dependent protein kinases of types I and II. In this case, in intact animals more than 80% of the binding activity was detected in peaks II, whereas in rats subjected to water deprivation, more than 60% of the binding was observed in peak I. The general regulatory activity of the cytosol was unchanged in the experimental animals in comparison with intact animals. It is suggested that during dehydration there is an induction of the synthesis of the regulatory subunit of type I cAMP-dependent protein kinase in the renal papilla