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Sample records for 9-mer-based protein binding

  1. Cellulose binding domain proteins

    SciTech Connect

    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.

  2. Cellulose binding domain proteins

    DOEpatents

    Shoseyov, O.; Shpiegl, I.; Goldstein, M.; Doi, R.

    1998-11-17

    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. 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. 16 figs.

  3. The prion protein binds thiamine.

    PubMed

    Perez-Pineiro, Rolando; Bjorndahl, Trent C; Berjanskii, Mark V; Hau, David; Li, Li; Huang, Alan; Lee, Rose; Gibbs, Ebrima; Ladner, Carol; Dong, Ying Wei; Abera, Ashenafi; Cashman, Neil R; Wishart, David S

    2011-11-01

    Although highly conserved throughout evolution, the exact biological function of the prion protein is still unclear. In an effort to identify the potential biological functions of the prion protein we conducted a small-molecule screening assay using the Syrian hamster prion protein [shPrP(90-232)]. The screen was performed using a library of 149 water-soluble metabolites that are known to pass through the blood-brain barrier. Using a combination of 1D NMR, fluorescence quenching and surface plasmon resonance we identified thiamine (vitamin B1) as a specific prion ligand with a binding constant of ~60 μM. Subsequent studies showed that this interaction is evolutionarily conserved, with similar binding constants being seen for mouse, hamster and human prions. Various protein construct lengths, both with and without the unstructured N-terminal region in the presence and absence of copper, were examined. This indicates that the N-terminus has no influence on the protein's ability to interact with thiamine. In addition to thiamine, the more biologically abundant forms of vitamin B1 (thiamine monophosphate and thiamine diphosphate) were also found to bind the prion protein with similar affinity. Heteronuclear NMR experiments were used to determine thiamine's interaction site, which is located between helix 1 and the preceding loop. These data, in conjunction with computer-aided docking and molecular dynamics, were used to model the thiamine-binding pharmacophore and a comparison with other thiamine binding proteins was performed to reveal the common features of interaction.

  4. Cellulose binding domain fusion proteins

    SciTech Connect

    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.

  5. Cellulose binding domain fusion proteins

    DOEpatents

    Shoseyov, O.; Yosef, K.; Shpiegl, I.; Goldstein, M.A.; Doi, R.H.

    1998-02-17

    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. 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. 16 figs.

  6. Protein binding assay for hyaluronate

    SciTech Connect

    Lacy, B.E.; Underhill, C.B.

    1986-11-01

    A relatively quick and simple assay for hyaluronate was developed using the specific binding protein, hyaluronectin. The hyaluronectin was obtained by homogenizing the brains of Sprague-Dawley rats, and then centrifuging the homogenate. The resulting supernatant was used as a source of crude hyaluronectin. In the binding assay, the hyaluronectin was mixed with (/sup 3/H)hyaluronate, followed by an equal volume of saturated (NH/sub 4/)/sub 2/SO/sub 4/, which precipitated the hyaluronectin and any (/sup 3/H)hyaluronate associated with it, but left free (/sup 3/H)hyaluronate in solution. The mixture was then centrifuged, and the amount of bound (/sup 3/H)hyaluronate in the precipitate was determined. Using this assay, the authors found that hyaluronectin specifically bound hyaluronate, since other glycosaminoglycans failed to compete for the binding protein. In addition, the interaction between hyaluronectin and hyaluronate was of relatively high affinity, and the size of the hyaluronate did not appear to substantially alter the amount of binding. To determine the amount of hyaluronate in an unknown sample, they used a competition assay in which the binding of a set amount of (/sup 3/H)hyaluronate was blocked by the addition of unlabeled hyaluronate. By comparing the degree of competition of the unknown samples with that of known amounts of hyaluronate, it was possible to determine the amount of hyaluronate in the unknowns. They have found that this method is sensitive to 1 ..mu..g or less of hyaluronate, and is unaffected by the presence of proteins.

  7. Data of protein-RNA binding sites.

    PubMed

    Lee, Wook; Park, Byungkyu; Choi, Daesik; Han, Kyungsook

    2017-02-01

    Despite the increasing number of protein-RNA complexes in structure databases, few data resources have been made available which can be readily used in developing or testing a method for predicting either protein-binding sites in RNA sequences or RNA-binding sites in protein sequences. The problem of predicting protein-binding sites in RNA has received much less attention than the problem of predicting RNA-binding sites in protein. The data presented in this paper are related to the article entitled "PRIdictor: Protein-RNA Interaction predictor" (Tuvshinjargal et al. 2016) [1]. PRIdictor can predict protein-binding sites in RNA as well as RNA-binding sites in protein at the nucleotide- and residue-levels. This paper presents four datasets that were used to test four prediction models of PRIdictor: (1) model RP for predicting protein-binding sites in RNA from protein and RNA sequences, (2) model RaP for predicting protein-binding sites in RNA from RNA sequence alone, (3) model PR for predicting RNA-binding sites in protein from protein and RNA sequences, and (4) model PaR for predicting RNA-binding sites in protein from protein sequence alone. The datasets supplied in this article can be used as a valuable resource to evaluate and compare different methods for predicting protein-RNA binding sites.

  8. Erythropoietin binding protein from mammalian serum

    DOEpatents

    Clemons, G.K.

    1997-04-29

    Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described. 11 figs.

  9. Erythropoietin binding protein from mammalian serum

    DOEpatents

    Clemons, Gisela K.

    1997-01-01

    Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described.

  10. Engineering RNA-binding proteins for biology.

    PubMed

    Chen, Yu; Varani, Gabriele

    2013-08-01

    RNA-binding proteins play essential roles in the regulation of gene expression. Many have modular structures and combine relatively few common domains in various arrangements to recognize RNA sequences and/or structures. Recent progress in engineering the specificity of the PUF class RNA-binding proteins has shown that RNA-binding domains may be combined with various effector or functional domains to regulate the metabolism of targeted RNAs. Designer RNA-binding proteins with tailored sequence specificity will provide valuable tools for biochemical research as well as potential therapeutic applications. In this review, we discuss the suitability of various RNA-binding domains for engineering RNA-binding specificity, based on the structural basis for their recognition. We also compare various protein engineering and design methods applied to RNA-binding proteins, and discuss future applications of these proteins.

  11. Calcium-binding proteins and development

    NASA Technical Reports Server (NTRS)

    Beckingham, K.; Lu, A. Q.; Andruss, B. F.; McIntire, L. V. (Principal Investigator)

    1998-01-01

    The known roles for calcium-binding proteins in developmental signaling pathways are reviewed. Current information on the calcium-binding characteristics of three classes of cell-surface developmental signaling proteins (EGF-domain proteins, cadherins and integrins) is presented together with an overview of the intracellular pathways downstream of these surface receptors. The developmental roles delineated to date for the universal intracellular calcium sensor, calmodulin, and its targets, and for calcium-binding regulators of the cytoskeleton are also reviewed.

  12. Monobodies and other synthetic binding proteins for expanding protein science.

    PubMed

    Sha, Fern; Salzman, Gabriel; Gupta, Ankit; Koide, Shohei

    2017-03-01

    Synthetic binding proteins are constructed using nonantibody molecular scaffolds. Over the last two decades, in-depth structural and functional analyses of synthetic binding proteins have improved combinatorial library designs and selection strategies, which have resulted in potent platforms that consistently generate binding proteins to diverse targets with affinity and specificity that rival those of antibodies. Favorable attributes of synthetic binding proteins, such as small size, freedom from disulfide bond formation and ease of making fusion proteins, have enabled their unique applications in protein science, cell biology and beyond. Here, we review recent studies that illustrate how synthetic binding proteins are powerful probes that can directly link structure and function, often leading to new mechanistic insights. We propose that synthetic proteins will become powerful standard tools in diverse areas of protein science, biotechnology and medicine.

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

    PubMed Central

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

    2015-01-01

    Proteins and RNA interaction have vital roles in many cellular processes such as protein synthesis, sequence encoding, RNA transfer, and gene regulation at the transcriptional and post-transcriptional levels. Approximately 6%–8% of all proteins are RNA-binding proteins (RBPs). Distinguishing these RBPs or their binding residues is a major aim of structural biology. Previously, a number of experimental methods were developed for the determination of protein–RNA interactions. However, these experimental methods are expensive, time-consuming, and labor-intensive. Alternatively, researchers have developed many computational approaches to predict RBPs and protein–RNA binding sites, by combining various machine learning methods and abundant sequence and/or structural features. There are three kinds of computational approaches, which are prediction from protein sequence, prediction from protein structure, and protein-RNA docking. In this paper, we review all existing studies of predictions of RNA-binding sites and RBPs and complexes, including data sets used in different approaches, sequence and structural features used in several predictors, prediction method classifications, performance comparisons, evaluation methods, and future directions. PMID:26540053

  14. Structure and Function of Lipopolysaccharide Binding Protein

    NASA Astrophysics Data System (ADS)

    Schumann, Ralf R.; Leong, Steven R.; Flaggs, Gail W.; Gray, Patrick W.; Wright, Samuel D.; Mathison, John C.; Tobias, Peter S.; Ulevitch, Richard J.

    1990-09-01

    The primary structure of lipopolysaccharide binding protein (LBP), a trace plasma protein that binds to the lipid A moiety of bacterial lipopolysaccharides (LPSs), was deduced by sequencing cloned complementary DNA. LBP shares sequence identity with another LPS binding protein found in granulocytes, bactericidal/permeability-increasing protein, and with cholesterol ester transport protein of the plasma. LBP may control the response to LPS under physiologic conditions by forming high-affinity complexes with LPS that bind to monocytes and macrophages, which then secrete tumor necrosis factor. The identification of this pathway for LPS-induced monocyte stimulation may aid in the development of treatments for diseases in which Gram-negative sepsis or endotoxemia are involved.

  15. SVOP Is a Nucleotide Binding Protein

    PubMed Central

    Yao, Jia; Bajjalieh, Sandra M.

    2009-01-01

    Background Synaptic Vesicle Protein 2 (SV2) and SV2-related protein (SVOP) are transporter-like proteins that localize to neurotransmitter-containing vesicles. Both proteins share structural similarity with the major facilitator (MF) family of small molecule transporters. We recently reported that SV2 binds nucleotides, a feature that has also been reported for another MF family member, the human glucose transporter 1 (Glut1). In the case of Glut1, nucleotide binding affects transport activity. In this study, we determined if SVOP also binds nucleotides and assessed its nucleotide binding properties. Methodology/Principal Findings We performed in vitro photoaffinity labeling experiments with the photoreactive ATP analogue, 8-azido-ATP[γ] biotin and purified recombinant SVOP-FLAG fusion protein. We found that SVOP is a nucleotide-binding protein, although both its substrate specificity and binding site differ from that of SV2. Within the nucleotides tested, ATP, GTP and NAD show same level of inhibition on SVOP-FLAG labeling. Dose dependent studies indicated that SVOP demonstrates the highest affinity for NAD, in contrast to SV2, which binds both NAD and ATP with equal affinity. Mapping of the binding site revealed a single region spanning transmembrane domains 9–12, which contrasts to the two binding sites in the large cytoplasmic domains in SV2A. Conclusions/Significance SVOP is the third MF family member to be found to bind nucleotides. Given that the binding sites are unique in SVOP, SV2 and Glut1, this feature appears to have arisen separately. PMID:19390693

  16. Surface-Based Protein Binding Pocket Similarity

    PubMed Central

    Spitzer, Russell; Cleves, Ann E.; Jain, Ajay N.

    2011-01-01

    Protein similarity comparisons may be made on a local or global basis and may consider sequence information or differing levels of structural information. We present a local 3D method that compares protein binding site surfaces in full atomic detail. The approach is based on the morphological similarity method which has been widely applied for global comparison of small molecules. We apply the method to all-by-all comparisons two sets of human protein kinases, a very diverse set of ATP-bound proteins from multiple species, and three heterogeneous benchmark protein binding site data sets. Cases of disagreement between sequence-based similarity and binding site similarity yield informative examples. Where sequence similarity is very low, high pocket similarity can reliably identify important binding motifs. Where sequence similarity is very high, significant differences in pocket similarity are related to ligand binding specificity and similarity. Local protein binding pocket similarity provides qualitatively complementary information to other approaches, and it can yield quantitative information in support of functional annotation. PMID:21769944

  17. Lipid binding proteins from parasitic platyhelminthes.

    PubMed

    Alvite, Gabriela; Esteves, Adriana

    2012-01-01

    TWO MAIN FAMILIES OF LIPID BINDING PROTEINS HAVE BEEN IDENTIFIED IN PARASITIC PLATYHELMINTHES: hydrophobic ligand binding proteins (HLBPs) and fatty acid binding proteins (FABPs). Members of the former family of proteins are specific to the Cestoda class, while FABPs are conserved across a wide range of animal species. Because Platyhelminthes are unable to synthesize their own lipids, these lipid-binding proteins are important molecules in these organisms. HLBPs are a high molecular mass complex of proteins and lipids. They are composed of subunits of low molecular mass proteins and a wide array of lipid molecules ranging from CoA esters to cholesterol. These proteins are excretory-secretory molecules and are key serological tools for diagnosis of diseases caused by cestodes. FABPs are mainly intracellular proteins of low molecular weight. They are also vaccine candidates. Despite that the knowledge of their function is scarce, the differences in their molecular organization, ligand preferences, intra/extracellular localization, evolution, and phylogenetic distribution, suggest that platyhelminths HLBPs and FABPs should play different functions. FABPs might be involved in the removal of fatty acids from the inner surface of the cell membrane and in their subsequent targeting to specific cellular destinations. In contrast, HLBPs might be involved in fatty acid uptake from the host environment.

  18. The binding domain structure of retinoblastoma-binding proteins.

    PubMed Central

    Figge, J.; Breese, K.; Vajda, S.; Zhu, Q. L.; Eisele, L.; Andersen, T. T.; MacColl, R.; Friedrich, T.; Smith, T. F.

    1993-01-01

    The retinoblastoma gene product (Rb), a cellular growth suppressor, complexes with viral and cellular proteins that contain a specific binding domain incorporating three invariant residues: Leu-X-Cys-X-Glu, where X denotes a nonconserved residue. Hydrophobic and electrostatic properties are strongly conserved in this segment even though the nonconserved amino acids vary considerably from one Rb-binding protein to another. In this report, we present a diagnostic computer pattern for a high-affinity Rb-binding domain featuring the three conserved residues as well as the conserved physico-chemical properties. Although the pattern encompasses only 10 residues (with only 4 of these explicitly defined), it exhibits 100% sensitivity and 99.95% specificity in database searches. This implies that a certain pattern of structural and physico-chemical properties encoded by this short sequence is sufficient to govern specific Rb binding. We also present evidence that the secondary structural conformation through this region is important for effective Rb binding. PMID:8382993

  19. Folding funnels, binding funnels, and protein function.

    PubMed Central

    Tsai, C. J.; Kumar, S.; Ma, B.; Nussinov, R.

    1999-01-01

    Folding funnels have been the focus of considerable attention during the last few years. These have mostly been discussed in the general context of the theory of protein folding. Here we extend the utility of the concept of folding funnels, relating them to biological mechanisms and function. In particular, here we describe the shape of the funnels in light of protein synthesis and folding; flexibility, conformational diversity, and binding mechanisms; and the associated binding funnels, illustrating the multiple routes and the range of complexed conformers. Specifically, the walls of the folding funnels, their crevices, and bumps are related to the complexity of protein folding, and hence to sequential vs. nonsequential folding. Whereas the former is more frequently observed in eukaryotic proteins, where the rate of protein synthesis is slower, the latter is more frequent in prokaryotes, with faster translation rates. The bottoms of the funnels reflect the extent of the flexibility of the proteins. Rugged floors imply a range of conformational isomers, which may be close on the energy landscape. Rather than undergoing an induced fit binding mechanism, the conformational ensembles around the rugged bottoms argue that the conformers, which are most complementary to the ligand, will bind to it with the equilibrium shifting in their favor. Furthermore, depending on the extent of the ruggedness, or of the smoothness with only a few minima, we may infer nonspecific, broad range vs. specific binding. In particular, folding and binding are similar processes, with similar underlying principles. Hence, the shape of the folding funnel of the monomer enables making reasonable guesses regarding the shape of the corresponding binding funnel. Proteins having a broad range of binding, such as proteolytic enzymes or relatively nonspecific endonucleases, may be expected to have not only rugged floors in their folding funnels, but their binding funnels will also behave similarly

  20. Aspects of Protein, Chemistry, Part II: Oxygen-Binding Proteins

    ERIC Educational Resources Information Center

    Nixon, J. E.

    1977-01-01

    Compares differences in function and behavior of two oxygen-binding proteins, myoglobin found in muscle and hemoglobin found in blood. Describes the mechanism of oxygen-binding and allosteric effect in hemoglobin; also describes the effect of pH on the affinity of hemoglobin for oxygen. (CS)

  1. Computational analysis of maltose binding protein translocation

    NASA Astrophysics Data System (ADS)

    Chinappi, Mauro; Cecconi, Fabio; Massimo Casciola, Carlo

    2011-05-01

    We propose a computational model for the study of maltose binding protein translocation across α-hemolysin nanopores. The phenomenological approach simplifies both the pore and the polypeptide chain; however it retains the basic structural protein-like properties of the maltose binding protein by promoting the correct formation of its native key interactions. By considering different observables characterising the channel blockade and molecule transport, we verified that MD simulations reproduce qualitatively the behaviour observed in a recent experiment. Simulations reveal that blockade events consist of a capture stage, to some extent related to the unfolding kinetics, and a single file translocation process in the channel. A threshold mechanics underlies the process activation with a critical force depending on the protein denaturation state. Finally, our results support the simple interpretation of translocation via first-passage statistics of a driven diffusion process of a single reaction coordinate.

  2. ABP: a novel AMPA receptor binding protein.

    PubMed

    Srivastava, S; Ziff, E B

    1999-04-30

    We review the cloning of a novel AMPA receptor binding protein (ABP) that interacts with GluR2/3 and is homologous to GRIP. ABP is enriched in the PSD with GluR2 and is localized to the PSD by EM. ABP binds GluR2 via the C-terminal VXI motif through a Class I PDZ interaction. ABP and GRIP can also homo- and heteromultimerize. Thus, ABP and GRIP may be involved in AMPA receptor regulation and localization, by linking it to other cytoskeletal or signaling molecules. We suggest that the ABP/GRIP and PSD-95 families form distinct scaffolds that anchor, respectively, AMPA and NMDA receptors. We are currently investigating proteins that bind ABP and that may regulate the AMPA receptor.

  3. Exploring the binding dynamics of BAR proteins.

    PubMed

    Kabaso, Doron; Gongadze, Ekaterina; Jorgačevski, Jernej; Kreft, Marko; Van Rienen, Ursula; Zorec, Robert; Iglič, Aleš

    2011-09-01

    We used a continuum model based on the Helfrich free energy to investigate the binding dynamics of a lipid bilayer to a BAR domain surface of a crescent-like shape of positive (e.g. I-BAR shape) or negative (e.g. F-BAR shape) intrinsic curvature. According to structural data, it has been suggested that negatively charged membrane lipids are bound to positively charged amino acids at the binding interface of BAR proteins, contributing a negative binding energy to the system free energy. In addition, the cone-like shape of negatively charged lipids on the inner side of a cell membrane might contribute a positive intrinsic curvature, facilitating the initial bending towards the crescent-like shape of the BAR domain. In the present study, we hypothesize that in the limit of a rigid BAR domain shape, the negative binding energy and the coupling between the intrinsic curvature of negatively charged lipids and the membrane curvature drive the bending of the membrane. To estimate the binding energy, the electric potential at the charged surface of a BAR domain was calculated using the Langevin-Bikerman equation. Results of numerical simulations reveal that the binding energy is important for the initial instability (i.e. bending of a membrane), while the coupling between the intrinsic shapes of lipids and membrane curvature could be crucial for the curvature-dependent aggregation of negatively charged lipids near the surface of the BAR domain. In the discussion, we suggest novel experiments using patch clamp techniques to analyze the binding dynamics of BAR proteins, as well as the possible role of BAR proteins in the fusion pore stability of exovesicles.

  4. Quantifying drug-protein binding in vivo.

    SciTech Connect

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

    2004-02-17

    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.

  5. Phylointeractomics reconstructs functional evolution of protein binding

    PubMed Central

    Kappei, Dennis; Scheibe, Marion; Paszkowski-Rogacz, Maciej; Bluhm, Alina; Gossmann, Toni Ingolf; Dietz, Sabrina; Dejung, Mario; Herlyn, Holger; Buchholz, Frank; Mann, Matthias; Butter, Falk

    2017-01-01

    Molecular phylogenomics investigates evolutionary relationships based on genomic data. However, despite genomic sequence conservation, changes in protein interactions can occur relatively rapidly and may cause strong functional diversification. To investigate such functional evolution, we here combine phylogenomics with interaction proteomics. We develop this concept by investigating the molecular evolution of the shelterin complex, which protects telomeres, across 16 vertebrate species from zebrafish to humans covering 450 million years of evolution. Our phylointeractomics screen discovers previously unknown telomere-associated proteins and reveals how homologous proteins undergo functional evolution. For instance, we show that TERF1 evolved as a telomere-binding protein in the common stem lineage of marsupial and placental mammals. Phylointeractomics is a versatile and scalable approach to investigate evolutionary changes in protein function and thus can provide experimental evidence for phylogenomic relationships. PMID:28176777

  6. Gene encoding herbicide safener binding protein

    SciTech Connect

    Walton, J.D.; Scott-Craig, J.S.

    1999-10-26

    The cDNA encoding safener binding protein (SafBP), also referred to as SBP1, is presented. The deduced amino acid sequence is provided. Methods of making and using SBP1 and SafBP to alter a plant's sensitivity to certain herbicides or a plant's responsiveness to certain safeners are also provided, as well as expression vectors, transgenic plants or other organisms transfected with vectors and seeds from the plants.

  7. Polynucleotides encoding TRF1 binding proteins

    DOEpatents

    Campisi, Judith; Kim, Sahn-Ho

    2002-01-01

    The present invention provides a novel telomere associated protein (Trf1-interacting nuclear protein 2 "Tin2") that hinders the binding of Trf1 to its specific telomere repeat sequence and mediates the formation of a Tin2-Trf1-telomeric DNA complex that limits telomerase access to the telomere. Also included are the corresponding nucleic acids that encode the Tin2 of the present invention, as well as mutants of Tin2. Methods of making, purifying and using Tin2 of the present invention are described. In addition, drug screening assays to identify drugs that mimic and/or complement the effect of Tin2 are presented.

  8. Novel retinoid-binding proteins from filarial parasites.

    PubMed Central

    Sani, B P; Vaid, A; Comley, J C; Montgomery, J A

    1985-01-01

    The present study deals with the discovery and partial characterization of specific binding proteins for retinol and retinoic acid from filarial parasites (worms of the superfamily Filarioidea), including those from two species of Onchocerca. These binding proteins, which are distinct in their physicochemical properties and in the mode of ligand interactions from the host-tissue retinoid-binding proteins, may be involved in the mediation of the putative biological roles of retinoids in the control of parasitic growth, differentiation and reproduction. Parasite retinol-binding protein and retinoic acid-binding protein exhibited specificity for binding retinol and retinoic acid respectively. Both the binding proteins showed an s20,w value of 2.0 S. On gel filtration, both proteins were retarded to a position corresponding to the same molecular size (19.0 kDa). On preparative columns, the parasite binding proteins exhibited isoelectric points at pH 5.7 and 5.75. Unlike the retinoid-binding proteins of mammalian and avian origin, the parasite retinoid-binding proteins showed a lack of mercurial sensitivity in ligand binding. The comparative amounts of retinoic acid-binding protein in five parasites, Onchocerca volvulus, Onchocerca gibsoni, Dipetalonema viteae, Brugia pahangi and Dirofilaria immitis, were between 2.7 and 3.1 pmol of retinoic acid bound/mg of extractable protein. However, the levels of parasite retinol-binding protein were between 4.8 and 5.8 pmol/mg, which is considerably higher than the corresponding levels of cellular retinol-binding protein of mammalian and avian origin. Both retinol- and retinoic acid-binding-protein levels in O. volvulus-infected human nodules and O. gibsoni-infected bovine nodules were similar to their levels in mammalian tissues. Also, these nodular binding proteins, like the host-binding proteins, exhibited mercurial sensitivity to ligand interactions. PMID:3004410

  9. Binding of transition metals to S100 proteins

    PubMed Central

    Gilston, Benjamin A.; Skaar, Eric P.; Chazin, Walter J.

    2016-01-01

    The S100 proteins are a unique class of EF-hand Ca2+ binding proteins distributed in a cell-specific, tissue-specific, and cell cycle-specific manner in humans and other vertebrates. These proteins are distinguished by their distinctive homodimeric structure, both intracellular and extracellular functions, and the ability to bind transition metals at the dimer interface. Here we summarize current knowledge of S100 protein binding of Zn2+, Cu2+ and Mn2+ ions, focusing on binding affinities, conformational changes that arise from metal binding, and the roles of transition metal binding in S100 protein function. PMID:27430886

  10. Phosphorylation of native porcine olfactory binding proteins.

    PubMed

    Nagnan-Le Meillour, Patricia; Le Danvic, Chrystelle; Brimau, Fanny; Chemineau, Philippe; Michalski, Jean-Claude

    2009-07-01

    The identification of various isoforms of olfactory binding proteins is of major importance to elucidate their involvement in detection of pheromones and other odors. Here, we report the characterization of the phosphorylation of OBP (odorant binding protein) and Von Ebner's gland protein (VEG) from the pig, Sus scrofa. After labeling with specific antibodies raised against the three types of phosphorylation (Ser, Thr, Tyr), the phosphate-modified residues were mapped by using the beta-elimination followed by Michael addition of dithiothreitol (BEMAD) method. Eleven phosphorylation sites were localized in the pOBP sequence and nine sites in the VEG sequence. OBPs are secreted by Bowman's gland cells in the extracellular mucus lining the nasal cavity. After tracking the secretion pathway in the rough endoplasmic reticulum of these cells, we hypothesize that these proteins may be phosphorylated by ectokinases that remain to be characterized. The existence of such a regulatory mechanism theoretically increases the number of OBP variants, and it suggests a more specific role for OBPs in odorant coding than the one of odorant solubilizer and transporter.

  11. Novel stereospecificity of the L-arabinose-binding protein

    NASA Astrophysics Data System (ADS)

    Quiocho, Florante A.; Vyas, Nand K.

    1984-08-01

    Tertiary structure refinement at 1.7 Å resolution of the liganded form of L-arabinose-binding protein from Escherichia coli has revealed a novel binding site geometry which accommodates both α- and β-anomers of L-arabinose. This detailed structure analysis provides new understanding of protein-sugar interaction, the process by which the binding protein minimizes the difference in the stability of the two bound sugar anomers, and the roles of periplasmic binding proteins in active transport

  12. Gene encoding herbicide safener binding protein

    SciTech Connect

    Walton, Jonathan D.; Scott-Craig, John S.

    1999-01-01

    The cDNA encoding safener binding protein (SafBP), also referred to as SBP1, is set forth in FIG. 5 and SEQ ID No. 1. The deduced amino acid sequence is provided in FIG. 5 and SEQ ID No. 2. Methods of making and using SBP1 and SafBP to alter a plant's sensitivity to certain herbicides or a plant's responsiveness to certain safeners are also provided, as well as expression vectors, transgenic plants or other organisms transfected with said vectors and seeds from said plants.

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

    ERIC Educational Resources Information Center

    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…

  14. Neurodegeneration and RNA-binding proteins.

    PubMed

    De Conti, Laura; Baralle, Marco; Buratti, Emanuele

    2017-03-01

    In the eukaryotic nucleus, RNA-binding proteins (RBPs) play a very important role in the life cycle of both coding and noncoding RNAs. As soon as they are transcribed, in fact, all RNA molecules within a cell are bound by distinct sets of RBPs that have the task of regulating its correct processing, transport, stability, and function/translation up to its final degradation. These tasks are particularly important in cells that have a complex RNA metabolism, such as neurons. Not surprisingly, therefore, recent findings have shown that the misregulation of genes involved in RNA metabolism or the autophagy/proteasome pathway plays an important role in the onset and progression of several neurodegenerative diseases. In this article, we aim to review the recent advances that link neurodegenerative processes and RBP proteins. WIREs RNA 2017, 8:e1394. doi: 10.1002/wrna.1394 For further resources related to this article, please visit the WIREs website.

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

    PubMed

    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. Comparison of the Folding Mechanism of Highly Homologous Proteins in the Lipid-binding Protein Family

    EPA Science Inventory

    The folding mechanism of two closely related proteins in the intracellular lipid binding protein family, human bile acid binding protein (hBABP) and rat bile acid binding protein (rBABP) were examined. These proteins are 77% identical (93% similar) in sequence Both of these singl...

  17. BindML/BindML+: Detecting Protein-Protein Interaction Interface Propensity from Amino Acid Substitution Patterns.

    PubMed

    Wei, Qing; La, David; Kihara, Daisuke

    2017-01-01

    Prediction of protein-protein interaction sites in a protein structure provides important information for elucidating the mechanism of protein function and can also be useful in guiding a modeling or design procedures of protein complex structures. Since prediction methods essentially assess the propensity of amino acids that are likely to be part of a protein docking interface, they can help in designing protein-protein interactions. Here, we introduce BindML and BindML+ protein-protein interaction sites prediction methods. BindML predicts protein-protein interaction sites by identifying mutation patterns found in known protein-protein complexes using phylogenetic substitution models. BindML+ is an extension of BindML for distinguishing permanent and transient types of protein-protein interaction sites. We developed an interactive web-server that provides a convenient interface to assist in structural visualization of protein-protein interactions site predictions. The input data for the web-server are a tertiary structure of interest. BindML and BindML+ are available at http://kiharalab.org/bindml/ and http://kiharalab.org/bindml/plus/ .

  18. Neuronal calcium-binding proteins and schizophrenia.

    PubMed

    Eyles, D W; McGrath, J J; Reynolds, G P

    2002-09-01

    Calcium-binding proteins (CBPs) such as calbindin, parvalbumin and calretinin are used as immunohistochemical markers for discrete neuronal subpopulations. They are particularly useful in identifying the various subpopulations of GABAergic interneurons that control output from prefrontal and cingulate cortices as well as from the hippocampus. The strategic role these interneurons play in regulating output from these three crucial brain regions has made them a focus for neuropathological investigation in schizophrenia. The number of pathological reports detailing subtle changes in these CBP-containing interneurons in patients with schizophrenia is rapidly growing. These proteins however are more than convenient neuronal markers. They confer survival advantages to neurons and can increase the neuron's ability to sustain firing. These properties may be important in the subtle pathophysiology of nondegenerative phenomena such as schizophrenia. The aim of this review is to introduce the reader to the functional properties of CBPs and to examine the emerging literature reporting alterations in these proteins in schizophrenia as well as draw some conclusions about the significance of these findings.

  19. Glycan Masking of Plasmodium vivax Duffy Binding Protein for Probing Protein Binding Function and Vaccine Development

    PubMed Central

    Janes, Joel; Gurumoorthy, Sairam; Gibson, Claire; Melcher, Martin; Chitnis, Chetan E.; Wang, Ruobing; Schief, William R.; Smith, Joseph D.

    2013-01-01

    Glycan masking is an emerging vaccine design strategy to focus antibody responses to specific epitopes, but it has mostly been evaluated on the already heavily glycosylated HIV gp120 envelope glycoprotein. Here this approach was used to investigate the binding interaction of Plasmodium vivax Duffy Binding Protein (PvDBP) and the Duffy Antigen Receptor for Chemokines (DARC) and to evaluate if glycan-masked PvDBPII immunogens would focus the antibody response on key interaction surfaces. Four variants of PVDBPII were generated and probed for function and immunogenicity. Whereas two PvDBPII glycosylation variants with increased glycan surface coverage distant from predicted interaction sites had equivalent binding activity to wild-type protein, one of them elicited slightly better DARC-binding-inhibitory activity than wild-type immunogen. Conversely, the addition of an N-glycosylation site adjacent to a predicted PvDBP interaction site both abolished its interaction with DARC and resulted in weaker inhibitory antibody responses. PvDBP is composed of three subdomains and is thought to function as a dimer; a meta-analysis of published PvDBP mutants and the new DBPII glycosylation variants indicates that critical DARC binding residues are concentrated at the dimer interface and along a relatively flat surface spanning portions of two subdomains. Our findings suggest that DARC-binding-inhibitory antibody epitope(s) lie close to the predicted DARC interaction site, and that addition of N-glycan sites distant from this site may augment inhibitory antibodies. Thus, glycan resurfacing is an attractive and feasible tool to investigate protein structure-function, and glycan-masked PvDBPII immunogens might contribute to P. vivax vaccine development. PMID:23853575

  20. Calcyclin Binding Protein/Siah-1 Interacting Protein Is a Hsp90 Binding Chaperone

    PubMed Central

    Góral, Agnieszka; Bieganowski, Paweł; Prus, Wiktor; Krzemień-Ojak, Łucja; Kądziołka, Beata; Fabczak, Hanna; Filipek, Anna

    2016-01-01

    The Hsp90 chaperone activity is tightly regulated by interaction with many co-chaperones. Since CacyBP/SIP shares some sequence homology with a known Hsp90 co-chaperone, Sgt1, in this work we performed a set of experiments in order to verify whether CacyBP/SIP can interact with Hsp90. By applying the immunoprecipitation assay we have found that CacyBP/SIP binds to Hsp90 and that the middle (M) domain of Hsp90 is responsible for this binding. Furthermore, the proximity ligation assay (PLA) performed on HEp-2 cells has shown that the CacyBP/SIP-Hsp90 complexes are mainly localized in the cytoplasm of these cells. Using purified proteins and applying an ELISA we have shown that Hsp90 interacts directly with CacyBP/SIP and that the latter protein does not compete with Sgt1 for the binding to Hsp90. Moreover, inhibitors of Hsp90 do not perturb CacyBP/SIP-Hsp90 binding. Luciferase renaturation assay and citrate synthase aggregation assay with the use of recombinant proteins have revealed that CacyBP/SIP exhibits chaperone properties. Also, CacyBP/SIP-3xFLAG expression in HEp-2 cells results in the appearance of more basic Hsp90 forms in 2D electrophoresis, which may indicate that CacyBP/SIP dephosphorylates Hsp90. Altogether, the obtained results suggest that CacyBP/SIP is involved in regulation of the Hsp90 chaperone machinery. PMID:27249023

  1. Characterizing the morphology of protein binding patches.

    PubMed

    Malod-Dognin, Noël; Bansal, Achin; Cazals, Frédéric

    2012-12-01

    Let the patch of a partner in a protein complex be the collection of atoms accounting for the interaction. To improve our understanding of the structure-function relationship, we present a patch model decoupling the topological and geometric properties. While the geometry is classically encoded by the atomic positions, the topology is recorded in a graph encoding the relative position of concentric shells partitioning the interface atoms. The topological-geometric duality provides the basis of a generic dynamic programming-based algorithm comparing patches at the shell level, which may favor topological or geometric features. On the biological side, we address four questions, using 249 cocrystallized heterodimers organized in biological families. First, we dissect the morphology of binding patches and show that Nature enjoyed the topological and geometric degrees of freedom independently while retaining a finite set of qualitatively distinct topological signatures. Second, we argue that our shell-based comparison is effective to perform atomic-level comparisons and show that topological similarity is a less stringent than geometric similarity. We also use the topological versus geometric duality to exhibit topo-rigid patches, whose topology (but not geometry) remains stable upon docking. Third, we use our comparison algorithms to infer specificity-related information amidst a database of complexes. Finally, we exhibit a descriptor outperforming its contenders to predict the binding affinities of the affinity benchmark. The softwares developed with this article are availablefrom http://team.inria.fr/abs/vorpatch_compatch/.

  2. Prediction of zinc finger DNA binding protein.

    PubMed

    Nakata, K

    1995-04-01

    Using the neural network algorithm with back-propagation training procedure, we analysed the zinc finger DNA binding protein sequences. We incorporated the characteristic patterns around the zinc finger motifs TFIIIA type (Cys-X2-5-Cys-X12-13-His-X2-5-His) and the steroid hormone receptor type (Cys-X2-5-Cys-X12-15-Cys-X2-5-Cys-X15-16-Cys-X4-5-Cys-X8-10- Cys-X2-3-Cys) in the neural network algorithm. The patterns used in the neural network were the amino acid pattern, the electric charge and polarity pattern, the side-chain chemical property and subproperty patterns, the hydrophobicity and hydrophilicity patterns and the secondary structure propensity pattern. Two consecutive patterns were also considered. Each pattern was incorporated in the single layer perceptron algorithm and the combinations of patterns were considered in the two-layer perceptron algorithm. As for the TFIIIA type zinc finger DNA binding motifs, the prediction results of the two-layer perceptron algorithm reached up to 96.9% discrimination, and the prediction results of the discriminant analysis using the combination of several characters reached up to 97.0%. As for the steroid hormone receptor type zinc finger, the prediction results of neural network algorithm and the discriminant analyses reached up to 96.0%.

  3. Penicillin-binding proteins in Actinobacteria.

    PubMed

    Ogawara, Hiroshi

    2015-04-01

    Because some Actinobacteria, especially Streptomyces species, are β-lactam-producing bacteria, they have to have some self-resistant mechanism. The β-lactam biosynthetic gene clusters include genes for β-lactamases and penicillin-binding proteins (PBPs), suggesting that these are involved in self-resistance. However, direct evidence for the involvement of β-lactamases does not exist at the present time. Instead, phylogenetic analysis revealed that PBPs in Streptomyces are distinct in that Streptomyces species have much more PBPs than other Actinobacteria, and that two to three pairs of similar PBPs are present in most Streptomyces species examined. Some of these PBPs bind benzylpenicillin with very low affinity and are highly similar in their amino-acid sequences. Furthermore, other low-affinity PBPs such as SCLAV_4179 in Streptomyces clavuligerus, a β-lactam-producing Actinobacterium, may strengthen further the self-resistance against β-lactams. This review discusses the role of PBPs in resistance to benzylpenicillin in Streptomyces belonging to Actinobacteria.

  4. Protein function annotation by local binding site surface similarity.

    PubMed

    Spitzer, Russell; Cleves, Ann E; Varela, Rocco; Jain, Ajay N

    2014-04-01

    Hundreds of protein crystal structures exist for proteins whose function cannot be confidently determined from sequence similarity. Surflex-PSIM, a previously reported surface-based protein similarity algorithm, provides an alternative method for hypothesizing function for such proteins. The method now supports fully automatic binding site detection and is fast enough to screen comprehensive databases of protein binding sites. The binding site detection methodology was validated on apo/holo cognate protein pairs, correctly identifying 91% of ligand binding sites in holo structures and 88% in apo structures where corresponding sites existed. For correctly detected apo binding sites, the cognate holo site was the most similar binding site 87% of the time. PSIM was used to screen a set of proteins that had poorly characterized functions at the time of crystallization, but were later biochemically annotated. Using a fully automated protocol, this set of 8 proteins was screened against ∼60,000 ligand binding sites from the PDB. PSIM correctly identified functional matches that predated query protein biochemical annotation for five out of the eight query proteins. A panel of 12 currently unannotated proteins was also screened, resulting in a large number of statistically significant binding site matches, some of which suggest likely functions for the poorly characterized proteins.

  5. An ent-kaurene that inhibits mitotic chromosome movement and binds the kinetochore protein ran-binding protein 2.

    PubMed

    Rundle, Natalie T; Nelson, Jim; Flory, Mark R; Joseph, Jomon; Th'ng, John; Aebersold, Ruedi; Dasso, Mary; Andersen, Raymond J; Roberge, Michel

    2006-08-22

    Using a chemical genetics screen, we have identified ent-15-oxokaurenoic acid (EKA) as a chemical that causes prolonged mitotic arrest at a stage resembling prometaphase. EKA inhibits the association of the mitotic motor protein centromeric protein E with kinetochores and inhibits chromosome movement. Unlike most antimitotic agents, EKA does not inhibit the polymerization or depolymerization of tubulin. To identify EKA-interacting proteins, we used a cell-permeable biotinylated form that retains biological activity to isolate binding proteins from living cells. Mass spectrometric analysis identified six EKA-binding proteins, including Ran-binding protein 2, a kinetochore protein whose depletion by small interfering RNA causes a similar mitotic arrest phenotype.

  6. Lipopolysaccharide binding protein in preterm infants

    PubMed Central

    Behrendt, D; Dembinski, J; Heep, A; Bartmann, P

    2004-01-01

    Objective: To assess serum concentrations of lipopolysaccharide binding protein (LBP) in preterm infants with neonatal bacterial infection (NBI). Methods: Blood samples were analysed of 57 preterm (28+1 to 36+6, median 33+2 weeks gestation) and 17 term infants admitted to the neonatal intensive care unit within the first 72 hours of life with suspicion of NBI. Samples were obtained at first suspicion of sepsis and after 12 and 24 hours. Diagnosis of NBI was confirmed by raised concentrations of C reactive protein and/or interleukin 6. The influence of gestational age and labour was analysed. Results: Maximum LBP concentrations in infants with NBI were greatly increased compared with infants without NBI (13.0–46.0 µg/ml (median 20.0 µg/ml) v 0.6–17.4 µg/ml (median 4.2 µg/ml)). LBP concentrations in infected infants were not yet significantly raised when NBI was first suspected. The LBP concentrations of preterm infants were comparable to those of term infants. Regression analysis revealed no significant effect of labour or gestational age on LBP. Conclusions: Raised LBP concentrations indicate NBI in preterm and term infants. Preterm infants of > 28 weeks gestation seem to be capable of producing LBP as efficiently as term infants. Neonatal LBP concentrations are not influenced by labour. LBP may be a useful diagnostic marker of NBI in preterm infants. PMID:15499153

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

    PubMed

    Figueroa-Angulo, Elisa E; Calla-Choque, Jaeson S; Mancilla-Olea, Maria Inocente; Arroyo, Rossana

    2015-11-26

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

  8. Identification of a fibronectin-binding protein from Staphylococcus epidermidis.

    PubMed

    Williams, Rachel J; Henderson, Brian; Sharp, Lindsay J; Nair, Sean P

    2002-12-01

    Staphylococcus epidermidis has been reported to bind to a number of host cell extracellular matrix proteins, including fibronectin. Here we report the identification of a fibronectin-binding protein from S. epidermidis. A phage display library of S. epidermidis genomic DNA was constructed and panned against immobilized fibronectin. A number of phagemid clones containing overlapping inserts were identified, and one of these clones, pSE109FN, contained a 1.4-kb insert. Phage pSE109FN was found to bind to fibronectin but not to collagen, fibrinogen, laminin, or vitronectin. However, pSE109FN also bound to heparin, hyaluronate, and plasminogen, although to a lesser extent than it bound to fibronectin. Analysis of The Institute for Genomic Research S. epidermidis genome sequence database revealed a 1.85-kb region within a putative 30.5-kb open reading frame, to which the overlapping DNA inserts contained within the fibronectin-binding phagemids mapped. We have designated the gene encoding the fibronectin-binding domain embp. A recombinant protein, Embp32, which encompassed the fibronectin-binding domain of Embp, blocked the binding of S. epidermidis, but not the binding of Staphylococcus aureus, to fibronectin. In contrast, a recombinant protein, FnBPB[D1-D4], spanning the fibronectin-binding domain of the S. aureus fibronectin-binding protein FnBPB, blocked binding of S. aureus to fibronectin but had a negligible effect on the binding of S. epidermidis.

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

    SciTech Connect

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

    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.

  10. Discovery of binding proteins for a protein target using protein-protein docking-based virtual screening.

    PubMed

    Zhang, Changsheng; Tang, Bo; Wang, Qian; Lai, Luhua

    2014-10-01

    Target structure-based virtual screening, which employs protein-small molecule docking to identify potential ligands, has been widely used in small-molecule drug discovery. In the present study, we used a protein-protein docking program to identify proteins that bind to a specific target protein. In the testing phase, an all-to-all protein-protein docking run on a large dataset was performed. The three-dimensional rigid docking program SDOCK was used to examine protein-protein docking on all protein pairs in the dataset. Both the binding affinity and features of the binding energy landscape were considered in the scoring function in order to distinguish positive binding pairs from negative binding pairs. Thus, the lowest docking score, the average Z-score, and convergency of the low-score solutions were incorporated in the analysis. The hybrid scoring function was optimized in the all-to-all docking test. The docking method and the hybrid scoring function were then used to screen for proteins that bind to tumor necrosis factor-α (TNFα), which is a well-known therapeutic target for rheumatoid arthritis and other autoimmune diseases. A protein library containing 677 proteins was used for the screen. Proteins with scores among the top 20% were further examined. Sixteen proteins from the top-ranking 67 proteins were selected for experimental study. Two of these proteins showed significant binding to TNFα in an in vitro binding study. The results of the present study demonstrate the power and potential application of protein-protein docking for the discovery of novel binding proteins for specific protein targets.

  11. Partial characterization of GTP-binding proteins in Neurospora

    SciTech Connect

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

    1987-08-14

    Six fractions of GTP-binding proteins separated by gel filtration of a mycelial extract containing membrane components of Neurospora crassa were partially characterized. (/sup 35/S)GTP gamma S bound to GTP-binding protein was assayed by repeated treatments with a Norit solution and centrifugation. The binding of (/sup 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.

  12. Exploring NMR ensembles of calcium binding proteins: Perspectives to design inhibitors of protein-protein interactions

    PubMed Central

    2011-01-01

    Background Disrupting protein-protein interactions by small organic molecules is nowadays a promising strategy employed to block protein targets involved in different pathologies. However, structural changes occurring at the binding interfaces make difficult drug discovery processes using structure-based drug design/virtual screening approaches. Here we focused on two homologous calcium binding proteins, calmodulin and human centrin 2, involved in different cellular functions via protein-protein interactions, and known to undergo important conformational changes upon ligand binding. Results In order to find suitable protein conformations of calmodulin and centrin for further structure-based drug design/virtual screening, we performed in silico structural/energetic analysis and molecular docking of terphenyl (a mimicking alpha-helical molecule known to inhibit protein-protein interactions of calmodulin) into X-ray and NMR ensembles of calmodulin and centrin. We employed several scoring methods in order to find the best protein conformations. Our results show that docking on NMR structures of calmodulin and centrin can be very helpful to take into account conformational changes occurring at protein-protein interfaces. Conclusions NMR structures of protein-protein complexes nowadays available could efficiently be exploited for further structure-based drug design/virtual screening processes employed to design small molecule inhibitors of protein-protein interactions. PMID:21569443

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

    PubMed

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

    2016-10-20

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

  14. Actin binding proteins, spermatid transport and spermiation*

    PubMed Central

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

    2014-01-01

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

  15. Informing the Human Plasma Protein Binding of ...

    EPA Pesticide Factsheets

    The free fraction of a xenobiotic in plasma (Fub) is an important determinant of chemical adsorption, distribution, metabolism, elimination, and toxicity, yet experimental plasma protein binding data is scarce for environmentally relevant chemicals. The presented work explores the merit of utilizing available pharmaceutical data to predict Fub for environmentally relevant chemicals via machine learning techniques. Quantitative structure-activity relationship (QSAR) models were constructed with k nearest neighbors (kNN), support vector machines (SVM), and random forest (RF) machine learning algorithms from a training set of 1045 pharmaceuticals. The models were then evaluated with independent test sets of pharmaceuticals (200 compounds) and environmentally relevant ToxCast chemicals (406 total, in two groups of 238 and 168 compounds). The selection of a minimal feature set of 10-15 2D molecular descriptors allowed for both informative feature interpretation and practical applicability domain assessment via a bounded box of descriptor ranges and principal component analysis. The diverse pharmaceutical and environmental chemical sets exhibit similarities in terms of chemical space (99-82% overlap), as well as comparable bias and variance in constructed learning curves. All the models exhibit significant predictability with mean absolute errors (MAE) in the range of 0.10-0.18 Fub. The models performed best for highly bound chemicals (MAE 0.07-0.12), neutrals (MAE 0

  16. Plasma protein binding: from discovery to development.

    PubMed

    Bohnert, Tonika; Gan, Liang-Shang

    2013-09-01

    The importance of plasma protein binding (PPB) in modulating the effective drug concentration at pharmacological target sites has been the topic of significant discussion and debate amongst drug development groups over the past few decades. Free drug theory, which states that in absence of energy-dependent processes, after steady state equilibrium has been attained, free drug concentration in plasma is equal to free drug concentration at the pharmacologic target receptor(s) in tissues, has been used to explain pharmacokinetics/pharmacodynamics relationships in a large number of cases. Any sudden increase in free concentration of a drug could potentially cause toxicity and may need dose adjustment. Free drug concentration is also helpful to estimate the effective concentration of drugs that potentially can precipitate metabolism (or transporter)-related drug-drug interactions. Disease models are extensively validated in animals to progress a compound into development. Unbound drug concentration, and therefore PPB information across species is very informative in establishing safety margins and guiding selection of First in Human (FIH) dose and human efficacious dose. The scope of this review is to give an overview of reported role of PPB in several therapeutic areas, highlight cases where PPB changes are clinically relevant, and provide drug metabolism and pharmacokinetics recommendations in discovery and development settings.

  17. RNA binding protein and binding site useful for expression of recombinant molecules

    DOEpatents

    Mayfield, Stephen P.

    2006-10-17

    The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

  18. RNA binding protein and binding site useful for expression of recombinant molecules

    DOEpatents

    Mayfield, Stephen

    2000-01-01

    The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

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

    SciTech Connect

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

    1987-08-01

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

  20. Acyl-CoA binding proteins: multiplicity and function.

    PubMed

    Gossett, R E; Frolov, A A; Roths, J B; Behnke, W D; Kier, A B; Schroeder, F

    1996-09-01

    The physiological role of long-chain fatty acyl-CoA is thought to be primarily in intermediary metabolism of fatty acids. However, recent data show that nM to microM levels of these lipophilic molecules are potent regulators of cell functions in vitro. Although long-chain fatty acyl-CoA are present at several hundred microM concentration in the cell, very little long-chain fatty acyl-CoA actually exists as free or unbound molecules, but rather is bound with high affinity to membrane lipids and/or proteins. Recently, there is growing awareness that cytosol contains nonenzymatic proteins also capable of binding long-chain fatty acyl-CoA with high affinity. Although the identity of the cytosolic long-chain fatty acyl-CoA binding protein(s) has been the subject of some controversy, there is growing evidence that several diverse nonenzymatic cytosolic proteins will bind long-chain fatty acyl-CoA. Not only does acyl-CoA binding protein specifically bind medium and long-chain fatty acyl-CoA (LCFA-CoA), but ubiquitous proteins with multiple ligand specificities such as the fatty acid binding proteins and sterol carrier protein-2 also bind LCFA-CoA with high affinity. The potential of these acyl-CoA binding proteins to influence the level of free LCFA-CoA and thereby the amount of LCFA-CoA bound to regulatory sites in proteins and enzymes is only now being examined in detail. The purpose of this article is to explore the identity, nature, function, and pathobiology of these fascinating newly discovered long-chain fatty acyl-CoA binding proteins. The relative contributions of these three different protein families to LCFA-CoA utilization and/or regulation of cellular activities are the focus of new directions in this field.

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

    PubMed Central

    Teyra, Joan; Paszkowski-Rogacz, Maciej; Anders, Gerd; Pisabarro, M Teresa

    2008-01-01

    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. The hierarchical

  2. Characterization of the DNA binding properties of polyomavirus capsid protein

    NASA Technical Reports Server (NTRS)

    Chang, D.; Cai, X.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    The DNA binding properties of the polyomavirus structural proteins VP1, VP2, and VP3 were studied by Southwestern analysis. The major viral structural protein VP1 and host-contributed histone proteins of polyomavirus virions were shown to exhibit DNA binding activity, but the minor capsid proteins VP2 and VP3 failed to bind DNA. The N-terminal first five amino acids (Ala-1 to Lys-5) were identified as the VP1 DNA binding domain by genetic and biochemical approaches. Wild-type VP1 expressed in Escherichia coli (RK1448) exhibited DNA binding activity, but the N-terminal truncated VP1 mutants (lacking Ala-1 to Lys-5 and Ala-1 to Cys-11) failed to bind DNA. The synthetic peptide (Ala-1 to Cys-11) was also shown to have an affinity for DNA binding. Site-directed mutagenesis of the VP1 gene showed that the point mutations at Pro-2, Lys-3, and Arg-4 on the VP1 molecule did not affect DNA binding properties but that the point mutation at Lys-5 drastically reduced DNA binding affinity. The N-terminal (Ala-1 to Lys-5) region of VP1 was found to be essential and specific for DNA binding, while the DNA appears to be non-sequence specific. The DNA binding domain and the nuclear localization signal are located in the same N-terminal region.

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

    PubMed

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

    2016-11-16

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-11-01

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

  5. Clinical relevance of drug binding to plasma proteins

    NASA Astrophysics Data System (ADS)

    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.

  6. Identification of a common hyaluronan binding motif in the hyaluronan binding proteins RHAMM, CD44 and link protein.

    PubMed Central

    Yang, B; Yang, B L; Savani, R C; Turley, E A

    1994-01-01

    We have previously identified two hyaluronan (HA) binding domains in the HA receptor, RHAMM, that occur near the carboxyl-terminus of this protein. We show here that these two HA binding domains are the only HA binding regions in RHAMM, and that they contribute approximately equally to the HA binding ability of this receptor. Mutation of domain II using recombinant polypeptides of RHAMM demonstrates that K423 and R431, spaced seven amino acids apart, are critical for HA binding activity. Domain I contains two sets of two basic amino acids, each spaced seven residues apart, and mutation of these basic amino acids reduced their binding to HA--Sepharose. These results predict that two basic amino acids flanking a seven amino acid stretch [hereafter called B(X7)B] are minimally required for HA binding activity. To assess whether this motif predicts HA binding in the intact RHAMM protein, we mutated all basic amino acids in domains I and II that form part of these motifs using site-directed mutagenesis and prepared fusion protein from the mutated cDNA. The altered RHAMM protein did not bind HA, confirming that the basic amino acids and their spacing are critical for binding. A specific requirement for arginine or lysine residues was identified since mutation of K430, R431 and K432 to histidine residues abolished binding. Clustering of basic amino acids either within or at either end of the motif enhanced HA binding activity while the occurrence of acidic residues between the basic amino acids reduced binding. The B(X7)B motif, in which B is either R or K and X7 contains no acidic residues and at least one basic amino acid, was found in all HA binding proteins molecularly characterized to date. Recombinant techniques were used to generate chimeric proteins containing either the B(X7)B motifs present in CD44 or link protein, with the amino-terminus of RHAMM (amino acids 1-238) that does not bind HA. All chimeric proteins containing the motif bound HA in transblot analyses

  7. Molecular simulations of multimodal ligand-protein binding: elucidation of binding sites and correlation with experiments.

    PubMed

    Freed, Alexander S; Garde, Shekhar; Cramer, Steven M

    2011-11-17

    Multimodal chromatography, which employs more than one mode of interaction between ligands and proteins, has been shown to have unique selectivity and high efficacy for protein purification. To test the ability of free solution molecular dynamics (MD) simulations in explicit water to identify binding regions on the protein surface and to shed light on the "pseudo affinity" nature of multimodal interactions, we performed MD simulations of a model protein ubiquitin in aqueous solution of free ligands. Comparisons of MD with NMR spectroscopy of ubiquitin mutants in solutions of free ligands show a good agreement between the two with regard to the preferred binding region on the surface of the protein and several binding sites. MD simulations also identify additional binding sites that were not observed in the NMR experiments. "Bound" ligands were found to be sufficiently flexible and to access a number of favorable conformations, suggesting only a moderate loss of ligand entropy in the "pseudo affinity" binding of these multimodal ligands. Analysis of locations of chemical subunits of the ligand on the protein surface indicated that electrostatic interaction units were located on the periphery of the preferred binding region on the protein. The analysis of the electrostatic potential, the hydrophobicity maps, and the binding of both acetate and benzene probes were used to further study the localization of individual ligand moieties. These results suggest that water-mediated electrostatic interactions help the localization and orientation of the MM ligand to the binding region with additional stability provided by nonspecific hydrophobic interactions.

  8. Identification of an imidazoline binding protein: Creatine kinase and an imidazoline-2 binding site

    PubMed Central

    Kimura, Atsuko; Tyacke, Robin J.; Robinson, James J.; Husbands, Stephen M.; Minchin, Michael C.W.; Nutt, David J.; Hudson, Alan L.

    2009-01-01

    Drugs that bind to imidazoline binding proteins have major physiological actions. To date, three subtypes of such proteins, I1, I2 and I3, have been proposed, although characterisations of these binding proteins are lacking. I2 binding sites are found throughout the brain, particularly dense in the arcuate nucleus of the hypothalamus. Selective I2 ligands demonstrate antidepressant-like activity and the identity of the proteins that respond to such ligands remained unknown until now. Here we report the isolation of a ∼ 45 kDa imidazoline binding protein from rabbit and rat brain using a high affinity ligand for the I2 subtype, 2-BFI, to generate an affinity column. Following protein sequencing of the isolated ∼ 45 kDa imidazoline binding protein, we identified it to be brain creatine kinase (B-CK). B-CK shows high binding capacity to selective I2 ligands; [3H]-2-BFI (5 nM) specifically bound to B-CK (2330 ± 815 fmol mg protein− 1). We predicted an I2 binding pocket near the active site of B-CK using molecular modelling. Furthermore, B-CK activity was inhibited by a selective I2 irreversible ligand, where 20 μM BU99006 reduced the enzyme activity by 16%, confirming the interaction between B-CK and the I2 ligand. In summary, we have identified B-CK to be the ∼ 45 kDa imidazoline binding protein and we have demonstrated the existence of an I2 binding site within this enzyme. The importance of B-CK in regulating neuronal activity and neurotransmitter release may well explain the various actions of I2 ligands in brain and the alterations in densities of I2 binding sites in psychiatric disorders. PMID:19410564

  9. Fibrinogen and Fibronectin Binding Activity and Immunogenic Nature of Choline Binding Protein M

    PubMed Central

    AFSHAR, Davoud; POURMAND, Mohammad Reza; JEDDI-TEHRANI, Mahmood; SABOOR YARAGHI, Ali Akbar; AZARSA, Mohammad; SHOKRI, Fazel

    2016-01-01

    Background: Choline-binding proteins (CBPs) are a group of surface-exposed proteins, which play crucial and physiological roles in Streptococcus pneumoniae. The novel member of CBPs, choline-binding protein M (CbpM) may have binding activity to plasma proteins. This study aimed to clone and express CbpM and demonstrate its interaction with plasma proteins and patients’ sera. Methods: The total length of cbpM gene was cloned in pET21a vector and expressed in BL21 expression host. Verification of recombinant protein was evaluated by Western blot using anti-His tag monoclonal antibody. Binding ability of the recombinant protein to plasma proteins and the interaction with patients’ sera were assessed by Western blot and ELISA methods. Results: The cbpM gene was successfully cloned into pET21a and expressed in BL21 host. Binding activity to fibronectin and fibrinogen and antibody reaction of CbpM to patients’ sera was demonstrated by Western blot and ELISA methods, respectively. Conclusion: CbpM is one of the pneumococcal surface-exposed proteins, which mediates pneumococcal binding to fibronectin and fibrinogen proteins. PMID:28053927

  10. Binding of globular proteins to DNA from surface tension measurement.

    PubMed

    Mitra, A; Chattoraj, D K; Chakraborty, P

    2001-10-01

    Extent of binding (gammap) of globular proteins to calf-thymus DNA have been measured in mole per mole of nucleotide as function of equilibrium protein concentration. We have exploited measurement of the surface tension of the protein solution in the presence and absence of DNA to calculate the binding ration (gammap). Interaction of bovine serum albumin with DNA has been studied at different pH. Interaction of bovine serum albumin with DNA has been studied at different pH, ionic strength and in presence of Ca2+. Interaction of BSA with denatured DNA has also been investigated. Binding isotherms for other globular proteins like beta-lactoglobulin, alpha-lactalbumin and lysozyme have been compared under identical physicochemical condition. It has been noted with considerable interest that globular form of protein is important to some extent in protein-DNA interaction. An attempt has been made to explain the significance of difference in binding ratios of these two biopolymers in aqueous medium for different systems in the light of electrostatic and hydrophobic effects. Values of maximum binding ration (gammap(m)) at saturated level for different systems have been also presented. The Gibb's free energy decrease (-deltaG0) of the binding of proteins to DNA has been compared more precisely for the saturation of binding sites in the DNA with the change of activity of protein in solution from zero to unity in the rational mole fraction scale.

  11. Concentration-dependent Cu(II) binding to prion protein

    NASA Astrophysics Data System (ADS)

    Hodak, Miroslav; Lu, Wenchang; Bernholc, Jerry

    2008-03-01

    The prion protein plays a causative role in several neurodegenerative diseases, including mad cow disease in cattle and Creutzfeldt-Jakob disease in humans. The normal function of the prion protein is unknown, but it has been linked to its ability to bind copper ions. Experimental evidence suggests that copper can be bound in three distinct modes depending on its concentration, but only one of those binding modes has been fully characterized experimentally. Using a newly developed hybrid DFT/DFT method [1], which combines Kohn-Sham DFT with orbital-free DFT, we have examined all the binding modes and obtained their detailed binding geometries and copper ion binding energies. Our results also provide explanation for experiments, which have found that when the copper concentration increases the copper binding mode changes, surprisingly, from a stronger to a weaker one. Overall, our results indicate that prion protein can function as a copper buffer. 1. Hodak, Lu, Bernholc, JCP, in press.

  12. Leukocyte protease binding to nucleic acids promotes nuclear localization and cleavage of nucleic acid binding proteins.

    PubMed

    Thomas, Marshall P; Whangbo, Jennifer; McCrossan, Geoffrey; Deutsch, Aaron J; Martinod, Kimberly; Walch, Michael; Lieberman, Judy

    2014-06-01

    Killer lymphocyte granzyme (Gzm) serine proteases induce apoptosis of pathogen-infected cells and tumor cells. Many known Gzm substrates are nucleic acid binding proteins, and the Gzms accumulate in the target cell nucleus by an unknown mechanism. In this study, we show that human Gzms bind to DNA and RNA with nanomolar affinity. Gzms cleave their substrates most efficiently when both are bound to nucleic acids. RNase treatment of cell lysates reduces Gzm cleavage of RNA binding protein targets, whereas adding RNA to recombinant RNA binding protein substrates increases in vitro cleavage. Binding to nucleic acids also influences Gzm trafficking within target cells. Preincubation with competitor DNA and DNase treatment both reduce Gzm nuclear localization. The Gzms are closely related to neutrophil proteases, including neutrophil elastase (NE) and cathepsin G. During neutrophil activation, NE translocates to the nucleus to initiate DNA extrusion into neutrophil extracellular traps, which bind NE and cathepsin G. These myeloid cell proteases, but not digestive serine proteases, also bind DNA strongly and localize to nuclei and neutrophil extracellular traps in a DNA-dependent manner. Thus, high-affinity nucleic acid binding is a conserved and functionally important property specific to leukocyte serine proteases. Furthermore, nucleic acid binding provides an elegant and simple mechanism to confer specificity of these proteases for cleavage of nucleic acid binding protein substrates that play essential roles in cellular gene expression and cell proliferation.

  13. Cooperative binding modes of Cu(II) in prion protein

    NASA Astrophysics Data System (ADS)

    Hodak, Miroslav; Chisnell, Robin; Lu, Wenchang; Bernholc, Jerry

    2007-03-01

    The misfolding of the prion protein, PrP, is responsible for a group of neurodegenerative diseases including mad cow disease and Creutzfeldt-Jakob disease. It is known that the PrP can efficiently bind copper ions; four high-affinity binding sites located in the octarepeat region of PrP are now well known. Recent experiments suggest that at low copper concentrations new binding modes, in which one copper ion is shared between two or more binding sites, are possible. Using our hybrid Thomas-Fermi/DFT computational scheme, which is well suited for simulations of biomolecules in solution, we investigate the geometries and energetics of two, three and four binding sites cooperatively binding one copper ion. These geometries are then used as inputs for classical molecular dynamics simulations. We find that copper binding affects the secondary structure of the PrP and that it stabilizes the unstructured (unfolded) part of the protein.

  14. The RNA-binding protein Gemin5 binds directly to the ribosome and regulates global translation

    PubMed Central

    Francisco-Velilla, Rosario; Fernandez-Chamorro, Javier; Ramajo, Jorge; Martinez-Salas, Encarnación

    2016-01-01

    RNA-binding proteins (RBPs) play crucial roles in all organisms. The protein Gemin5 harbors two functional domains. The N-terminal domain binds to snRNAs targeting them for snRNPs assembly, while the C-terminal domain binds to IRES elements through a non-canonical RNA-binding site. Here we report a comprehensive view of the Gemin5 interactome; most partners copurified with the N-terminal domain via RNA bridges. Notably, Gemin5 sediments with the subcellular ribosome fraction, and His-Gemin5 binds to ribosome particles via its N-terminal domain. The interaction with the ribosome was lost in F381A and Y474A Gemin5 mutants, but not in W14A and Y15A. Moreover, the ribosomal proteins L3 and L4 bind directly with Gemin5, and conversely, Gemin5 mutants impairing the binding to the ribosome are defective in the interaction with L3 and L4. The overall polysome profile was affected by Gemin5 depletion or overexpression, concomitant to an increase or a decrease, respectively, of global protein synthesis. Gemin5, and G5-Nter as well, were detected on the polysome fractions. These results reveal the ribosome-binding capacity of the N-ter moiety, enabling Gemin5 to control global protein synthesis. Our study uncovers a crosstalk between this protein and the ribosome, and provides support for the view that Gemin5 may control translation elongation. PMID:27507887

  15. Stereoselective binding of chiral drugs to plasma proteins.

    PubMed

    Shen, Qi; Wang, Lu; Zhou, Hui; Jiang, Hui-di; Yu, Lu-shan; Zeng, Su

    2013-08-01

    Chiral drugs show distinct biochemical and pharmacological behaviors in the human body. The binding of chiral drugs to plasma proteins usually exhibits stereoselectivity, which has a far-reaching influence on their pharmacological activities and pharmacokinetic profiles. In this review, the stereoselective binding of chiral drugs to human serum albumin (HSA), α1-acid glycoprotein (AGP) and lipoprotein, three most important proteins in human plasma, are detailed. Furthermore, the application of AGP variants and recombinant fragments of HSA for studying enantiomer binding properties is also discussed. Apart from the stereoselectivity of enantiomer-protein binding, enantiomer-enantiomer interactions that may induce allosteric effects are also described. Additionally, the techniques and methods used to determine drug-protein binding parameters are briefly reviewed.

  16. Stereoselective binding of chiral drugs to plasma proteins

    PubMed Central

    Shen, Qi; Wang, Lu; Zhou, Hui; Jiang, Hui-di; Yu, Lu-shan; Zeng, Su

    2013-01-01

    Chiral drugs show distinct biochemical and pharmacological behaviors in the human body. The binding of chiral drugs to plasma proteins usually exhibits stereoselectivity, which has a far-reaching influence on their pharmacological activities and pharmacokinetic profiles. In this review, the stereoselective binding of chiral drugs to human serum albumin (HSA), α1-acid glycoprotein (AGP) and lipoprotein, three most important proteins in human plasma, are detailed. Furthermore, the application of AGP variants and recombinant fragments of HSA for studying enantiomer binding properties is also discussed. Apart from the stereoselectivity of enantiomer-protein binding, enantiomer-enantiomer interactions that may induce allosteric effects are also described. Additionally, the techniques and methods used to determine drug-protein binding parameters are briefly reviewed. PMID:23852086

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

    NASA Technical Reports Server (NTRS)

    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.

  18. Structure and Function of Nematode RNA-Binding Proteins

    PubMed Central

    Kaymak, Ebru; Wee, L.M.; Ryder, Sean P.

    2010-01-01

    RNA-binding proteins are critical effectors of gene expression. They guide mRNA localization, translation, and stability, and potentially play a role in regulating mRNA synthesis. The structural basis for RNA recognition by RNA-binding proteins is the key to understanding how they target specific transcripts for regulation. Compared to other metazoans, nematode genomes contain a significant expansion in several RNA-binding protein families, including Pumilio-FBF (PUF), TTP-like zinc finger (TZF), and argonaute-like (AGO) proteins. Genetic data suggest that individual members of each family have distinct functions, presumably due to sequence variations that alter RNA binding specificity or protein interaction partners. In this review, we highlight example structures and identify the variable regions that likely contribute to functional divergence in nematodes. PMID:20418095

  19. Guardian of Genetic Messenger-RNA-Binding Proteins

    PubMed Central

    Anji, Antje; Kumari, Meena

    2016-01-01

    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. PMID:26751491

  20. Guardian of Genetic Messenger-RNA-Binding Proteins.

    PubMed

    Anji, Antje; Kumari, Meena

    2016-01-06

    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.

  1. Comparative serum protein binding of anthracycline derivatives.

    PubMed

    Chassany, O; Urien, S; Claudepierre, P; Bastian, G; Tillement, J P

    1996-01-01

    The binding of doxorubicin, iododoxorubicin, daunorubicin, epirubicin, pirarubicin, zorubicin, aclarubicin, and mitoxantrone to 600 microM human serum albumin and 50 microM alpha 1-acid glycoprotein was studied by ultrafiltration at 37 degrees C and pH 7.4. Anthracycline concentrations (total and free) were determined by high-performance liquid chromatography (HPLC) with fluorometric detection. Binding to albumin (600 microM) varied from 61% (daunorubicin) to 94% (iododoxorubicin). The binding to alpha 1-acid glycoprotein (50 microM) was more variable, ranging from 31% (epirubicin) to 64% (zorubicin), and was essentially related to the hydrophobicity of the derivatives. Simulations showed that the total serum binding varied over a broad range from 71% (doxorubicin) to 96% (iododoxorubicin). We recently reported that the binding to lipoproteins of a series of eight anthracycline analogues could be ascribed to chemicophysical determinants of lipophilicity [2]. The present study was conducted to evaluate in vitro the contribution of albumin and alpha 1-acid glycoprotein to the total serum binding of these drugs.

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

    PubMed

    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

  3. HTLV-1 Tax Protein Stimulation of DNA Binding of bZIP Proteins by Enhancing Dimerization

    NASA Astrophysics Data System (ADS)

    Wagner, Susanne; Green, Michael R.

    1993-10-01

    The Tax protein of human T cell leukemia virus type-1 (HTLV-I) transcriptionally activates the HTLV-I promoter. This activation requires binding sites for activating transcription factor (ATF) proteins, a family of cellular proteins that contain basic region-leucine zipper (bZIP) DNA binding domains. Data are presented showing that Tax increases the in vitro DNA binding activity of multiple ATF proteins. Tax also stimulated DNA binding by other bZIP proteins, but did not affect DNA binding proteins that lack a bZIP domain. The increase in DNA binding occurred because Tax promotes dimerization of the bZIP domain in the absence of DNA, and the elevated concentration of the bZIP homodimer then facilitates the DNA binding reaction. These results help explain how Tax activates viral transcription and transforms cells.

  4. 9S binding protein for androgens and progesterone.

    PubMed

    Wilson, E M; Lea, O A; French, F S

    1977-05-01

    A steroid binding protein fraction with a sedimentation coefficient of approximately 9 S (molecular weight approximately equal to 200,000) has been identified in 105,000 X g supernatants of several androgen-responsive organs. Highest concentrations were found in epididymis and testis, but small amounts were detected in prostate, seminal vesicle, kidney, submandibular gland, and lung. The 9S protein binds [3H]dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one) and [3H]progesterone (4-pregnene-3,20-dione) with equilibrium binding constants of approximately 10(5) M-1 and 10(6) M-1, respectively. The concentration of 9S binding sites in epididymis is approximately 10(-11) mol/mg of supernatant protein, which is at least 10(5) times greater than the concentration of androgen receptor. 9S binding protein appears to be a nonsecretory, intracellular protein and has properties different from the andorgen receptor. It is unretarded on DEAE-Sephadex chromatography at pH 8.0, and its sedimentation rate on sucrose gradients is not altered at high ionic strength (0.4 M KCl). Like the androgen receptor, its binding activity, which is maximal between pH 7 and 9.5, is heat labile, decreased by sulfhydryl reagents, and enhanced by 2-mercaptoethanol. It is suggested that because of its high concentration and low affinity, 9S binding protein may function in the intracellular accumulation of compartmentalization of androgens or progesterone.

  5. Protein surface-distribution and protein-protein interactions in the binding of peripheral proteins to charged lipid membranes.

    PubMed Central

    Heimburg, T; Marsh, D

    1995-01-01

    The binding of native cytochrome c to negatively charged lipid dispersions of dioleoyl phosphatidylglycerol has been studied over a wide range of ionic strengths. Not only is the strength of protein binding found to decrease rapidly with increasing ionic strength, but also the binding curves reach an apparent saturation level that decreases rapidly with increasing ionic strength. Analysis of the binding isotherms with a general statistical thermodynamic model that takes into account not only the free energy of the electrostatic double layer, but also the free energy of the surface distribution of the protein, demonstrates that the apparent saturation effects could arise from a competition between the out-of-plane binding reaction and the lateral in-plane interactions between proteins at the surface. It is found that association with nonlocalized sites results in binding isotherms that display the apparent saturation effect to a much more pronounced extent than does the Langmuir adsorption isotherm for binding to localized sites. With the model for nonlocalized sites, the binding isotherms of native cytochrome c can be described adequately by taking into account only the entropy of the surface distribution of the protein, without appreciable enthalpic interactions between the bound proteins. The binding of cytochrome c to dioleoyl phosphatidylglycerol dispersions at a temperature at which the bound protein is denatured on the lipid surface, but is nondenatured when free in solution, has also been studied. The binding curves for the surface-denatured protein differ from those for the native protein in that the apparent saturation at high ionic strength is less pronounced. This indicates the tendency of the denatured protein to aggregate on the lipid surface, and can be described by the binding isotherms for nonlocalized sites only if attractive interactions between the surface-bound proteins are included in addition to the distributional entropic terms. Additionally

  6. Paramagnetic Ligand Tagging To Identify Protein Binding Sites

    PubMed Central

    2015-01-01

    Transient biomolecular interactions are the cornerstones of the cellular machinery. The identification of the binding sites for low affinity molecular encounters is essential for the development of high affinity pharmaceuticals from weakly binding leads but is hindered by the lack of robust methodologies for characterization of weakly binding complexes. We introduce a paramagnetic ligand tagging approach that enables localization of low affinity protein–ligand binding clefts by detection and analysis of intermolecular protein NMR pseudocontact shifts, which are invoked by the covalent attachment of a paramagnetic lanthanoid chelating tag to the ligand of interest. The methodology is corroborated by identification of the low millimolar volatile anesthetic interaction site of the calcium sensor protein calmodulin. It presents an efficient route to binding site localization for low affinity complexes and is applicable to rapid screening of protein–ligand systems with varying binding affinity. PMID:26289584

  7. Diversity of Cyclic Di-GMP-Binding Proteins and Mechanisms

    PubMed Central

    2015-01-01

    ABSTRACT Cyclic di-GMP (c-di-GMP) synthetases and hydrolases (GGDEF, EAL, and HD-GYP domains) can be readily identified in bacterial genome sequences by using standard bioinformatic tools. In contrast, identification of c-di-GMP receptors remains a difficult task, and the current list of experimentally characterized c-di-GMP-binding proteins is likely incomplete. Several classes of c-di-GMP-binding proteins have been structurally characterized; for some others, the binding sites have been identified; and for several potential c-di-GMP receptors, the binding sites remain to be determined. We present here a comparative structural analysis of c-di-GMP-protein complexes that aims to discern the common themes in the binding mechanisms that allow c-di-GMP receptors to bind it with (sub)micromolar affinities despite the 1,000-fold excess of GTP. The available structures show that most receptors use their Arg and Asp/Glu residues to bind c-di-GMP monomers, dimers, or tetramers with stacked guanine bases. The only exception is the EAL domains that bind c-di-GMP monomers in an extended conformation. We show that in c-di-GMP-binding signature motifs, Arg residues bind to the O-6 and N-7 atoms at the Hoogsteen edge of the guanine base, while Asp/Glu residues bind the N-1 and N-2 atoms at its Watson-Crick edge. In addition, Arg residues participate in stacking interactions with the guanine bases of c-di-GMP and the aromatic rings of Tyr and Phe residues. This may account for the presence of Arg residues in the active sites of every receptor protein that binds stacked c-di-GMP. We also discuss the implications of these structural data for the improved understanding of the c-di-GMP signaling mechanisms. PMID:26055114

  8. De-novo protein function prediction using DNA binding and RNA binding proteins as a test case

    PubMed Central

    Peled, Sapir; Leiderman, Olga; Charar, Rotem; Efroni, Gilat; Shav-Tal, Yaron; Ofran, Yanay

    2016-01-01

    Of the currently identified protein sequences, 99.6% have never been observed in the laboratory as proteins and their molecular function has not been established experimentally. Predicting the function of such proteins relies mostly on annotated homologs. However, this has resulted in some erroneous annotations, and many proteins have no annotated homologs. Here we propose a de-novo function prediction approach based on identifying biophysical features that underlie function. Using our approach, we discover DNA and RNA binding proteins that cannot be identified based on homology and validate these predictions experimentally. For example, FGF14, which belongs to a family of secreted growth factors was predicted to bind DNA. We verify this experimentally and also show that FGF14 is localized to the nucleus. Mutating the predicted binding site on FGF14 abrogated DNA binding. These results demonstrate the feasibility of automated de-novo function prediction based on identifying function-related biophysical features. PMID:27869118

  9. De-novo protein function prediction using DNA binding and RNA binding proteins as a test case.

    PubMed

    Peled, Sapir; Leiderman, Olga; Charar, Rotem; Efroni, Gilat; Shav-Tal, Yaron; Ofran, Yanay

    2016-11-21

    Of the currently identified protein sequences, 99.6% have never been observed in the laboratory as proteins and their molecular function has not been established experimentally. Predicting the function of such proteins relies mostly on annotated homologs. However, this has resulted in some erroneous annotations, and many proteins have no annotated homologs. Here we propose a de-novo function prediction approach based on identifying biophysical features that underlie function. Using our approach, we discover DNA and RNA binding proteins that cannot be identified based on homology and validate these predictions experimentally. For example, FGF14, which belongs to a family of secreted growth factors was predicted to bind DNA. We verify this experimentally and also show that FGF14 is localized to the nucleus. Mutating the predicted binding site on FGF14 abrogated DNA binding. These results demonstrate the feasibility of automated de-novo function prediction based on identifying function-related biophysical features.

  10. Prediction of DNA-binding proteins from relational features

    PubMed Central

    2012-01-01

    Background The process of protein-DNA binding has an essential role in the biological processing of genetic information. We use relational machine learning to predict DNA-binding propensity of proteins from their structures. Automatically discovered structural features are able to capture some characteristic spatial configurations of amino acids in proteins. Results Prediction based only on structural relational features already achieves competitive results to existing methods based on physicochemical properties on several protein datasets. Predictive performance is further improved when structural features are combined with physicochemical features. Moreover, the structural features provide some insights not revealed by physicochemical features. Our method is able to detect common spatial substructures. We demonstrate this in experiments with zinc finger proteins. Conclusions We introduced a novel approach for DNA-binding propensity prediction using relational machine learning which could potentially be used also for protein function prediction in general. PMID:23146001

  11. Ca2+ signaling and intracellular Ca2+ binding proteins.

    PubMed

    Niki, I; Yokokura, H; Sudo, T; Kato, M; Hidaka, H

    1996-10-01

    Changes in cytosolic Ca2+ concentrations evoke a wide range of cellular responses and intracellular Ca(2+)-binding proteins are the key molecules to transduce Ca2+ signaling via enzymatic reactions or modulation of protein/protein interations (Fig.1). The EF hand proteins, like calmodulin and S100 proteins, are considered to exert Ca(2+)-dependent actions in the nucleus or the cytoplasm. The Ca2+/phospholipid binding proteins are classified into two groups, the annexins and the C2 region proteins. These proteins, distributed mainly in the cytoplasm, translocate to the plasma membrane in response to an increase in cytosolic Ca2+ and function in the vicinity of the membrane. Ca2+ storage proteins in the endoplasmic or sarcoplasmic reticulum provide the high Ca2+ capacity of the Ca2+ store sites, which regulate intracellular Ca2+ distribution. The variety and complexity of Ca2+ signaling result from the cooperative actions of specific Ca(2+)-binding proteins. This review describes biochemical properties of intracellular Ca(2+)-binding proteins and their proposed roles in mediating Ca2+ signaling.

  12. A new aspect of serum protein binding of tolbutamide.

    PubMed

    Ayanoğlu, G; Uihlein, M; Grigoleit, H G

    1986-02-01

    Tolbutamide is known to bind highly to serum proteins. Quite different values have, however, been reported for binding, ranging from 80 to 99 percent. In this study, in vivo and in vitro binding of increasing concentrations of tolbutamide to human serum proteins were evaluated. In vitro studies were done serum from three healthy males and for in vivo studies serum samples from eight healthy males who had received 1,000 mg tolbutamide were used. Protein binding was determined by equilibrium dialysis, using DIANORM system. Tolbutamide concentrations were determined by HPLC method of Uihlein and Hack. The results suggest that there is an increase in percent tolbutamide bound with increasing concentrations of tolbutamide. Generally, an inverse relationship between the total concentration of a drug in serum and its bound fraction is observed. Our findings seem to be contrary to this, at least within the concentration range studied. There exist at least two binding sites on albumin with different affinities for tolbutamide and most probably, at low concentrations, the drug binds mainly to the high affinity sites, whereas at higher concentrations additional drug will bind to the lower affinity sites leading to the observed increase in fraction bound with concentration. In conclusion it may be said that serum protein binding is a much more complicated phenomenon than generally stated and that the normal observations are only true for some ideal compounds where only one site of adsorption has to be taken into account.

  13. Nucleic acid-binding specificity of human FUS protein

    PubMed Central

    Wang, Xueyin; Schwartz, Jacob C.; Cech, Thomas R.

    2015-01-01

    FUS, a nuclear RNA-binding protein, plays multiple roles in RNA processing. Five specific FUS-binding RNA sequence/structure motifs have been proposed, but their affinities for FUS have not been directly compared. Here we find that human FUS binds all these sequences with Kdapp values spanning a 10-fold range. Furthermore, some RNAs that do not contain any of these motifs bind FUS with similar affinity. FUS binds RNA in a length-dependent manner, consistent with a substantial non-specific component to binding. Finally, investigation of FUS binding to different nucleic acids shows that it binds single-stranded DNA with three-fold lower affinity than ssRNA of the same length and sequence, while binding to double-stranded nucleic acids is weaker. We conclude that FUS has quite general nucleic acid-binding activity, with the various proposed RNA motifs being neither necessary for FUS binding nor sufficient to explain its diverse binding partners. PMID:26150427

  14. Erythrocyte Protein 4.1 Binds and Regulates Myosin

    NASA Astrophysics Data System (ADS)

    Pasternack, Gary R.; Racusen, Richard H.

    1989-12-01

    Myosin was recently identified in erythrocytes and was shown to partition both with membrane and cytosolic fractions, suggesting that it may be loosely bound to membranes [Fowler, V. M., Davis, J. Q. & Bennett, V. (1985) J. Cell Biol. 100, 47-55, and Wong, A. J., Kiehart, D. P. & Pollard, T. D. (1985) J. Biol. Chem. 260, 46-49]; however, the molecular basis for this binding was unclear. The present studies employed immobilized monomeric myosin to examine the interaction of myosin with erythrocyte protein 4.1. In human erythrocytes, protein 4.1 binds to integral membrane proteins and mediates spectrin-actin assembly. Protein 4.1 binds to rabbit skeletal muscle myosin with a Kd = 140 nM and a stoichiometry consistent with 1:1 binding. Heavy meromyosin competes for protein 4.1 binding with Ki = 36-54 nM; however, the S1 fragment (the myosin head) competes less efficiently. Affinity chromatography of partial chymotryptic digests of protein 4.1 on immobilized myosin identified a 10-kDa domain of protein 4.1 as the myosin-binding site. In functional studies, protein 4.1 partially inhibited the actin-activated Mg2+-ATPase activity of rabbit skeletal muscle myosin with Ki = 51 nM. Liver cytosolic and erythrocyte myosins preactivated with myosin light-chain kinase were similarly inhibited by protein 4.1. These studies show that protein 4.1 binds, modulates, and thus may regulate myosin. This interaction might serve to generate the contractile forces involved in Mg2+-ATP-dependent shape changes in erythrocytes and may additionally serve as a model for myosin organization and regulation in non-muscle cells.

  15. Carbohydrate-binding protein identification by coupling structural similarity searching with binding affinity prediction.

    PubMed

    Zhao, Huiying; Yang, Yuedong; von Itzstein, Mark; Zhou, Yaoqi

    2014-11-15

    Carbohydrate-binding proteins (CBPs) are potential biomarkers and drug targets. However, the interactions between carbohydrates and proteins are challenging to study experimentally and computationally because of their low binding affinity, high flexibility, and the lack of a linear sequence in carbohydrates as exists in RNA, DNA, and proteins. Here, we describe a structure-based function-prediction technique called SPOT-Struc that identifies carbohydrate-recognizing proteins and their binding amino acid residues by structural alignment program SPalign and binding affinity scoring according to a knowledge-based statistical potential based on the distance-scaled finite-ideal gas reference state (DFIRE). The leave-one-out cross-validation of the method on 113 carbohydrate-binding domains and 3442 noncarbohydrate binding proteins yields a Matthews correlation coefficient of 0.56 for SPalign alone and 0.63 for SPOT-Struc (SPalign + binding affinity scoring) for CBP prediction. SPOT-Struc is a technique with high positive predictive value (79% correct predictions in all positive CBP predictions) with a reasonable sensitivity (52% positive predictions in all CBPs). The sensitivity of the method was changed slightly when applied to 31 APO (unbound) structures found in the protein databank (14/31 for APO versus 15/31 for HOLO). The result of SPOT-Struc will not change significantly if highly homologous templates were used. SPOT-Struc predicted 19 out of 2076 structural genome targets as CBPs. In particular, one uncharacterized protein in Bacillus subtilis (1oq1A) was matched to galectin-9 from Mus musculus. Thus, SPOT-Struc is useful for uncovering novel carbohydrate-binding proteins. SPOT-Struc is available at http://sparks-lab.org.

  16. In Vitro Biochemical Characterization of Cytokinesis Actin-Binding Proteins.

    PubMed

    Zimmermann, Dennis; Morganthaler, Alisha N; Kovar, David R; Suarez, Cristian

    2016-01-01

    Characterizing the biochemical and biophysical properties of purified proteins is critical to understand the underlying molecular mechanisms that facilitate complicated cellular processes such as cytokinesis. Here we outline in vitro assays to investigate the effects of cytokinesis actin-binding proteins on actin filament dynamics and organization. We describe (1) multicolor single-molecule TIRF microscopy actin assembly assays, (2) "bulk" pyrene actin assembly/disassembly assays, and (3) "bulk" sedimentation actin filament binding and bundling assays.

  17. Cellular Retinoic Acid Binding Protein and Breast Cancer

    DTIC Science & Technology

    2006-05-01

    fatty acid probe anilinonaphtalene-8- sulphonic acid (ANS) was measured. ANS readily associates with various FABPs and its fluorescence is highly...DAMD17-03-1-0249 TITLE: Cellular Retinoic Acid Binding Protein and Breast Cancer PRINCIPAL INVESTIGATOR: Leslie J. (Willmert) Donato...DATES COVERED (From - To) 14 Apr 03 – 13 Apr 06 5a. CONTRACT NUMBER Cellular Retinoic Acid Binding Protein and Breast Cancer 5b. GRANT NUMBER

  18. Theoretical studies of protein-protein and protein-DNA binding rates

    NASA Astrophysics Data System (ADS)

    Alsallaq, Ramzi A.

    Proteins are folded chains of amino acids. Some of the amino acids (e.g. Lys, Arg, His, Asp, and Glu) carry charges under physiological conditions. Proteins almost always function through binding to other proteins or ligands, for example barnase is a ribonuclease protein, found in the bacterium Bacillus amyloliquefaceus. Barnase degrades RNA by hydrolysis. For the bacterium to inhibit the potentially lethal action of Barnase within its own cell it co-produces another protein called barstar which binds quickly, and tightly, to barnase. The biological function of this binding is to block the active site of barnase. The speeds (rates) at which proteins associate are vital to many biological processes. They span a wide range (from less than 103 to 108 M-1s-1 ). Rates greater than ˜ 106 M -1s-1 are typically found to be manifestations of enhancements by long-range electrostatic interactions between the associating proteins. A different paradigm appears in the case of protein binding to DNA. The rate in this case is enhanced through attractive surface potential that effectively reduces the dimensionality of the available search space for the diffusing protein. This thesis presents computational and theoretical models on the rate of association of ligands/proteins to other proteins or DNA. For protein-protein association we present a general strategy for computing protein-protein rates of association. The main achievements of this strategy is the ability to obtain a stringent reaction criteria based on the landscape of short-range interactions between the associating proteins, and the ability to compute the effect of the electrostatic interactions on the rates of association accurately using the best known solvers for Poisson-Boltzmann equation presently available. For protein-DNA association we present a mathematical model for proteins targeting specific sites on a circular DNA topology. The main achievements are the realization that a linear DNA with reflecting ends

  19. Being a binding site: characterizing residue composition of binding sites on proteins.

    PubMed

    Iván, Gábor; Szabadka, Zoltán; Grolmusz, Vince

    2007-12-30

    The Protein Data Bank contains the description of more than 45,000 three-dimensional protein and nucleic-acid structures today. Started to exist as the computer-readable depository of crystallographic data complementing printed articles, the proper interpretation of the content of the individual files in the PDB still frequently needs the detailed information found in the citing publication. This fact implies that the fully automatic processing of the whole PDB is a very hard task. We first cleaned and re-structured the PDB data, then analyzed the residue composition of the binding sites in the whole PDB for frequency and for hidden association rules. Main results of the paper: (i) the cleaning and repairing algorithm (ii) redundancy elimination from the data (iii) application of association rule mining to the cleaned non-redundant data set. We have found numerous significant relations of the residue-composition of the ligand binding sites on protein surfaces, summarized in two figures. One of the classical data-mining methods for exploring implication-rules, the association-rule mining, is capable to find previously unknown residue-set preferences of bind ligands on protein surfaces. Since protein-ligand binding is a key step in enzymatic mechanisms and in drug discovery, these uncovered preferences in the study of more than 19,500 binding sites may help in identifying new binding protein-ligand pairs.

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

    SciTech Connect

    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 (/sup 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/sub 2/ or CO:O/sub 2/ (8:2) significantly decreased the NADPH-dependent binding of (/sup 14/C)NNN to liver microsomal protein by 40% or 27% respectively. Extensive covalent binding of (/sup 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.

  1. 21 CFR 866.5765 - Retinol-binding protein immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Retinol-binding protein immunological test system....5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... the retinol-binding protein that binds and transports vitamin A in serum and urine. Measurement...

  2. 21 CFR 866.5765 - Retinol-binding protein immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Retinol-binding protein immunological test system....5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... the retinol-binding protein that binds and transports vitamin A in serum and urine. Measurement...

  3. 21 CFR 866.5765 - Retinol-binding protein immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Retinol-binding protein immunological test system....5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... the retinol-binding protein that binds and transports vitamin A in serum and urine. Measurement...

  4. 21 CFR 866.5765 - Retinol-binding protein immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Retinol-binding protein immunological test system....5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... the retinol-binding protein that binds and transports vitamin A in serum and urine. Measurement...

  5. 21 CFR 866.5765 - Retinol-binding protein immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Retinol-binding protein immunological test system....5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... the retinol-binding protein that binds and transports vitamin A in serum and urine. Measurement...

  6. Discodermolide interferes with the binding of tau protein to microtubules.

    PubMed

    Kar, Santwana; Florence, Gordon J; Paterson, Ian; Amos, Linda A

    2003-03-27

    We investigated whether discodermolide, a novel antimitotic agent, affects the binding to microtubules of tau protein repeat motifs. Like taxol, the new drug reduces the proportion of tau that pellets with microtubules. Despite their differing structures, discodermolide, taxol and tau repeats all bind to a site on beta-tubulin that lies within the microtubule lumen and is crucial in controlling microtubule assembly. Low concentrations of tau still bind strongly to the outer surfaces of preformed microtubules when the acidic C-terminal regions of at least six tubulin dimers are available for interaction with each tau molecule; otherwise binding is very weak.

  7. Protein-DNA binding in high-resolution

    PubMed Central

    Mahony, Shaun; Pugh, B. Franklin

    2015-01-01

    Recent advances in experimental and computational methodologies are enabling ultra-high resolution genome-wide profiles of protein-DNA binding events. For example, the ChIP-exo protocol precisely characterizes protein-DNA crosslinking patterns by combining chromatin immunoprecipitation (ChIP) with 5′ → 3′ exonuclease digestion. Similarly, deeply sequenced chromatin accessibility assays (e.g. DNase-seq and ATACseq) enable the detection of protected footprints at protein-DNA binding sites. With these techniques and others, we have the potential to characterize the individual nucleotides that interact with transcription factors, nucleosomes, RNA polymerases, and other regulatory proteins in a particular cellular context. In this review, we explain the experimental assays and computational analysis methods that enable high-resolution profiling of protein-DNA binding events. We discuss the challenges and opportunities associated with such approaches. PMID:26038153

  8. A sliding selectivity scale for lipid binding to membrane proteins

    PubMed Central

    Landreh, Michael; Marty, Michael T.; Gault, Joseph; Robinson, Carol V.

    2017-01-01

    Biological membranes form barriers that are essential for cellular integrity and compartmentalisation. Proteins that reside in the membrane have co-evolved with their hydrophobic lipid environment which serves as a solvent for proteins with very diverse requirements. As a result, membrane protein-lipid interactions range from completely non-selective to highly discriminating. Mass spectrometry (MS), in combination with X-ray crystallography and molecular dynamics simulations, enables us to monitor how lipids interact with intact membrane protein complexes and assess their effects on structure and dynamics. Recent studies illustrate the ability to differentiate specific lipid binding, preferential interactions with lipid subsets, and nonselective annular contacts. In this review, we consider the biological implications of different lipid-binding scenarios and propose that binding occurs on a sliding selectivity scale, in line with the view of biological membranes as facilitators of dynamic protein and lipid organization. PMID:27155089

  9. HIGH AFFINITY, DSRNA BINDING BY DISCONNECTED INTERACTING PROTEIN 1†

    PubMed Central

    Catanese, Daniel J.; Matthews, Kathleen S.

    2010-01-01

    Disconnected Interacting Protein 1 (DIP1) appears from sequence analysis and preliminary binding studies to be a member of the dsRNA-binding protein family. Of interest, DIP1 was shown previously to interact with and influence multiple proteins involved in transcription regulation in Drosophila melanogaster. We show here that the longest isoform of this protein, DIP1-c, exhibits a 500-fold preference for dsRNA over dsDNA of similar nucleotide sequence. Further, DIP1-c demonstrated very high affinity for a subset of dsRNA ligands, with binding in the picomolar range for VA1 RNA and miR-iab-4 precursor stem-loop, a potential physiological RNA target involved in regulating expression of its protein partner, Ultrabithorax. PMID:20643095

  10. The presence of zinc-binding proteins in brain.

    PubMed

    Itoh, M; Ebadi, M; Swanson, S

    1983-09-01

    Zinc is one of the most abundant divalent metal ions in the brain, its concentration being greater than those of copper and manganese. Since free zinc ion is a potent inhibitor of sulfhydryl enzymes, we postulated that zinc in the brain most probably exists bound to macromolecules. As zinc-binding proteins in brain have not been characterized, we attempted to discover the occurrence and properties of these proteins. By using Sephadex G-75 column chromatography calibrated with proteins of known molecular weights, and by other techniques, we detected separate zinc-binding proteins, with apparent estimated molecular weights ranging from 15,000 to 210,000. Unlike the hepatic or renal zinc thioneins, the zinc-binding proteins in brain are not inducible following administration of zinc. Our interpretation of the results is that the major portion of the existing zinc in the brain is bound, and does not exist in free form.

  11. Pulmonary surfactant protein A (SP-A) specifically binds dipalmitoylphosphatidylcholine

    SciTech Connect

    Kuroki, Y.; Akino, T. )

    1991-02-15

    Phospholipids are the major components of pulmonary surfactant. Dipalmitoylphosphatidylcholine is believed to be especially essential for the surfactant function of reducing the surface tension at the air-liquid interface. Surfactant protein A (SP-A) with a reduced denatured molecular mass of 26-38 kDa, characterized by a collagen-like structure and N-linked glycosylation, interacts strongly with a mixture of surfactant-like phospholipids. In the present study the direct binding of SP-A to phospholipids on a thin layer chromatogram was visualized using 125I-SP-A as a probe, so that the phospholipid specificities of SP-A binding and the structural requirements of SP-A and phospholipids for the binding could be examined. Although 125I-SP-A bound phosphatidylcholine and sphingomyeline, it was especially strong in binding dipalmitoylphosphatidylcholine, but failed to bind phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, and phosphatidylserine. Labeled SP-A also exhibited strong binding to distearoylphosphatidylcholine, but weak binding to dimyristoyl-, 1-palmitoyl-2-linoleoyl-, and dilinoleoylphosphatidylcholine. Unlabeled SP-A readily competed with labeled SP-A for phospholipid binding. SP-A strongly bound dipalmitoylglycerol produced by phospholipase C treatment of dipalmitoylphosphatidylcholine, but not palmitic acid. This protein also failed to bind lysophosphatidylcholine produced by phospholipase A2 treatment of dipalmitoylphosphatidylcholine. 125I-SP-A shows almost no binding to dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylethanolamine. The addition of 10 mM EGTA into the binding buffer reduced much of the 125I-SP-A binding to phospholipids. Excess deglycosylated SP-A competed with labeled SP-A for binding to dipalmitoylphosphatidylcholine, but the excess collagenase-resistant fragment of SP-A failed.

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

    PubMed

    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.

  13. Interaction of ruthenium red with Ca2(+)-binding proteins

    SciTech Connect

    Charuk, J.H.; Pirraglia, C.A.; Reithmeier, R.A. )

    1990-07-01

    The interaction of ruthenium red, ((NH3)5Ru-O-Ru(NH3)4-O-Ru(NH3)5)Cl6.4H2O, with various Ca2(+)-binding proteins was studied. Ruthenium red inhibited Ca2+ binding to the sarcoplasmic reticulum protein, calsequestrin, immobilized on Sepharose 4B. Furthermore, ruthenium red bound to calsequestrin with high affinity (Kd = 0.7 microM; Bmax = 218 nmol/mg protein). The dye stained calsequestrin in sodium dodecyl sulfate-polyacrylamide gels or on nitrocellulose paper and was displaced by Ca2+ (Ki = 1.4 mM). The specificity of ruthenium red staining of several Ca2(+)-binding proteins was investigated by comparison with two other detection methods, 45Ca2+ autoradiography and the Stains-all reaction. Ruthenium red bound to the same proteins detected by the 45Ca2+ overlay technique. Ruthenium red stained both the erythrocyte Band 3 anion transporter and the Ca2(+)-ATPase of skeletal muscle sarcoplasmic reticulum. Ruthenium red also stained the EF hand conformation Ca2(+)-binding proteins, calmodulin, troponin C, and S-100. This inorganic dye provides a simple, rapid method for detecting various types of Ca2(+)-binding proteins following electrophoresis.

  14. Modulation of Auxin-Binding Proteins in Cell Suspensions 1

    PubMed Central

    LoSchiavo, Fiorella; Filippini, Francesco; Cozzani, Fabrizio; Vallone, Daniela; Terzi, Mario

    1991-01-01

    This paper shows that the level of 2,4-dichlorophenoxyacetic acid (2,4-D) in the medium determines the level of auxin-binding proteins in the membranes of carrot, Daucus carota, cells grown in suspension. This induction takes slightly more than 2 hours to complete and can be elicited by natural as well as synthetic auxins. The auxin binding sites thus generated, which are pronase-sensitive, bind 2,4-D, indoleacetic acid, and naphthalene-acetic acid (NAA) equally well. However both α- and β-NAA bind, whereas only α-NAA is effective in the inductive process. Cells committed to embryogeny (proembryogenic masses) do not respond to auxin, i.e. their level of auxin-binding proteins remains very low, and they do not seem to synthesize the hormone, as indicated by inhibitor studies. Sensitivity to, and production of, auxin, begins when the embryo becomes polarized, i.e. at postglobular stage. PMID:16668416

  15. Binding Mechanisms of Intrinsically Disordered Proteins: Theory, Simulation, and Experiment

    PubMed Central

    Mollica, Luca; Bessa, Luiza M.; Hanoulle, Xavier; Jensen, Malene Ringkjøbing; Blackledge, Martin; Schneider, Robert

    2016-01-01

    In recent years, protein science has been revolutionized by the discovery of intrinsically disordered proteins (IDPs). In contrast to the classical paradigm that a given protein sequence corresponds to a defined structure and an associated function, we now know that proteins can be functional in the absence of a stable three-dimensional structure. In many cases, disordered proteins or protein regions become structured, at least locally, upon interacting with their physiological partners. Many, sometimes conflicting, hypotheses have been put forward regarding the interaction mechanisms of IDPs and the potential advantages of disorder for protein-protein interactions. Whether disorder may increase, as proposed, e.g., in the “fly-casting” hypothesis, or decrease binding rates, increase or decrease binding specificity, or what role pre-formed structure might play in interactions involving IDPs (conformational selection vs. induced fit), are subjects of intense debate. Experimentally, these questions remain difficult to address. Here, we review experimental studies of binding mechanisms of IDPs using NMR spectroscopy and transient kinetic techniques, as well as the underlying theoretical concepts and numerical methods that can be applied to describe these interactions at the atomic level. The available literature suggests that the kinetic and thermodynamic parameters characterizing interactions involving IDPs can vary widely and that there may be no single common mechanism that can explain the different binding modes observed experimentally. Rather, disordered proteins appear to make combined use of features such as pre-formed structure and flexibility, depending on the individual system and the functional context. PMID:27668217

  16. Metal-binding proteins as metal pollution indicators.

    PubMed Central

    Hennig, H F

    1986-01-01

    The fact that metal-binding proteins are a consequence of elevated metal concentration in organisms is well known. What has been overlooked is that the presence of these proteins provides a unique opportunity to reformulate the criteria of metal pollution. The detoxification effect of metal-binding proteins in animals from polluted areas has been cited, but there have been only very few studies relating metal-binding proteins to pollution. This lack is due partly to the design of most experiments, which were aimed at isolation of metal-binding proteins and hence were of too short duration to allow for correlation to adverse physiological effects on the organism. In this study metal-binding proteins were isolated and characterized from five different marine animals (rock lobster, Jasus lalandii; hermit crab, Diogenes brevirostris; sandshrimp, Palaemon pacificus; black mussel, Choromytilus meridionalis; and limpet, Patella granularis). These animals were kept under identical metal-enriched conditions, hence eliminating differences in method and seasons. The study animals belonged to different phyla; varied in size, mass, age, behavior, food requirements and life stages; and accumulated metals at different rates. It is possible to link unseasonal moulting in crustacea, a known physiological effect due to a metal-enriched environment, to the production of the metal-binding protein without evidence of obvious metal body burden. Thus a new concept of pollution is defined: the presence of metal-binding proteins confirms toxic metal pollution. This concept was then tested under field conditions in the whelk Bullia digitalis and in metal-enriched grass. PMID:3709437

  17. Metal-binding proteins as metal pollution indicators

    SciTech Connect

    Hennig, H.F.

    1986-03-01

    The fact that metal-binding proteins are a consequence of elevated metal concentration in organisms is well known. What has been overlooked is that the presence of these proteins provides a unique opportunity to reformulate the criteria of metal pollution. The detoxification effect of metal-binding proteins in animals from polluted areas has been cited, but there have been only very few studies relating metal-binding proteins to pollution. This lack is due partly to the design of most experiments, which were aimed at isolation of metal-binding proteins and hence were of too short duration to allow for correlation to adverse physiological effects on the organism. In this study metal-binding proteins were isolated and characterized from five different marine animals (rock lobster, Jasus lalandii; hermit crab, Diogenes brevirostris; sandshrimp, Palaemon pacificus; black mussel, Choromytilus meridionalis; and limpet, Patella granularis). These animals were kept under identical metal-enriched conditions, hence eliminating differences in method and seasons. The study animals belonged to different phyla; varied in size, mass, age, behavior, food requirements and life stages; and accumulated metals at different rates. It is possible to link unseasonal moulting in crustacea, a known physiological effect due to a metal-enriched environment, to the production of the metal-binding protein without evidence of obvious metal body burden. Thus a new concept of pollution is defined: the presence of metal-binding proteins confirms toxic metal pollution. This concept was then tested under field conditions in the whelk Bullia digitalis and in metal-enriched grass.

  18. Protein binding elements in the human beta-polymerase promoter.

    PubMed Central

    Englander, E W; Wilson, S H

    1990-01-01

    The core promoter for human DNA polymerase beta contains discrete binding sites for mammalian nuclear proteins, as revealed by DNasel footprinting and gel mobility shift assays. Two sites correspond to sequences identical with the Sp1 factor binding element, and a third site includes an eight residue palindromic sequence, TGACGTCA, known as the CRE element of several cAMP responsive promoters; the 5 to 10 residues flanking this palindrome on each side have no apparent sequence homology with known elements in other promoters. Nuclear extract from a variety of tissues and cells were examined; these included rat liver and testes and cultured cells of human and hamster origin. The DNasel footprint is strong over and around the palindromic element for each of the extracts and is equivalent in size (approximately 22 residues); footprinting over the Sp1 binding sites is seen also. Two potential tissue-specific binding sites, present in liver but not in testes, were found corresponding to residues -13 to -10 and +33 to +48, respectively. Protein binding to the palindromic element was confirmed by an electrophoretic mobility shift assay with the core promoter as probe. Binding specificity of the 22 residue palindromic element, as revealed by oligonucleotide competition, is different from that of AP-1 binding element. Controlled proteolysis with trypsin was used to study structural properties of proteins forming the mobility shift bands. Following digestion with trypsin, most of the palindrome binding activity of each extract corresponded to a sharp, faster migrating band, potentially representing a DNA binding domain of the palindrome binding protein. Images PMID:2315044

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

    PubMed

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

    2004-01-01

    Cornstarch used as a donning powder on natural rubber latex (NRL) gloves adsorbs NRL proteins. During glove use, powder-carried proteins can be aerosolized and can cause allergic reactions in NRL sensitized individuals. The amount of NRL proteins bound to glove powder and its relative relationship to the total amount of proteins on the glove has not been studied, due to the difficulty in measuring proteins on powder. Using the ELISA inhibition assay for NRL proteins [Standard test method for the immunological measurement of antigenic protein in natural rubber and its products. In: The Annual Book of ASTM Standards; ASTM: West Conshohocken, PA, 2000; ASTM D 64-0] we have investigated possible protocol modifications in order to include measurement of proteins bound to glove powder, as well as the water-extractable glove proteins. Possible interference of the starch itself was evaluated by adding clean cornstarch to the assay. No significant interference was observed with powder concentrations below 5 mg/mL. We analyzed 19 extracts of powdered surgical and examination gloves before and after removal of the particulate component. Comparison of NRL glove extracts with, and without, the cornstarch powder fraction indicated significant variations in the ratios of powder-bound protein and corresponding water-extractable protein. The ratios did not appear to correlate with either the total protein on the glove, the glove weight, or the total amount of powder on the glove. However, when virgin glove powders were exposed to NRL proteins, binding was proportional to the protein concentration in the suspension. Temperature in the range from 4 degrees C to 37 degrees C, did not affect binding intensity, while a higher pH resulted in a higher level of protein associated with, or bound to, the starch. The major differences in the propensity for NRL protein binding were observed among different glove powders. The data indicate that the amount of protein that binds to glove powder

  20. Detecting O2 binding sites in protein cavities

    PubMed Central

    Kitahara, Ryo; Yoshimura, Yuichi; Xue, Mengjun; Kameda, Tomoshi; Mulder, Frans A. A.

    2016-01-01

    Internal cavities are important elements in protein structure, dynamics, stability and function. Here we use NMR spectroscopy to investigate the binding of molecular oxygen (O2) to cavities in a well-studied model for ligand binding, the L99A mutant of T4 lysozyme. On increasing the O2 concentration to 8.9 mM, changes in 1H, 15N, and 13C chemical shifts and signal broadening were observed specifically for backbone amide and side chain methyl groups located around the two hydrophobic cavities of the protein. O2-induced longitudinal relaxation enhancements for amide and methyl protons could be adequately accounted for by paramagnetic dipolar relaxation. These data provide the first experimental demonstration that O2 binds specifically to the hydrophobic, and not the hydrophilic cavities, in a protein. Molecular dynamics simulations visualized the rotational and translational motions of O2 in the cavities, as well as the binding and egress of O2, suggesting that the channel consisting of helices D, E, G, H, and J could be the potential gateway for ligand binding to the protein. Due to strong paramagnetic relaxation effects, O2 gas-pressure NMR measurements can detect hydrophobic cavities when populated to as little as 1%, and thereby provide a general and highly sensitive method for detecting oxygen binding in proteins. PMID:26830762

  1. Theoretical studies of binding of mannose-binding protein to monosaccharides

    NASA Astrophysics Data System (ADS)

    Aida-Hyugaji, Sachiko; Takano, Keiko; Takada, Toshikazu; Hosoya, Haruo; Kojima, Naoya; Mizuochi, Tsuguo; Inoue, Yasushi

    2004-11-01

    Binding properties of mannose-binding protein (MBP) to monosaccharides are discussed based on ab initio molecular orbital calculations for cluster models constructed. The calculated binding energies indicate that MBP has an affinity for N-acetyl- D-glucosamine, D-mannose, L-fucose, and D-glucose rather than D-galactose and N-acetyl- D-galactosamine, which is consistent with the biochemical experimental results. Electrostatic potential surfaces at the binding site of four monosaccharides having binding properties matched well with that of MBP. A vacant frontier orbital was found to be localized around the binding site of MBP, suggesting that MBP-monosaccharide interaction may occur through electrostatic and orbital interactions.

  2. Mining the characteristic interaction patterns on protein-protein binding interfaces.

    PubMed

    Li, Yan; Liu, Zhihai; Han, Li; Li, Chengke; Wang, Renxiao

    2013-09-23

    Protein-protein interactions are observed in various biological processes. They are important for understanding the underlying molecular mechanisms and can be potential targets for developing small-molecule regulators of such processes. Previous studies suggest that certain residues on protein-protein binding interfaces are "hot spots". As an extension to this concept, we have developed a residue-based method to identify the characteristic interaction patterns (CIPs) on protein-protein binding interfaces, in which each pattern is a cluster of four contacting residues. Systematic analysis was conducted on a nonredundant set of 1,222 protein-protein binding interfaces selected out of the entire Protein Data Bank. Favored interaction patterns across different protein-protein binding interfaces were retrieved by considering both geometrical and chemical conservations. As demonstrated on two test tests, our method was able to predict hot spot residues on protein-protein binding interfaces with good recall scores and acceptable precision scores. By analyzing the function annotations and the evolutionary tree of the protein-protein complexes in our data set, we also observed that protein-protein interfaces sharing common characteristic interaction patterns are normally associated with identical or similar biological functions.

  3. Assessing Energetic Contributions to Binding from a Disordered Region in a Protein-Protein Interaction

    SciTech Connect

    S Cho; C Swaminathan; D Bonsor; M Kerzic; R Guan; J Yang; C Kieke; P Anderson; D Kranz; et al.

    2011-12-31

    Many functional proteins are at least partially disordered prior to binding. Although the structural transitions upon binding of disordered protein regions can influence the affinity and specificity of protein complexes, their precise energetic contributions to binding are unknown. Here, we use a model protein-protein interaction system in which a locally disordered region has been modified by directed evolution to quantitatively assess the thermodynamic and structural contributions to binding of disorder-to-order transitions. Through X-ray structure determination of the protein binding partners before and after complex formation and isothermal titration calorimetry of the interactions, we observe a correlation between protein ordering and binding affinity for complexes along this affinity maturation pathway. Additionally, we show that discrepancies between observed and calculated heat capacities based on buried surface area changes in the protein complexes can be explained largely by heat capacity changes that would result solely from folding the locally disordered region. Previously developed algorithms for predicting binding energies of protein-protein interactions, however, are unable to correctly model the energetic contributions of the structural transitions in our model system. While this highlights the shortcomings of current computational methods in modeling conformational flexibility, it suggests that the experimental methods used here could provide training sets of molecular interactions for improving these algorithms and further rationalizing molecular recognition in protein-protein interactions.

  4. Ligand binding to a high-energy partially unfolded protein.

    PubMed

    Kasper, Joseph R; Park, Chiwook

    2015-01-01

    The conformational energy landscape of a protein determines populations of all possible conformations of the protein and also determines the kinetics of the conversion between the conformations. Interaction with ligands influences the conformational energy landscapes of proteins and shifts populations of proteins in different conformational states. To investigate the effect of ligand binding on partial unfolding of a protein, we use Escherichia coli dihydrofolate reductase (DHFR) and its functional ligand NADP(+) as a model system. We previously identified a partially unfolded form of DHFR that is populated under native conditions. In this report, we determined the free energy for partial unfolding of DHFR at varying concentrations of NADP(+) and found that NADP(+) binds to the partially unfolded form as well as the native form. DHFR unfolds partially without releasing the ligand, though the binding affinity for NADP(+) is diminished upon partial unfolding. Based on known crystallographic structures of NADP(+) -bound DHFR and the model of the partially unfolded protein we previously determined, we propose that the adenosine-binding domain of DHFR remains folded in the partially unfolded form and interacts with the adenosine moiety of NADP(+) . Our result demonstrates that ligand binding may affect the conformational free energy of not only native forms but also high-energy non-native forms.

  5. A Binding Model and Similarity for Flexible Modular Proteins

    NASA Astrophysics Data System (ADS)

    Máté, Gabriell; Feinauer, Christoph J.; Hofmann, Andreas; Goldt, Sebastian; Liu, Lei; Heermann, Dieter W.

    2013-03-01

    Modular proteins are one of the most commonly found disordered protein motifs. An example is CTCF, a protein that has been named the master waver of the genome i.e., the organizer of the 3D structure of the chromosomes. Using NMR and numerical simulations, much progress has been made in understanding their various functions and ways of binding. Modular proteins are often composed of protein modules interconnected by flexible linkers. They can be imagined as ``beads on a string.'' We argue that when the number of beads is small, these structures behave like a self avoiding random walk. Nevertheless, when binding to a target, linkers can fold in more ordered and stable states. At the same time, folding can influence functional roles. We show that the flexibility of the linkers can boost binding affinity. As a result of flexibility, the conformations of these proteins before and after binding are different. So this implies that generic binding site prediction methods may fail. To deal with this we introduce a new methodology to characterize and compare these flexible structures. Employing topological concepts we propose a method which intrinsically fuses topology and geometry. GM gratefully acknowledges support from the HGS-MathComp and the RTG 1653.

  6. Structural mechanism of the simultaneous binding of two drugs to a multidrug-binding protein

    PubMed Central

    Schumacher, Maria A; Miller, Marshall C; Brennan, Richard G

    2004-01-01

    The structural basis of simultaneous binding of two or more different drugs by any multidrug-binding protein is unknown and also how this can lead to a noncompetitive, uncompetitive or cooperative binding mechanism. Here, we describe the crystal structure of the Staphylococcus aureus multidrug-binding transcription repressor, QacR, bound simultaneously to ethidium (Et) and proflavin (Pf). The structure underscores the plasticity of the multidrug-binding pocket and reveals an alternative, Pf-induced binding mode for Et. To monitor the simultaneous binding of Pf and Et to QacR, as well as to determine the effects on the binding affinity of one drug when the other drug is prebound, a novel application of near-ultraviolet circular dichroism (UVCD) was developed. The UVCD equilibrium-binding studies revealed identical affinities of Pf for QacR in the presence or absence of Et, but significantly diminished affinity of Et for QacR when Pf is prebound, findings that are readily explicable by their structures. The principles for simultaneous binding of two different drugs discerned here are likely employed by the multidrug efflux transporters. PMID:15257299

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

    SciTech Connect

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

    1982-01-01

    A minor cow's whey protein associated with ..beta..-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 ..beta..-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 (lipids/detergents).

  8. Liver takes up retinol-binding protein from plasma

    SciTech Connect

    Gjoen, T.; Bjerkelund, T.; Blomhoff, H.K.; Norum, K.R.; Berg, T.; Blomhoff, R.

    1987-08-15

    Retinol is transported in plasma bound to a specific transport protein, retinol-binding protein. We prepared /sup 125/I-tyramine cellobiose-labeled rat retinol-binding protein and studied its tissue uptake 1, 5, and 24 h after intravenous injection into rats. The liver was the organ containing most radioactivity at all time points studied. After 5 and 24 h, 30 and 22% of the injected dose were recovered in liver, respectively. After separating the liver into parenchymal and nonparenchymal cells in the 5-h group, we found that both cell fractions contained approximately the same amount of radioactivity (per gram of liver). Most of the retinol-binding protein radioactivity in the nonparenchymal cell fraction was in the stellate cells. The implication of these results for a possible transfer mechanism for retinol between parenchymal and stellate cells is discussed.

  9. Single-stranded DNA-binding proteins regulate the abundance of LIM domain and LIM domain-binding proteins

    PubMed Central

    Xu, Zhixiong; Meng, Xianzhang; Cai, Ying; Liang, Hong; Nagarajan, Lalitha; Brandt, Stephen J.

    2007-01-01

    The LIM domain-binding protein Ldb1 is an essential cofactor of LIM-homeodomain (LIM-HD) and LIM-only (LMO) proteins in development. The stoichiometry of Ldb1, LIM-HD, and LMO proteins is tightly controlled in the cell and is likely a critical determinant of their biological actions. Single-stranded DNA-binding proteins (SSBPs) were recently shown to interact with Ldb1 and are also important in developmental programs. We establish here that two mammalian SSBPs, SSBP2 and SSBP3, contribute to an erythroid DNA-binding complex that contains the transcription factors Tal1 and GATA-1, the LIM domain protein Lmo2, and Ldb1 and binds a bipartite E-box-GATA DNA sequence motif. In addition, SSBP2 was found to augment transcription of the Protein 4.2 (P4.2) gene, a direct target of the E-box-GATA-binding complex, in an Ldb1-dependent manner and to increase endogenous Ldb1 and Lmo2 protein levels, E-box-GATA DNA-binding activity, and P4.2 and β-globin expression in erythroid progenitors. Finally, SSBP2 was demonstrated to inhibit Ldb1 and Lmo2 interaction with the E3 ubiquitin ligase RLIM, prevent RLIM-mediated Ldb1 ubiquitination, and protect Ldb1 and Lmo2 from proteasomal degradation. These results define a novel biochemical function for SSBPs in regulating the abundance of LIM domain and LIM domain-binding proteins. PMID:17437998

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

    PubMed Central

    Schroeder, Michael

    2013-01-01

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

  11. Determinants of the plasma protein binding of theophylline in health.

    PubMed Central

    Buss, D; Leopold, D; Smith, A P; Routledge, P A

    1983-01-01

    1 The plasma protein binding of theophylline was determined after addition of [14C]-theophylline (15 micrograms/ml) to plasma from 24 healthy drug-free volunteers and equilibrium dialysis for 2 h at 37 degrees C. 2 The percentage of drug unbound was 60.0% +/- 2.2% (s.d.) with very little variation between individuals. The binding ratio of theophylline was not significantly related to the plasma albumin or alpha 1-acid glycoprotein (AAG) concentrations but was significantly, although weakly, negatively related to the logarithm of the non-esterified fatty acid concentration (NEFA) (r = 0.443, P less than 0.05). 3 Intravenous administration of heparin (1000 units) caused a significant rise in plasma NEFA concentration and in the percentage of drug unbound in plasma after equilibrium dialysis. 4 In human serum albumin solutions, the binding ratio of theophylline was significantly related to the albumin concentration and at the albumin concentration seen in the 24 normal subjects, the percentage of drug unbound was almost identical. Addition of AAG in physiological concentrations did not enhance theophylline binding but oleic acid, and to a lesser extent palmitic acid, reduced binding significantly. 5 The percentage of theophylline unbound in plasma varied markedly with pH so that at pH7 the percentage unbound was 52% greater than at pH 8. There was no evidence of concentration dependence of binding up to 140 micrograms/ml theophylline. 6 Theophylline appears to bind almost exclusively to albumin and its plasma protein binding varies little in healthy subjects, showing no concentration-dependence over the therapeutic range of concentrations. The binding is affected by pH and by NEFA concentration, however, and these factors may be of greater importance in disease states. Caution should be employed in the use of heparin in studies of plasma protein binding of theophylline. PMID:6849774

  12. FRET in a Synthetic Flavin- and Bilin-binding Protein.

    PubMed

    Simon, Julian; Losi, Aba; Zhao, Kai-Hong; Gärtner, Wolfgang

    2017-01-05

    The last decade has seen development and application of a large number of novel fluorescence-based techniques that have revolutionized fluorescence microscopy in life sciences. Preferred tags for such applications are genetically encoded fluorescent proteins (FP), mostly derivatives of the green fluorescent protein (GFP). Combinations of FPs with wavelength-separated absorption/fluorescence properties serve as excellent tools for molecular interaction studies, for example, protein-protein complexes or enzyme-substrate interactions, based on the FRET phenomenon (Förster resonance energy transfer). However, alternatives are requested for experimental conditions where FP proteins or FP couples are not or less efficiently applicable. We here report as a "proof of principle" a specially designed, non-naturally occurring protein (LG1) carrying a combination of a flavin-binding LOV- and a photochromic bilin-binding GAF domain and demonstrate a FRET process between both chromophores.

  13. Leucine/isoleucine/valine-binding protein contracts upon binding of ligand.

    PubMed

    Olah, G A; Trakhanov, S; Trewhella, J; Quiocho, F A

    1993-08-05

    Small-angle x-ray scattering and computer modeling have been used to study the effects of ligand binding to the leucine/isoleucine/valine-binding protein, an initial component of the high-affinity active transport system for branched-chain aliphatic amino acids in Escherichia coli. Measurements were made with no ligand present and with either L-leucine or L-valine present. Upon binding of either leucine or valine, there is a decrease in the radius of gyration, from 23.2 +/- 0.2 to 22.2 +/- 0.2 A, and in the maximum particle dimension, from 82 +/- 3 to 73 +/- 3 A. The x-ray structure of the unbound form has been determined and gives a radius of gyration and a maximum dimension consistent with the values found for the solution structure in this study (Sack, J. S., Saper, M. A., and Quiocho, F. A. (1989) J. Mol. Biol. 206, 171-191). The reduction in the radius of gyration and maximum dimension upon ligand binding can be accounted for by a substrate-induced cleft closure in a combined "hinge-twist" motion. Modeling of the substrate-bound state was done by comparison of this protein with another periplasmic binding protein (L-arabinose-binding protein), which possesses a similar two-lobe structure and for which the x-ray structure is known in its ligand-bound form.

  14. The biotin repressor: thermodynamic coupling of corepressor binding, protein assembly, and sequence-specific DNA binding.

    PubMed

    Streaker, Emily D; Gupta, Aditi; Beckett, Dorothy

    2002-12-03

    The Escherichia coli biotin repressor, an allosteric transcriptional regulator, is activated for binding to the biotin operator by the small molecule biotinyl-5'-AMP. Results of combined thermodynamic, kinetic, and structural studies of the protein have revealed that corepressor binding results in disorder to order transitions in the protein monomer that facilitate tighter dimerization. The enhanced stability of the dimer leads to stabilization of the resulting biotin repressor-biotin operator complex. It is not clear, however, that the allosteric response in the system is transmitted solely through the protein-protein interface. In this work, the allosteric mechanism has been quantitatively probed by measuring the biotin operator binding and dimerization properties of three biotin repressor species: the apo or unliganded form, the biotin-bound form, and the holo or bio-5'-AMP-bound form. Comparisons of the pairwise differences in the bioO binding and dimerization energetics for the apo and holo species reveal that the enhanced DNA binding energetics resulting from adenylate binding track closely with the enhanced assembly energetics. However, when the results for repressor pairs that include the biotin-bound species are compared, no such equivalence is observed.

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

    PubMed

    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).

  16. Natural history of S-adenosylmethionine-binding proteins

    PubMed Central

    Kozbial, Piotr Z; Mushegian, Arcady R

    2005-01-01

    Background S-adenosylmethionine is a source of diverse chemical groups used in biosynthesis and modification of virtually every class of biomolecules. The most notable reaction requiring S-adenosylmethionine, transfer of methyl group, is performed by a large class of enzymes, S-adenosylmethionine-dependent methyltransferases, which have been the focus of considerable structure-function studies. Evolutionary trajectories of these enzymes, and especially of other classes of S-adenosylmethionine-binding proteins, nevertheless, remain poorly understood. We addressed this issue by computational comparison of sequences and structures of various S-adenosylmethionine-binding proteins. Results Two widespread folds, Rossmann fold and TIM barrel, have been repeatedly used in evolution for diverse types of S-adenosylmethionine conversion. There were also cases of recruitment of other relatively common folds for S-adenosylmethionine binding. Several classes of proteins have unique unrelated folds, specialized for just one type of chemistry and unified by the theme of internal domain duplications. In several cases, functional divergence is evident, when evolutionarily related enzymes have changed the mode of binding and the type of chemical transformation of S-adenosylmethionine. There are also instances of functional convergence, when biochemically similar processes are performed by drastically different classes of S-adenosylmethionine-binding proteins. Comparison of remote sequence similarities and analysis of phyletic patterns suggests that the last universal common ancestor of cellular life had between 10 and 20 S-adenosylmethionine-binding proteins from at least 5 fold classes, providing for S-adenosylmethionine formation, polyamine biosynthesis, and methylation of several substrates, including nucleic acids and peptide chain release factor. Conclusion We have observed several novel relationships between families that were not known to be related before, and defined 15

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

    PubMed Central

    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.

  18. Lactation-induced cadmium-binding proteins

    SciTech Connect

    Bhattacharyya, M.H.; Solaiman, D.; Garvey, J.S.; Miyazaki, W.Y.

    1987-01-01

    Previously we have demonstrated an increase during midlactation in /sup 109/Cd adsorption and increased retention by the duodenum, kidney, and mammary tissue of mouse dams receiving environmental levels of cadmium//sup 109/Cd via drinking water, with little change in /sup 109/Cd retention in liver and jejunum compared to nonpregnant controls. Results are reported here of a study of cadmium deposition during midlactation as associated with induction of metallothionein (MT). A cadmium/hemoglobin (Cd/Hb) assay and radioimmunoassay for MT which measures heat-stable cadmium binding capacity in tissues was used to determine MT concentrations in fractions of kidney, liver, duodenum, and jejunum from female mice. Both assays demonstrated clear lactation-induced increases in MT concentrations in liver, kidney, and duodenum, with MT concentrations falling rapidly to control levels after weaning. 4 refs., 1 tab.

  19. Camptothecin-binding site in human serum albumin and protein transformations induced by drug binding.

    PubMed

    Fleury, F; Ianoul, A; Berjot, M; Feofanov, A; Alix, A J; Nabiev, I

    1997-07-14

    Circular dichroism (CD) and Raman spectroscopy were employed in order to locate a camptothecin (CPT)-binding site within human serum albumin (HSA) and to identify protein structural transformations induced by CPT binding. A competitive binding of CPT and 3'-azido-3'-deoxythymidine (a ligand occupying IIIA structural sub-domain of the protein) to HSA does not show any competition and demonstrates that the ligands are located in the different binding sites, whereas a HSA-bound CPT may be replaced by warfarin, occupying IIA structural sub-domain of the protein. Raman and CD spectra of HSA and HSA/CPT complexes show that the CPT-binding does not induce changes of the global protein secondary structure. On the other hand, Raman spectra reveal pronounced CPT-induced local structural modifications of the HSA molecule, involving changes in configuration of the two disulfide bonds and transfer of a single Trp-residue to hydrophilic environment. These data suggest that CPT is bound in the region of interdomain connections within the IIA structural domain of HSA and it induces relative movement of the protein structural domains.

  20. Plasmodium falciparum AMA-1 erythrocyte binding peptides implicate AMA-1 as erythrocyte binding protein.

    PubMed

    Urquiza, M; Suarez, J E; Cardenas, C; Lopez, R; Puentes, A; Chavez, F; Calvo, J C; Patarroyo, M E

    2000-10-15

    The role of AMA-1 during merozoite invasion has not yet been determined. However, reported experimental evidence suggests that this protein can be used, in particular as erythrocyte-binding protein, since, Fab fragments against this protein are able to block merozoite invasion. Using a previously described methodology, eight peptides with high binding activity to human erythrocyte, scattered along the different domains and having around 130 nM affinity constants, were identified in the Plasmodium falciparum AMA-1 protein. Their binding activity was sialic acid independent. Some of these peptides showed homology with the erythrocyte binding domains of one of the apical organelle protein family, MAEBL, identified in rodent malarial parasites. One of these peptides shares amino acid sequence with a previously reported B-cell epitope which induces antibodies to block parasite growth. The critical residues were identified for erythrocyte binding conserved peptides 4313 (DAEVAGTQYRLPSGKCPVFG), 4321 (VVDNWEKVCPRKNLQNAKFG), 4325 (MIKSAFLPTGAFKADRYKSH) and 4337 (WGEEKRASHTTPVLMEKPYY). All conserved peptides were able to block merozoite invasion of new RBC and development, suggesting that these peptides are involved in P. falciparum invasion.

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

    PubMed Central

    Liu, Jiajian; Stormo, Gary D

    2005-01-01

    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. PMID:16014175

  2. Metal binding proteins, recombinant host cells and methods

    DOEpatents

    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.

  3. Characterization of the cellulose-binding domain of the Clostridium cellulovorans cellulose-binding protein A.

    PubMed Central

    Goldstein, M A; Takagi, M; Hashida, S; Shoseyov, O; Doi, R H; Segel, I H

    1993-01-01

    Cellulose-binding protein A (CbpA), a component of the cellulase complex of Clostridium cellulovorans, contains a unique sequence which has been demonstrated to be a cellulose-binding domain (CBD). The DNA coding for this putative CBD was subcloned into pET-8c, an Escherichia coli expression vector. The protein produced under the direction of the recombinant plasmid, pET-CBD, had a high affinity for crystalline cellulose. Affinity-purified CBD protein was used in equilibrium binding experiments to characterize the interaction of the protein with various polysaccharides. It was found that the binding capacity of highly crystalline cellulose samples (e.g., cotton) was greater than that of samples of low crystallinity (e.g., fibrous cellulose). At saturating CBD concentration, about 6.4 mumol of protein was bound by 1 g of cotton. Under the same conditions, fibrous cellulose bound only 0.2 mumol of CBD per g. The measured dissociation constant was in the 1 microM range for all cellulose samples. The results suggest that the CBD binds specifically to crystalline cellulose. Chitin, which has a crystal structure similar to that of cellulose, also was bound by the CBD. The presence of high levels of cellobiose or carboxymethyl cellulose in the assay mixture had no effect on the binding of CBD protein to crystalline cellulose. This result suggests that the CBD recognition site is larger than a simple cellobiose unit or more complex than a repeating cellobiose moiety. This CBD is of particular interest because it is the first CBD from a completely sequenced nonenzymatic protein shown to be an independently functional domain. Images PMID:8376323

  4. Mercury-binding proteins from the marine mussel, Mytilus edulis.

    PubMed Central

    Roesijadi, G

    1986-01-01

    The marine mussel, Mytilus edulis, possesses low molecular weight, metal-binding proteins which can be induced by and, in turn, bind mercury when individuals are exposed to low, but elevated concentrations of mercury as HgCl2. Induction of the proteins by exposure of mussels to copper, cadmium, or mercury is associated with enhanced tolerance to mercury toxicity. Mercury-binding proteins isolated from gills of mussels occur as two molecular weight variants of about 20-25 and 10-12 kdaltons, respectively, on Sephadex G-75. These have been designated as HgBP20 and HgBP10 following the nomenclature used for cadmium-binding proteins. HgBP20 represents the primary mercury-binding species. These exist as dimers which can be dissociated into subunits by treatment with 1% 2-mercaptoethanol. Further purification of HgBP20 by DEAE-cellulose ion-exchange chromatography resulted in the resolution of three major mercury-binding protein peaks; analysis of two of these showed that both had similar amino acid compositions with 26% half-cystine, 16% glycine, and very low levels of the aromatic amino acids phenylalanine and tyrosine (0.3-0.5%), histidine (0.4%), methionine (about 0.5%), and leucine (about 1%). These are similar to the compositions of proteins reported as mussel thioneins by others. Separation of HgBP20 by anion-exchange high-performance liquid chromatography resulted in the resolution of six peaks, indicating a more complex situation than was evident from DEAE-cellulose separations. Although not completely purified, these also contain cysteine- and glycine-rich proteins. PMID:3709464

  5. Evaluation of silica nanoparticle binding to major human blood proteins

    NASA Astrophysics Data System (ADS)

    Hata, Katsutomo; Higashisaka, Kazuma; Nagano, Kazuya; Mukai, Yohei; Kamada, Haruhiko; Tsunoda, Shin-ichi; Yoshioka, Yasuo; Tsutsumi, Yasuo

    2014-12-01

    Nanomaterials are used for various biomedical applications because they are often more effective than conventional materials. Recently, however, it has become clear that the protein corona that forms on the surface of nanomaterials when they make contact with biological fluids, such as blood, influences the pharmacokinetics and biological responses induced by the nanomaterials. Therefore, when evaluating nanomaterial safety and efficacy, it is important to analyze the interaction between nanomaterials and proteins in biological fluids and to evaluate the effects of the protein corona. Here, we evaluated the interaction of silica nanoparticles, a commonly used nanomaterial, with the human blood proteins albumin, transferrin, fibrinogen, and IgG. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the amount of albumin, transferrin, and IgG binding to the silica particles increased as the particle size decreased under conditions where the silica particle mass remained the same. However, under conditions in which the specific surface area remained constant, there were no differences in the binding of human plasma proteins to the silica particles tested, suggesting that the binding of silica particles with human plasma proteins is dependent on the specific surface area of the silica particles. Furthermore, the amount of albumin, transferrin, and IgG binding to silica nanoparticles with a diameter of 70 nm (nSP70) and a functional amino group was lower than that with unmodified nSP70, although there was no difference in the binding between nSP70 with the surface modification of a carboxyl functional group and nSP70. These results suggest that the characteristics of nanomaterials are important for binding with human blood proteins; this information may contribute to the development of safe and effective nanomaterials.

  6. Carotenoid Antenna Binding and Function in Retinal Proteins

    DTIC Science & Technology

    2012-08-13

    REPORT Carotenoid antenna binding and function in retinal proteins 14. ABSTRACT 16. SECURITY CLASSIFICATION OF: Xanthorhodopsin, a proton pump from the...eubacterium Salinibacter ruber, is a unique dual chromophore system that contains, in addition to retinal, the carotenoid salinixanthin as a light... carotenoid ring near the retinal ring. Substitution of the small glycine with bulky tryptophan in this site eliminates binding. The second factor is the 4

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

    PubMed

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

    2014-02-21

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

  8. Protein D of Haemophilus influenzae is not a universal immunoglobulin D-binding protein.

    PubMed Central

    Sasaki, K; Munson, R S

    1993-01-01

    Haemophilus influenzae type b and nontypeable H. influenzae have been reported to bind human immunoglobulin D (IgD). IgD myeloma sera from five patients were tested for the ability of IgD to bind to H. influenzae. Serotype b strains bound human IgD in four of the five sera tested. IgD in the fifth serum bound strongly to type b strain MinnA but poorly to other type b strains. Additionally, IgD binding was not observed when nontypeable strains were tested. The gene for protein D, the putative IgD-binding protein, was cloned from the IgD-binding H. influenzae type b strain MinnA and expressed in Escherichia coli. IgD binding to E. coli expressing protein D was not demonstrable. Recombinant protein D was purified, and antisera were generated in rabbits. Using these rabbit sera, we detected protein D in nontypeable as well as serotype b strains by Western blotting (immunoblotting). In contrast, IgD myeloma protein 4490, which was previously reported to bind to protein D by Ruan and coworkers (M. Ruan, M. Akkoyunlu, A. Grubb, and A. Forsgren, J. Immunol. 145:3379-3384), bound strongly to both type b and nontypeable H. influenzae as well as to E. coli expressing protein D. Thus, IgD binding is a general property of H. influenzae type b strains but not a general property of nontypeable strains, although both type b and nontypeable strains produce protein D. With the exception of IgD myeloma protein 4490 binding, we have no evidence for a role of protein D in IgD binding to H. influenzae. Images PMID:8514409

  9. Universal protein binding microarrays for the comprehensive characterization of the DNA binding specificities of transcription factors

    PubMed Central

    Berger, Michael F.; Bulyk, Martha L.

    2010-01-01

    Protein binding microarray (PBM) technology provides a rapid, high-throughput means of characterizing the in vitro DNA binding specificities of transcription factors (TFs). Using high-density, custom-designed microarrays containing all 10-mer sequence variants, one can obtain comprehensive binding site measurements for any TF, regardless of its structural class or species of origin. Here, we present a protocol for the examination and analysis of TF binding specificities at high resolution using such ‘all 10-mer’ universal PBMs. This procedure involves double-stranding a commercially synthesized DNA oligonucleotide array, binding a TF directly to the double-stranded DNA microarray, and labeling the protein-bound microarray with a fluorophore-conjugated antibody. We describe how to computationally extract the relative binding preferences of the examined TF for all possible contiguous and gapped 8-mers over the full range of affinities, from highest affinity sites to nonspecific sites. Multiple proteins can be tested in parallel in separate chambers on a single microarray, enabling the processing of a dozen or more TFs in a single day. PMID:19265799

  10. DNA-binding properties of ARID family proteins

    PubMed Central

    Patsialou, Antonia; Wilsker, Deborah; Moran, Elizabeth

    2005-01-01

    The ARID (A–T Rich Interaction Domain) is a helix–turn–helix motif-based DNA-binding domain, conserved in all eukaryotes and diagnostic of a family that includes 15 distinct human proteins with important roles in development, tissue-specific gene expression and proliferation control. The 15 human ARID family proteins can be divided into seven subfamilies based on the degree of sequence identity between individual members. Most ARID family members have not been characterized with respect to their DNA-binding behavior, but it is already apparent that not all ARIDs conform to the pattern of binding AT-rich sequences. To understand better the divergent characteristics of the ARID proteins, we undertook a survey of DNA-binding properties across the entire ARID family. The results indicate that the majority of ARID subfamilies (i.e. five out of seven) bind DNA without obvious sequence preference. DNA-binding affinity also varies somewhat between subfamilies. Site-specific mutagenesis does not support suggestions made from structure analysis that specific amino acids in Loop 2 or Helix 5 are the main determinants of sequence specificity. Most probably, this is determined by multiple interacting differences across the entire ARID structure. PMID:15640446

  11. Escherchia coli ribose binding protein based bioreporters revisited

    PubMed Central

    Reimer, Artur; Yagur-Kroll, Sharon; Belkin, Shimshon; Roy, Shantanu; van der Meer, Jan Roelof

    2014-01-01

    Bioreporter bacteria, i.e., strains engineered to respond to chemical exposure by production of reporter proteins, have attracted wide interest because of their potential to offer cheap and simple alternative analytics for specified compounds or conditions. Bioreporter construction has mostly exploited the natural variation of sensory proteins, but it has been proposed that computational design of new substrate binding properties could lead to completely novel detection specificities at very low affinities. Here we reconstruct a bioreporter system based on the native Escherichia coli ribose binding protein RbsB and one of its computationally designed variants, reported to be capable of binding 2,4,6-trinitrotoluene (TNT). Our results show in vivo reporter induction at 50 nM ribose, and a 125 nM affinity constant for in vitro ribose binding to RbsB. In contrast, the purified published TNT-binding variant did not bind TNT nor did TNT cause induction of the E. coli reporter system. PMID:25005019

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

    PubMed

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

    2017-02-02

    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.

  13. Novel DNA-binding properties of the RNA-binding protein TIAR.

    PubMed

    Suswam, Esther A; Li, Yan Yan; Mahtani, Harry; King, Peter H

    2005-01-01

    TIA-1 related protein binds avidly to uridine-rich elements in mRNA and pre-mRNAs of a wide range of genes, including interleukin (IL)-8 and vascular endothelial growth factor (VEGF). The protein has diverse regulatory roles, which in part depend on the locus of binding within the transcript, including translational control, splicing and apoptosis. Here, we observed selective and potent inhibition of TIAR-RNP complex formation with IL-8 and VEGF 3'-untranslated regions (3'-UTRs) using thymidine-rich deoxyoligonucleotide (ODN) sequences derived from the VEFG 3'-UTR. We show by ultraviolet crosslinking and electrophoretic mobility shift assays that TIAR can bind directly to single-stranded, thymidine-rich ODNs but not to double-stranded ODNs containing the same sequence. TIAR had a nearly 6-fold greater affinity for DNA than RNA (K(d)app = 1.6x10(-9) M versus 9.4 x 10(-9) M). Truncation of TIAR indicated that the high affinity DNA-binding site overlaps with the RNA-binding site involving RNA recognition motif 2 (RRM2). However, RRM1 alone could also bind to DNA. Finally, we show that TIAR can be displaced from single-stranded DNA by active transcription through the binding site. These results provide a potential mechanism by which TIAR can shuttle between RNA and DNA ligands.

  14. Zinc-protein from rat prostate fluid binds epididymal spermatozoa.

    PubMed

    Sansone, G; Abrescia, P

    1991-09-01

    The detection and the isolation of a zinc-protein from the secretion of the rat dorsolateral prostate is described. The purification procedure, based on gel filtration and cationic exchange chromatography, allowed to separate a minor protein (Mr approximately 66,000) from free zinc ions and other secretory components. Two zinc ions were estimated to be associated with one molecule of isolated protein. The zinc-protein was labelled with 125I and then incubated at 37 degrees C with spermatozoa from rat epididymal cauda. Time-dependent in vitro binding of the radioactive protein to sperm cells was demonstrated. This binding was not affected by the presence of proteins from the seminal vesicle during the incubation, while it was blocked in the presence of an excess of unlabelled zinc-protein. After binding, the labelled spermatozoa were treated with a buffer containing 0.5% sodium deoxycholate and 40 mM EDTA; only very small amounts of label were removed from the cells, thus suggesting that the zinc-proteins were kept on the plasma membrane by interactions which do not involve merely hydrophobic bonds.

  15. Grafting odorant binding proteins on diamond bio-MEMS.

    PubMed

    Manai, R; Scorsone, E; Rousseau, L; Ghassemi, F; Possas Abreu, M; Lissorgues, G; Tremillon, N; Ginisty, H; Arnault, J-C; Tuccori, E; Bernabei, M; Cali, K; Persaud, K C; Bergonzo, P

    2014-10-15

    Odorant binding proteins (OBPs) are small soluble proteins found in olfactory systems that are capable of binding several types of odorant molecules. Cantilevers based on polycrystalline diamond surfaces are very promising as chemical transducers. Here two methods were investigated for chemically grafting porcine OBPs on polycrystalline diamond surfaces for biosensor development. The first approach resulted in random orientation of the immobilized proteins over the surface. The second approach based on complexing a histidine-tag located on the protein with nickel allowed control of the proteins' orientation. Evidence confirming protein grafting was obtained using electrochemical impedance spectroscopy, fluorescence imaging and X-ray photoelectron spectroscopy. The chemical sensing performances of these OBP modified transducers were assessed. The second grafting method led to typically 20% more sensitive sensors, as a result of better access of ligands to the proteins active sites and also perhaps a better yield of protein immobilization. This new grafting method appears to be highly promising for further investigation of the ligand binding properties of OBPs in general and for the development of arrays of non-specific biosensors for artificial olfaction applications.

  16. Bovine coronavirus nonstructural protein 1 (p28) is an RNA binding protein that binds terminal genomic cis-replication elements.

    PubMed

    Gustin, Kortney M; Guan, Bo-Jhih; Dziduszko, Agnieszka; Brian, David A

    2009-06-01

    Nonstructural protein 1 (nsp1), a 28-kDa protein in the bovine coronavirus (BCoV) and closely related mouse hepatitis coronavirus, is the first protein cleaved from the open reading frame 1 (ORF 1) polyprotein product of genome translation. Recently, a 30-nucleotide (nt) cis-replication stem-loop VI (SLVI) has been mapped at nt 101 to 130 within a 288-nt 5'-terminal segment of the 738-nt nsp1 cistron in a BCoV defective interfering (DI) RNA. Since a similar nsp1 coding region appears in all characterized groups 1 and 2 coronavirus DI RNAs and must be translated in cis for BCoV DI RNA replication, we hypothesized that nsp1 might regulate ORF 1 expression by binding this intra-nsp1 cistronic element. Here, we (i) establish by mutation analysis that the 72-nt intracistronic SLV immediately upstream of SLVI is also a DI RNA cis-replication signal, (ii) show by gel shift and UV-cross-linking analyses that cellular proteins of approximately 60 and 100 kDa, but not viral proteins, bind SLV and SLVI, (SLV-VI) and (iii) demonstrate by gel shift analysis that nsp1 purified from Escherichia coli does not bind SLV-VI but does bind three 5' untranslated region (UTR)- and one 3' UTR-located cis-replication SLs. Notably, nsp1 specifically binds SLIII and its flanking sequences in the 5' UTR with approximately 2.5 muM affinity. Additionally, under conditions enabling expression of nsp1 from DI RNA-encoded subgenomic mRNA, DI RNA levels were greatly reduced, but there was only a slight transient reduction in viral RNA levels. These results together indicate that nsp1 is an RNA-binding protein that may function to regulate viral genome translation or replication but not by binding SLV-VI within its own coding region.

  17. BindUP: a web server for non-homology-based prediction of DNA and RNA binding proteins.

    PubMed

    Paz, Inbal; Kligun, Efrat; Bengad, Barak; Mandel-Gutfreund, Yael

    2016-07-08

    Gene expression is a multi-step process involving many layers of regulation. The main regulators of the pathway are DNA and RNA binding proteins. While over the years, a large number of DNA and RNA binding proteins have been identified and extensively studied, it is still expected that many other proteins, some with yet another known function, are awaiting to be discovered. Here we present a new web server, BindUP, freely accessible through the website http://bindup.technion.ac.il/, for predicting DNA and RNA binding proteins using a non-homology-based approach. Our method is based on the electrostatic features of the protein surface and other general properties of the protein. BindUP predicts nucleic acid binding function given the proteins three-dimensional structure or a structural model. Additionally, BindUP provides information on the largest electrostatic surface patches, visualized on the server. The server was tested on several datasets of DNA and RNA binding proteins, including proteins which do not possess DNA or RNA binding domains and have no similarity to known nucleic acid binding proteins, achieving very high accuracy. BindUP is applicable in either single or batch modes and can be applied for testing hundreds of proteins simultaneously in a highly efficient manner.

  18. Behind the scenes of vitamin D binding protein: more than vitamin D binding.

    PubMed

    Delanghe, Joris R; Speeckaert, Reinhart; Speeckaert, Marijn M

    2015-10-01

    Although being discovered in 1959, the number of published papers in recent years reveals that vitamin D binding protein (DBP), a member of the albuminoid superfamily, is a hot research topic. Besides the three major phenotypes (DBP1F, DBP1S and DBP2), more than 120 unique variants have been described of this polymorphic protein. The presence of DBP has been demonstrated in different body fluids (serum, urine, breast milk, ascitic fluid, cerebrospinal fluid, saliva and seminal fluid) and organs (brain, heart, lungs, kidneys, placenta, spleen, testes and uterus). Although the major function is binding, solubilization and transport of vitamin D and its metabolites, the name of this glycoprotein hides numerous other important biological functions. In this review, we will focus on the analytical aspects of the determination of DBP and discuss in detail the multifunctional capacity [actin scavenging, binding of fatty acids, chemotaxis, binding of endotoxins, influence on T cell response and influence of vitamin D binding protein-macrophage activating factor (DBP-MAF) on bone metabolism and cancer] of this abundant plasma protein.

  19. Reprogramming cellular events by poly(ADP-ribose)-binding proteins

    PubMed Central

    Pic, Émilie; Ethier, Chantal; Dawson, Ted M.; Dawson, Valina L.; Masson, Jean-Yves; Poirier, Guy G.; Gagné, Jean-Philippe

    2013-01-01

    Poly(ADP-ribosyl)ation is a posttranslational modification catalyzed by the poly(ADP-ribose) polymerases (PARPs). These enzymes covalently modify glutamic, aspartic and lysine amino acid side chains of acceptor proteins by the sequential addition of ADP-ribose (ADPr) units. The poly(ADP-ribose) (pADPr) polymers formed alter the physico-chemical characteristics of the substrate with functional consequences on its biological activities. Recently, non-covalent binding to pADPr has emerged as a key mechanism to modulate and coordinate several intracellular pathways including the DNA damage response, protein stability and cell death. In this review, we describe the basis of non-covalent binding to pADPr that has led to the emerging concept of pADPr-responsive signaling pathways. This review emphasizes the structural elements and the modular strategies developed by pADPr-binding proteins to exert a fine-tuned control of a variety of pathways. Poly(ADP-ribosyl)ation reactions are highly regulated processes, both spatially and temporally, for which at least four specialized pADPr-binding modules accommodate different pADPr structures and reprogram protein functions. In this review, we highlight the role of well-characterized and newly discovered pADPr-binding modules in a diverse set of physiological functions. PMID:23268355

  20. Solvation structure of ice-binding antifreeze proteins

    NASA Astrophysics Data System (ADS)

    Hansen-Goos, Hendrik; Wettlaufer, John

    2009-03-01

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

  1. Disulfide bridge regulates ligand-binding site selectivity in liver bile acid-binding proteins.

    PubMed

    Cogliati, Clelia; Tomaselli, Simona; Assfalg, Michael; Pedò, Massimo; Ferranti, Pasquale; Zetta, Lucia; Molinari, Henriette; Ragona, Laura

    2009-10-01

    Bile acid-binding proteins (BABPs) are cytosolic lipid chaperones that play central roles in driving bile flow, as well as in the adaptation to various pathological conditions, contributing to the maintenance of bile acid homeostasis and functional distribution within the cell. Understanding the mode of binding of bile acids with their cytoplasmic transporters is a key issue in providing a model for the mechanism of their transfer from the cytoplasm to the nucleus, for delivery to nuclear receptors. A number of factors have been shown to modulate bile salt selectivity, stoichiometry, and affinity of binding to BABPs, e.g. chemistry of the ligand, protein plasticity and, possibly, the formation of disulfide bridges. Here, the effects of the presence of a naturally occurring disulfide bridge on liver BABP ligand-binding properties and backbone dynamics have been investigated by NMR. Interestingly, the disulfide bridge does not modify the protein-binding stoichiometry, but has a key role in modulating recognition at both sites, inducing site selectivity for glycocholic and glycochenodeoxycholic acid. Protein conformational changes following the introduction of a disulfide bridge are small and located around the inner binding site, whereas significant changes in backbone motions are observed for several residues distributed over the entire protein, both in the apo form and in the holo form. Site selectivity appears, therefore, to be dependent on protein mobility rather than being governed by steric factors. The detected properties further establish a parallelism with the behaviour of human ileal BABP, substantiating the proposal that BABPs have parallel functions in hepatocytes and enterocytes.

  2. Holo- And Apo- Structures of Bacterial Periplasmic Heme Binding Proteins

    SciTech Connect

    Ho, W.W.; Li, H.; Eakanunkul, S.; Tong, Y.; Wilks, A.; Guo, M.; Poulos, T.L.

    2009-06-01

    An essential component of heme transport in Gram-negative bacterial pathogens is the periplasmic protein that shuttles heme between outer and inner membranes. We have solved the first crystal structures of two such proteins, ShuT from Shigella dysenteriae and PhuT from Pseudomonas aeruginosa. Both share a common architecture typical of Class III periplasmic binding proteins. The heme binds in a narrow cleft between the N- and C-terminal binding domains and is coordinated by a Tyr residue. A comparison of the heme-free (apo) and -bound (holo) structures indicates little change in structure other than minor alterations in the heme pocket and movement of the Tyr heme ligand from an 'in' position where it can coordinate the heme iron to an 'out' orientation where it points away from the heme pocket. The detailed architecture of the heme pocket is quite different in ShuT and PhuT. Although Arg{sup 228} in PhuT H-bonds with a heme propionate, in ShuT a peptide loop partially takes up the space occupied by Arg{sup 228}, and there is no Lys or Arg H-bonding with the heme propionates. A comparison of PhuT/ShuT with the vitamin B{sub 12}-binding protein BtuF and the hydroxamic-type siderophore-binding protein FhuD, the only two other structurally characterized Class III periplasmic binding proteins, demonstrates that PhuT/ShuT more closely resembles BtuF, which reflects the closer similarity in ligands, heme and B{sub 12}, compared with ligands for FhuD, a peptide siderophore.

  3. Actin-binding proteins take the reins in growth cones.

    PubMed

    Pak, Chi W; Flynn, Kevin C; Bamburg, James R

    2008-02-01

    Higher-order actin-based networks (actin superstructures) are important for growth-cone motility and guidance. Principles for generating, organizing and remodelling actin superstructures have emerged from recent findings in cell-free systems, non-neuronal cells and growth cones. This Review examines how actin superstructures are initiated de novo at the leading-edge membrane and how the spontaneous organization of actin superstructures is driven by ensembles of actin-binding proteins. How the regulation of actin-binding proteins can affect growth-cone turning and axonal regeneration is also discussed.

  4. Functional interactions between polypyrimidine tract binding protein and PRI peptide ligand containing proteins.

    PubMed

    Coelho, Miguel B; Ascher, David B; Gooding, Clare; Lang, Emma; Maude, Hannah; Turner, David; Llorian, Miriam; Pires, Douglas E V; Attig, Jan; Smith, Christopher W J

    2016-08-15

    Polypyrimidine tract binding protein (PTBP1) is a heterogeneous nuclear ribonucleoprotein (hnRNP) that plays roles in most stages of the life-cycle of pre-mRNA and mRNAs in the nucleus and cytoplasm. PTBP1 has four RNA binding domains of the RNA recognition motif (RRM) family, each of which can bind to pyrimidine motifs. In addition, RRM2 can interact via its dorsal surface with proteins containing short peptide ligands known as PTB RRM2 interacting (PRI) motifs, originally found in the protein Raver1. Here we review our recent progress in understanding the interactions of PTB with RNA and with various proteins containing PRI ligands.

  5. Quantitative analysis of pheromone-binding protein specificity

    PubMed Central

    Katti, S.; Lokhande, N.; González, D.; Cassill, A.; Renthal, R.

    2012-01-01

    Many pheromones have very low water solubility, posing experimental difficulties for quantitative binding measurements. A new method is presented for determining thermodynamically valid dissociation constants for ligands binding to pheromone-binding proteins (OBPs), using β-cyclodextrin as a solubilizer and transfer agent. The method is applied to LUSH, a Drosophila OBP that binds the pheromone 11-cis vaccenyl acetate (cVA). Refolding of LUSH expressed in E. coli was assessed by measuring N-phenyl-1-naphthylamine (NPN) binding and Förster resonance energy transfer between LUSH tryptophan 123 (W123) and NPN. Binding of cVA was measured from quenching of W123 fluorescence as a function of cVA concentration. The equilibrium constant for transfer of cVA between β-cyclodextrin and LUSH was determined from a linked equilibria model. This constant, multiplied by the β-cyclodextrin-cVA dissociation constant, gives the LUSH-cVA dissociation constant: ~100 nM. It was also found that other ligands quench W123 fluorescence. The LUSH-ligand dissociation constants were determined to be ~200 nM for the silk moth pheromone bombykol and ~90 nM for methyl oleate. The results indicate that the ligand-binding cavity of LUSH can accommodate a variety ligands with strong binding interactions. Implications of this for the pheromone receptor model proposed by Laughlin et al. (Cell 133: 1255–65, 2008) are discussed. PMID:23121132

  6. Conserved Odorant-Binding Proteins from Aphids and Eavesdropping Predators

    PubMed Central

    Vandermoten, Sophie; Francis, Frédéric; Haubruge, Eric; Leal, Walter S.

    2011-01-01

    Background The sesquiterpene (E)-ß-farnesene is the main component of the alarm pheromone system of various aphid species studied to date, including the English grain aphid, Sitobion avenae. Aphid natural enemies, such as the marmalade hoverfly Episyrphus balteatus and the multicolored Asian lady beetle Harmonia axyridis, eavesdrop on aphid chemical communication and utilize (E)-ß-farnesene as a kairomone to localize their immediate or offspring preys. These aphid-predator systems are important models to study how the olfactory systems of distant insect taxa process the same chemical signal. We postulated that odorant-binding proteins (OBPs), which are highly expressed in insect olfactory tissues and involved in the first step of odorant reception, have conserved regions involved in binding (E)-ß-farnesene. Methodology We cloned OBP genes from the English grain aphid and two major predators of this aphid species. We then expressed these proteins and compare their binding affinities to the alarm pheromone/kairomone. By using a fluorescence reporter, we tested binding of (E)-ß-farnesene and other electrophysiologically and behaviorally active compounds, including a green leaf volatile attractant. Conclusion We found that OBPs from disparate taxa of aphids and their predators are highly conserved proteins, with apparently no orthologue genes in other insect species. Properly folded, recombinant proteins from the English grain aphid, SaveOBP3, and the marmalade hoverfly, EbalOBP3, specifically bind (E)-ß-farnesene with apparent high affinity. For the first time we have demonstrated that insect species belonging to distinct Orders have conserved OBPs, which specifically bind a common semiochemical and has no binding affinity for related compounds. PMID:21912599

  7. Human CAP18: a novel antimicrobial lipopolysaccharide-binding protein.

    PubMed Central

    Larrick, J W; Hirata, M; Balint, R F; Lee, J; Zhong, J; Wright, S C

    1995-01-01

    CAP18 (18-kDa cationic antimicrobial protein) is a protein originally identified and purified from rabbit leukocytes on the basis of its capacity to bind and inhibit various activities of lipopolysaccharide (LPS). Here we report the cloning of human CAP18 and characterize the anti-LPS activity of the C-terminal fragment. Oligonucleotide probes designed from the rabbit CAP18 cDNA were used to identify human CAP18 from a bone marrow cDNA library. The cDNA encodes a protein composed of a 30-amino-acid signal peptide, a 103-amino-acid N-terminal domain of unknown function, and a C-terminal domain of 37 amino acids homologous to the LPS-binding antimicrobial domain of rabbit CAP18, designated CAP18(104-140). A human CAP18-specific antiserum was generated by using CAP18 expressed as a fusion protein with the maltose-binding protein. Western blots (immunoblots) with this antiserum showed specific expression of human CAP18 in granulocytes. Synthetic human CAP18(104-140) and a more active truncated fragment, CAP18(104-135), were shown to (i) bind to erythrocytes coated with diverse strains of LPS, (ii) inhibit LPS-induced release of nitric oxide from macrophages, (iii) inhibit LPS-induced generation of tissue factor, and (iv) protect mice from LPS lethality. CAP18(104-140) may have therapeutic utility for conditions associated with elevated concentrations of LPS. PMID:7890387

  8. Drug-drug plasma protein binding interactions of ivacaftor.

    PubMed

    Schneider, Elena K; Huang, Johnny X; Carbone, Vincenzo; Baker, Mark; Azad, Mohammad A K; Cooper, Matthew A; Li, Jian; Velkov, Tony

    2015-06-01

    Ivacaftor is a novel cystic fibrosis (CF) transmembrane conductance regulator (CFTR) potentiator that improves the pulmonary function for patients with CF bearing a G551D CFTR-protein mutation. Because ivacaftor is highly bound (>97%) to plasma proteins, there is the strong possibility that co-administered CF drugs may compete for the same plasma protein binding sites and impact the free drug concentration. This, in turn, could lead to drastic changes in the in vivo efficacy of ivacaftor and therapeutic outcomes. This biochemical study compares the binding affinity of ivacaftor and co-administered CF drugs for human serum albumin (HSA) and α1 -acid glycoprotein (AGP) using surface plasmon resonance and fluorimetric binding assays that measure the displacement of site-selective probes. Because of their ability to strongly compete for the ivacaftor binding sites on HSA and AGP, drug-drug interactions between ivacaftor are to be expected with ducosate, montelukast, ibuprofen, dicloxacillin, omeprazole, and loratadine. The significance of these plasma protein drug-drug interactions is also interpreted in terms of molecular docking simulations. This in vitro study provides valuable insights into the plasma protein drug-drug interactions of ivacaftor with co-administered CF drugs. The data may prove useful in future clinical trials for a staggered treatment that aims to maximize the effective free drug concentration and clinical efficacy of ivacaftor.

  9. The RNA-binding protein repertoire of Arabidopsis thaliana

    PubMed Central

    Marondedze, Claudius; Thomas, Ludivine; Serrano, Natalia L.; Lilley, Kathryn S.; Gehring, Chris

    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, 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. PMID:27405932

  10. Structural and functional analysis of fatty acid-binding proteins

    PubMed Central

    Storch, Judith; McDermott, Lindsay

    2009-01-01

    The mammalian FA-binding proteins (FABPs) bind long-chain FA with high affinity. The large number of FABP types is suggestive of distinct functions in specific tissues. Multiple experimental approaches have shown that individual FABPs possess both unique and overlapping functions, some of which are based on specific elements in the protein structure. Although FA binding affinities for all FABPs tend to correlate directly with FA hydrophobicity, structure-function studies indicate that subtle three-dimensional changes that occur upon ligand binding may promote specific protein-protein or protein-membrane interactions that ultimately determine the function of each FABP. The conformational changes are focused in the FABP helical/portal domain, a region that was identified by in vitro studies to be vital for the FA transport properties of the FABPs. Thus, the FABPs modulate intracellular lipid homeostasis by regulating FA transport in the nuclear and extra-nuclear compartments of the cell; in so doing, they also impact systemic energy homeostasis. PMID:19017610

  11. The size and detergent binding of membrane proteins.

    PubMed

    Clarke, S

    1975-07-25

    Sucrose density gradient centrifugation has been used to measure the binding of Triton X-100 above its critical micellar concentration to a variety of purified membrane and non-membrane proteins. In addition, binding studies were done on the three proteins below the critical micellar concentration of detergent to distinguish between the interaction of proteins with detergent monomers and detergent micelles. A procedure is described for the calculation of the molecular weight of these Triton X-100 protein complexes and measurements were made for opsin, plasma low density lipoprotein, the (Na-+ plus K-+)-dependent adenosine triphosphatase, the human red blood cell major sialoglycoprotein (PAS-1) and the human red blood cell minor glycoprotein (bandIII). These proteins behave as monomers or dimers in detergent and bind between 0.28 and 1.12 g of detergent per g of protein. A general method is also present for calculating the molecular size and shape of impure membrane proteins in detergent. Finally, Triton X-100 was shown to replace bound Na dodecyl-SO4 on the minor glycoprotein of the red blood cell.

  12. Carotenoid-binding proteins; accessories to carotenoid function.

    PubMed

    Pilbrow, Jodi; Garama, Daniel; Carne, Alan

    2012-01-01

    Understanding of the widespread biological importance of carotenoids is increasing. Accompanying this is the developing recognition that the interaction of carotenoids with other molecules, such as proteins, is also essential. Here the significance of carotenoid-protein interactions with respect to biological function is reviewed for three well characterised carotenoprotein complexes; crustacyanin, the orange carotenoid protein and glutathione-S-transferase P1. In addition a preliminary report is made on the recent partial purification of an echinenone-binding protein extracted from a New Zealand sea urchin, Evechinus chloroticus.

  13. Molecular beacons for DNA binding proteins: an emerging technology for detection of DNA binding proteins and their ligands.

    PubMed

    Dummitt, Benjamin; Chang, Yie-Hwa

    2006-06-01

    Quantitation of the level or activity of specific proteins is one of the most commonly performed experiments in biomedical research. Protein detection has historically been difficult to adapt to high throughput platforms because of heavy reliance upon antibodies for protein detection. Molecular beacons for DNA binding proteins is a recently developed technology that attempts to overcome such limitations. Protein detection is accomplished using inexpensive, easy-to-synthesize oligonucleotides, accompanied by a fluorescence readout. Importantly, detection of the protein and reporting of the signal occur simultaneously, allowing for one-step protocols and increased potential for use in high throughput analysis. While the initial iteration of the technology allowed only for the detection of sequence-specific DNA binding proteins, more recent adaptations allow for the possibility of development of beacons for any protein, independent of native DNA binding activity. Here, we discuss the development of the technology, the mechanism of the reaction, and recent improvements and modifications made to improve the assay in terms of sensitivity, potential for multiplexing, and broad applicability.

  14. Translational repression by RNA-binding protein TIAR.

    PubMed

    Mazan-Mamczarz, Krystyna; Lal, Ashish; Martindale, Jennifer L; Kawai, Tomoko; Gorospe, Myriam

    2006-04-01

    The RNA-binding protein TIAR has been proposed to inhibit protein synthesis transiently by promoting the formation of translationally silent stress granules. Here, we report the selective binding of TIAR to several mRNAs encoding translation factors such as eukaryotic initiation factor 4A (eIF4A) and eIF4E (translation initiation factors), eEF1B (a translation elongation factor), and c-Myc (which transcriptionally controls the expression of numerous translation regulatory proteins). TIAR bound the 3'-untranslated regions of these mRNAs and potently suppressed their translation, particularly in response to low levels of short-wavelength UV (UVC) irradiation. The UVC-imposed global inhibition of the cellular translation machinery was significantly relieved after silencing of TIAR expression. We propose that the TIAR-mediated inhibition of translation factor expression elicits a sustained repression of protein biosynthesis in cells responding to stress.

  15. An Ancient Family of RNA-Binding Proteins: Still Important!

    PubMed

    Wells, Melissa L; Perera, Lalith; Blackshear, Perry J

    2017-04-01

    RNA-binding proteins are important modulators of mRNA stability, a crucial process that determines the ultimate cellular levels of mRNAs and their encoded proteins. The tristetraprolin (TTP) family of RNA-binding proteins appeared early in the evolution of eukaryotes, and has persisted in modern eukaryotes. The domain structures and biochemical functions of family members from widely divergent lineages are remarkably similar, but their mRNA 'targets' can be very different, even in closely related species. Recent gene knockout studies in species as distantly related as plants, flies, yeasts, and mice have demonstrated crucial roles for these proteins in a wide variety of physiological processes. Inflammatory and hematopoietic phenotypes in mice have suggested potential therapeutic approaches for analogous human disorders.

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

    NASA Astrophysics Data System (ADS)

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

    2012-12-01

    Water molecules in the binding pocket of a protein and their role in ligand binding have increasingly raised interest in recent years. Displacement of such water molecules by ligand atoms can be either favourable or unfavourable for ligand binding depending on the change in free enthalpy. In this study, we investigate the displacement of water molecules by an apolar probe in the binding pocket of two proteins, cyclin-dependent kinase 2 and tRNA-guanine transglycosylase, using the method of enveloping distribution sampling (EDS) to obtain free enthalpy differences. In both cases, a ligand core is placed inside the respective pocket and the remaining water molecules are converted to apolar probes, both individually and in pairs. The free enthalpy difference between a water molecule and a CH3 group at the same location in the pocket in comparison to their presence in bulk solution calculated from EDS molecular dynamics simulations corresponds to the binding free enthalpy of CH3 at this location. From the free enthalpy difference and the enthalpy difference, the entropic contribution of the displacement can be obtained too. The overlay of the resulting occupancy volumes of the water molecules with crystal structures of analogous ligands shows qualitative correlation between experimentally measured inhibition constants and the calculated free enthalpy differences. Thus, such an EDS analysis of the water molecules in the binding pocket may give valuable insight for potency optimization in drug design.

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

    PubMed

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

    2012-12-01

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

  18. High-affinity RNA binding by a hyperthermophilic single-stranded DNA-binding protein.

    PubMed

    Morten, Michael J; Gamsjaeger, Roland; Cubeddu, Liza; Kariawasam, Ruvini; Peregrina, Jose; Penedo, J Carlos; White, Malcolm F

    2017-03-01

    Single-stranded DNA-binding proteins (SSBs), including replication protein A (RPA) in eukaryotes, play a central role in DNA replication, recombination, and repair. SSBs utilise an oligonucleotide/oligosaccharide-binding (OB) fold domain to bind DNA, and typically oligomerise in solution to bring multiple OB fold domains together in the functional SSB. SSBs from hyperthermophilic crenarchaea, such as Sulfolobus solfataricus, have an unusual structure with a single OB fold coupled to a flexible C-terminal tail. The OB fold resembles those in RPA, whilst the tail is reminiscent of bacterial SSBs and mediates interaction with other proteins. One paradigm in the field is that SSBs bind specifically to ssDNA and much less strongly to RNA, ensuring that their functions are restricted to DNA metabolism. Here, we use a combination of biochemical and biophysical approaches to demonstrate that the binding properties of S. solfataricus SSB are essentially identical for ssDNA and ssRNA. These features may represent an adaptation to a hyperthermophilic lifestyle, where DNA and RNA damage is a more frequent event.

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

    SciTech Connect

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

    1991-06-04

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

  20. Importance of DNA stiffness in protein-DNA binding specificity

    NASA Astrophysics Data System (ADS)

    Hogan, M. E.; Austin, R. H.

    1987-09-01

    From the first high-resolution structure of a repressor bound specifically to its DNA recognition sequence1 it has been shown that the phage 434 repressor protein binds as a dimer to the helix. Tight, local interactions are made at the ends of the binding site, causing the central four base pairs (bp) to become bent and overtwisted. The centre of the operator is not in contact with protein but repressor binding affinity can be reduced at least 50-fold in response to a sequence change there2. This observation might be explained should the structure of the intervening DNA segment vary with its sequence, or if DNA at the centre of the operator resists the torsional and bending deformation necessary for complex formation in a sequence dependent fashion. We have considered the second hypothesis by demonstrating that DNA stiffness is sequence dependent. A method is formulated for calculating the stiffness of any particular DNA sequence, and we show that this predicted relationship between sequence and stiffness can explain the repressor binding data in a quantitative manner. We propose that the elastic properties of DNA may be of general importance to an understanding of protein-DNA binding specificity.

  1. Using the telobox to search for plant telomere binding proteins.

    PubMed

    Peška, Vratislav; Schrumpfová, Petra Procházková; Fajkus, Jiŕí

    2011-03-01

    Telobox is a Myb-related DNA-binding domain which is present in a number of yeast, plant and animal proteins. Its capacity to bind preferentially double-stranded telomeric DNA has been used in numerous studies to search for candidate telomeric proteins in various organisms, including plants. Here we provide an overview of these studies with a special emphasis on plants, where a specific subfamily of the proteins possessing the N-terminally positioned telobox is present in addition to more common C-terminal telobox proteins. We further demonstrate the presence of a telobox protein (CpTBP1) in Cestrum parqui, a plant lacking typical telomeres and telomerase. The protein shows nuclear localisation and association with chromatin. The role of this protein in ancestral and current telomere structure is discussed in the evolutionary context. Altogether, the present overview shows the importance of the telobox domain in a search for candidate telomere proteins but at the same time warns against oversimplified identification of any telobox protein with telomere structure without appropriate evidence of its telomeric localisation and function.

  2. Structural analysis of ibuprofen binding to human adipocyte fatty-acid binding protein (FABP4).

    PubMed

    González, Javier M; Fisher, S Zoë

    2015-02-01

    Inhibition of human adipocyte fatty-acid binding protein (FABP4) has been proposed as a treatment for type 2 diabetes, fatty liver disease and atherosclerosis. However, FABP4 displays a naturally low selectivity towards hydrophobic ligands, leading to the possibility of side effects arising from cross-inhibition of other FABP isoforms. In a search for structural determinants of ligand-binding selectivity, the binding of FABP4 towards a group of small molecules structurally related to the nonsteroidal anti-inflammatory drug ibuprofen was analyzed through X-ray crystallography. Several specific hydrophobic interactions are shown to enhance the binding affinities of these compounds, whereas an aromatic edge-to-face interaction is proposed to determine the conformation of bound ligands, highlighting the importance of aromatic interactions in hydrophobic environments.

  3. Asymmetric DNA binding by a homodimeric bHLH protein.

    PubMed

    Winston, R L; Ehley, J A; Baird, E E; Dervan, P B; Gottesfeld, J M

    2000-08-08

    Protein-DNA interactions that lie outside of the core recognition sequence for the Drosophila bHLH transcription factor Deadpan (Dpn) were investigated using minor groove binding pyrrole-imidazole polyamides. Electrophoretic mobility shift assays and DNase I footprinting demonstrate that hairpin polyamides bound immediately upstream, but not immediately downstream of the Dpn homodimer selectively inhibit protein-DNA complex formation. Mutation of the Dpn consensus binding site from the asymmetric sequence 5'-CACGCG-3' to the palindromic sequence 5'-CACGTG-3' abolishes asymmetric inhibition. A Dpn mutant containing the unnatural amino acid norleucine in place of lysine at position 80 in the bHLH loop region is not inhibited by the polyamide, suggesting that the epsilon amino group at this position is responsible for DNA contacts outside the major groove. We conclude that the nonpalindromic Dpn recognition site imparts binding asymmetry by providing unique contacts to the basic region of each monomer in the bHLH homodimer.

  4. Isothermal Titration Calorimetry Measurements of Metal Ions Binding to Proteins.

    PubMed

    Quinn, Colette F; Carpenter, Margaret C; Croteau, Molly L; Wilcox, Dean E

    2016-01-01

    ITC measurements involving metal ions are susceptible to a number of competing reactions (oxidation, precipitation, and hydrolysis) and coupled reactions involving the buffer and protons. Stabilization and delivery of the metal ion as a well-defined and well-characterized complex with the buffer, or a specific ligand, can suppress undesired solution chemistry and, depending on the stability of the metal complex, allow accurate measurements of higher affinity protein-binding sites. This requires, however, knowledge of the thermodynamics of formation of the metal complex and accounting for its contribution to the experimentally measured values (KITC and ΔHITC) through a post hoc analysis that provides the condition-independent binding thermodynamics (K, ΔG(o), ΔH, ΔS, and ΔCP). This analysis also quantifies the number of protons that are displaced when the metal ion binds to the protein.

  5. Effect of FGF-binding Protein 3 on Vascular Permeability*

    PubMed Central

    Zhang, Wentao; Chen, Yifan; Swift, Matthew R.; Tassi, Elena; Stylianou, Dora C.; Gibby, Krissa A.; Riegel, Anna T.; Wellstein, Anton

    2008-01-01

    Fibroblast growth factor-binding protein 1 (FGF-BP1 is BP1) is involved in the regulation of embryonic development, tumor growth, and angiogenesis by mobilizing endogenous FGFs from their extracellular matrix storage. Here we describe a new member of the FGF-BP family, human BP3. We show that the hBP3 protein is secreted from cells, binds to FGF2 in vitro and in intact cells, and inhibits FGF2 binding to heparin. To determine the function of hBP3 in vivo, hBP3 was transiently expressed in chicken embryos and resulted in >50% lethality within 24 h because of vascular leakage. The onset of vascular permeability was monitored by recording the extravasation kinetics of FITC-labeled 40-kDa dextran microperfused into the vitelline vein of 3-day-old embryos. Vascular permeability increased as early as 8 h after expression of hBP3. The increased vascular permeability caused by hBP3 was prevented by treatment of embryos with PD173074, a selective FGFR kinase inhibitor. Interestingly, a C-terminal 66-amino acid fragment (C66) of hBP3, which contains the predicted FGF binding domain, still inhibited binding of FGF2 to heparin similar to full-length hBP3. However, expression of the C66 fragment did not increase vascular permeability on its own, but required the administration of exogenous FGF2 protein. We conclude that the FGF binding domain and the heparin binding domain are necessary for the hBP3 interaction with endogenous FGF and the activation of FGFR signaling in vivo. PMID:18669637

  6. Urinary intestinal fatty acid binding protein predicts necrotizing enterocolitis.

    PubMed

    Gregory, Katherine E; Winston, Abigail B; Yamamoto, Hidemi S; Dawood, Hassan Y; Fashemi, Titilayo; Fichorova, Raina N; Van Marter, Linda J

    2014-06-01

    Necrotizing enterocolitis, characterized by sudden onset and rapid progression, remains the most significant gastrointestinal disorder among premature infants. In seeking a predictive biomarker, we found intestinal fatty acid binding protein, an indicator of enterocyte damage, was substantially increased within three and seven days before the diagnosis of necrotizing enterocolitis.

  7. Methods of use of cellulose binding domain proteins

    SciTech Connect

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

    1997-01-01

    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.

  8. Methods of use of cellulose binding domain proteins

    SciTech Connect

    Shoseyov, O.; Shpiegl, I.; Goldstein, M.A.; Doi, R.H.

    1997-09-23

    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. 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. 16 figs.

  9. Guanylate-binding proteins: niche recruiters for antimicrobial effectors.

    PubMed

    Dupont, Christopher D; Hunter, Christopher A

    2012-08-24

    There are fundamental questions regarding how IFN-γ activates host cells to eliminate intracellular pathogens. In this issue of Immunity, Yamamoto et al. (2012) demonstrate a critical role for the p65 guanylate-binding proteins (GBPs) in this process during infection with Toxoplasma gondii.

  10. JadX is a Disparate Natural Product Binding Protein.

    PubMed

    Robertson, Andrew W; Forget, Stephanie M; Martinez-Farina, Camilo F; McCormick, Nicole E; Syvitski, Raymond T; Jakeman, David L

    2016-02-24

    We report that JadX, a protein of previously undetermined function coded for in the jadomycin biosynthetic gene cluster of Streptomyces venezuelae ISP5230, affects both chloramphenicol and jadomycin production levels in blocked mutants. Characterization of recombinant JadX through protein-ligand interactions by chemical shift perturbation and WaterLOGSY NMR spectroscopy resulted in the observation of binding between JadX and a series of jadomycins and between JadX and chloramphenicol, another natural product produced by S. venezuelae ISP5230. These results suggest JadX to be an unusual class of natural product binding protein involved in binding structurally disparate natural products. The ability for JadX to bind two different natural products in vitro and the ability to affect production of these secondary metabolites in vivo suggest a potential role in regulation or signaling. This is the first example of functional characterization of these JadX-like proteins, and provides insight into a previously unobserved regulatory process.

  11. Differential DNA binding properties of three human homeodomain proteins.

    PubMed Central

    Corsetti, M T; Briata, P; Sanseverino, L; Daga, A; Airoldi, I; Simeone, A; Palmisano, G; Angelini, C; Boncinelli, E; Corte, G

    1992-01-01

    The products of three human homeobox containing (HOX) genes, 2C, 3C and 4B, were produced in insect cells using the Baculovirus expression system and purified to near homogeneity. In this system we observed that the DNA binding forms of the three proteins are not glycosylated. HOX 3C and 4B are phosphorylated in insect cells, while HOX 2C is not. The three HOX proteins bind to a DNA sequence known to be a target site for Antennapedia protein with a very similar affinity (Kd = 1-2 x 10(-9) M). We then measured their binding properties to four human sequences present in the HOX 3D, 4C, 1C and 4B promoters. Two of these sequences have been reported to be binding sites for HOX proteins. HOX 2C, 3C and 4B behaved quite differently, showing low affinity for promoters of genes located upstream from their own gene in the HOX clusters and a higher affinity for regulatory sequences of their own gene and downstream HOX genes. Images PMID:1357628

  12. Protein-Ligand Binding Detected by Terahertz Spectroscopy

    NASA Astrophysics Data System (ADS)

    Knab, J.; Chen, J. Y.; Mader, M.; Markelz, A.

    2004-03-01

    Established measures of protein flexibility through the B-factor use time intensive and facility limited techniques such as X-ray crystallography, NMR structure analysis and inelastic neutron scattering. We demonstrate a novel technique that may be used for determination of ligand binding for proteins as well as a measure of protein flexibility. Using the method of terahertz (THz) time domain spectroscopy, we measured the far infrared dielectric response as a function of the binding of N (1-4)-acetylglucosamine (NAG) to hen egg white lysozyme (HEWL). Vibrational modes associated with tertiary structure conformational motions lay in the THz frequency range. The THz dielectric response reflects the density and amplitude of these normal modes through dipole coupling. Transmission measurements on thin films show that while there is no change in the real part of the refractive index as a function of binding, there is a decrease in the absorbance for the HEWL+NAG thin films relative to HEWL films. This decrease can be attributed to a reduction in the flexibility of the protein with binding. These results are compared to calculated absorbance spectra.

  13. Capacitance-modulated transistor detects odorant binding protein chiral interactions

    NASA Astrophysics Data System (ADS)

    Mulla, Mohammad Yusuf; Tuccori, Elena; Magliulo, Maria; Lattanzi, Gianluca; Palazzo, Gerardo; Persaud, Krishna; Torsi, Luisa

    2015-01-01

    Peripheral events in olfaction involve odorant binding proteins (OBPs) whose role in the recognition of different volatile chemicals is yet unclear. Here we report on the sensitive and quantitative measurement of the weak interactions associated with neutral enantiomers differentially binding to OBPs immobilized through a self-assembled monolayer to the gate of an organic bio-electronic transistor. The transduction is remarkably sensitive as the transistor output current is governed by the small capacitance of the protein layer undergoing minute changes as the ligand-protein complex is formed. Accurate determination of the free-energy balances and of the capacitance changes associated with the binding process allows derivation of the free-energy components as well as of the occurrence of conformational events associated with OBP ligand binding. Capacitance-modulated transistors open a new pathway for the study of ultra-weak molecular interactions in surface-bound protein-ligand complexes through an approach that combines bio-chemical and electronic thermodynamic parameters.

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

    SciTech Connect

    Kanjilal, S.

    1992-01-01

    The primary objective of the study was to detect and characterize factors from human cells that bind DNA damaged by ultraviolet radiation. An application of the gel-shift assay was devised in which a DNA probe was UV-irradiated and compared with non-irradiated probe DNA for the ability to bind to such factors in cell extracts. UV-dose dependent binding proteins were identified. Formation of the DNA-protein complexes was independent of the specific sequence, form or source of the DNA. There was a marked preference for lesions on double stranded DNA over those on single stranded DNA. DNA irradiated with gamma rays did not compete with UV-irradiated DNA for the binding activities. Cell lines from patients with genetic diseases associated with disorders of the DNA repair system were screened for the presence of damaged-DNA-binding activities. Simultaneous occurrence of the clinical symptoms of some of these diseases had been previously documented and possible links between the syndromes proposed. However, supporting biochemical or molecular evidence for such associations were lacking. The data from the present investigations indicate that some cases of Xeroderma Pigmentosum group A, Cockayne's Syndrome, Bloom's Syndrome and Ataxia Telangiectasia, all of which exhibit sensitivity to UV or gamma radiation, share an aberrant damaged-DNA-binding factor. These findings support the hypothesis that some of the repair disorder diseases are closely related and may have arisen from a common defect. Partial purification of the binding activities from HeLa cells was achieved. Size-exclusion chromatography resolved the activities into various peaks, one of which was less damage-specific than the others as determined by competition studies using native or UV-irradiated DNA. Some of the activities were further separated by ion-exchange chromatography. On using affinity chromatography methods, the major damage-binding factor could be eluted in the presence of 2 M KCl and 1% NP-40.

  15. Sequence specific binding of chlamydial histone H1-like protein.

    PubMed Central

    Kaul, R; Allen, M; Bradbury, E M; Wenman, W M

    1996-01-01

    Chlamydia trachomatis is one of the few prokaryotic organisms known to contain proteins that bear homology to eukaryotic histone H1. Changes in macromolecular conformation of DNA mediated by the histone H1-like protein (Hc1) appear to regulate stage specific differentiation. We have developed a cross-linking immunoprecipitation protocol to examine in vivo protein-DNA interaction by immune precipitating chlamydial Hc1 cross linked to DNA. Our results strongly support the presence of sequence specific binding sites on the chlamydial plasmid and hc1 gene upstream of its open reading frame. The preferential binding sites were mapped to 520 bp BamHI-XhoI and 547 bp BamHI-DraI DNA fragments on the plasmid and hc1 respectively. Comparison of these two DNA sequences using Bestfit program has identified a 24 bp region with >75% identity that is unique to the chlamydial genome. Double-stranded DNA prepared by annealing complementary oligonucleotides corresponding to the conserved 24 bp region bind Hc1, in contrast to control sequences with similar A+T ratios. Further, Hc1 binds to DNA in a strand specific fashion, with preferential binding for only one strand. The site specific affinity to plasmid DNA was also demonstrated by atomic force microscopy data images. Binding was always followed by coiling, shrinking and aggregation of the affected DNA. Very low protein-DNA ratio was required if incubations were carried out in solution. However, if DNA was partially immobilized on mica substrate individual strands with dark foci were still visible even after the addition of excess Hc1. PMID:8760883

  16. Major coat protein and single-stranded DNA-binding protein of filamentous virus Pf3.

    PubMed Central

    Putterman, D G; Casadevall, A; Boyle, P D; Yang, H L; Frangione, B; Day, L A

    1984-01-01

    The region of the Pf3 virus genome encoding its major coat protein and its single-stranded DNA-binding protein is organized somewhat like the corresponding region of the fd (M13, f1) genome. Nevertheless, the major coat protein is unique among the major coat proteins of fd and the other filamentous phages studied in that it lacks a signal sequence and appears to be a direct translation product and in that it has fewer basic amino acid residues than its equivalent of DNA phosphates in the virion. These features are relevant to considerations of both protein insertion into membranes and DNA structure in filamentous viruses. The single-stranded DNA-binding protein also has a sequence that is different from the sequences of single-stranded DNA-binding proteins from other filamentous viruses. Images PMID:6422463

  17. The Overlap of Small Molecule and Protein Binding Sites within Families of Protein Structures

    PubMed Central

    Davis, Fred P.; Sali, Andrej

    2010-01-01

    Protein–protein interactions are challenging targets for modulation by small molecules. Here, we propose an approach that harnesses the increasing structural coverage of protein complexes to identify small molecules that may target protein interactions. Specifically, we identify ligand and protein binding sites that overlap upon alignment of homologous proteins. Of the 2,619 protein structure families observed to bind proteins, 1,028 also bind small molecules (250–1000 Da), and 197 exhibit a statistically significant (p<0.01) overlap between ligand and protein binding positions. These “bi-functional positions”, which bind both ligands and proteins, are particularly enriched in tyrosine and tryptophan residues, similar to “energetic hotspots” described previously, and are significantly less conserved than mono-functional and solvent exposed positions. Homology transfer identifies ligands whose binding sites overlap at least 20% of the protein interface for 35% of domain–domain and 45% of domain–peptide mediated interactions. The analysis recovered known small-molecule modulators of protein interactions as well as predicted new interaction targets based on the sequence similarity of ligand binding sites. We illustrate the predictive utility of the method by suggesting structural mechanisms for the effects of sanglifehrin A on HIV virion production, bepridil on the cellular entry of anthrax edema factor, and fusicoccin on vertebrate developmental pathways. The results, available at http://pibase.janelia.org, represent a comprehensive collection of structurally characterized modulators of protein interactions, and suggest that homologous structures are a useful resource for the rational design of interaction modulators. PMID:20140189

  18. Phage display screen for peptides that bind Bcl-2 protein.

    PubMed

    Park, Hye-Yeon; Kim, Joungmok; Cho, June-Haeng; Moon, Ji Young; Lee, Su-Jae; Yoon, Moon-Young

    2011-01-01

    Bcl-2 family proteins are key regulators of apoptosis associated with human disease, including cancer. Bcl-2 protein has been found to be overexpressed in many cancer cells. Therefore, Bcl-2 protein is a potential diagnostic target for cancer detection. In the present study, the authors have identified several Bcl-2 binding peptides with high affinity (picomolar range) from a 5-round M13 phage display library screening. These peptides can be used to develop novel diagnostic probes or potent inhibitors with diverse polyvalencies.

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

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  20. Resolving protein structure-function-binding site relationships from a binding site similarity network perspective.

    PubMed

    Mudgal, Richa; Srinivasan, Narayanaswamy; Chandra, Nagasuma

    2017-03-25

    Functional annotation is seldom straightforward with complexities arising due to functional divergence in protein families or functional convergence between non-homologous protein families, leading to mis-annotations. An enzyme may contain multiple domains and not all domains may be involved in a given function, adding to the complexity in function annotation. To address this, we use binding site information from bound cognate ligands and catalytic residues, since it can help in resolving fold-function relationships at a finer level and with higher confidence. A comprehensive database of 2,020 fold-function-binding site relationships has been systematically generated. A network-based approach is employed to capture the complexity in these relationships, from which different types of associations are deciphered, that identify versatile protein folds performing diverse functions, same function associated with multiple folds and one-to-one relationships. Binding site similarity networks integrated with fold, function and ligand similarity information are generated to understand the depth of these relationships. Apart from the observed continuity in the functional site space, network properties of these revealed versatile families with topologically different or dissimilar binding sites and structural families that perform very similar functions. As a case study, subtle changes in the active site of a set of evolutionarily related superfamilies are studied using these networks. Tracing of such similarities in evolutionarily related proteins provide clues into the transition and evolution of protein functions. Insights from this study will be helpful in accurate and reliable functional annotations of uncharacterized proteins, poly-pharmacology and designing enzymes with new functional capabilities. This article is protected by copyright. All rights reserved.

  1. Calcium binding to Procambarus clarkii sarcoplasmic calcium binding protein splice variants.

    PubMed

    E Rohrback, Suzanne; Wheatly, Michele G; Gillen, Christopher M

    2015-01-01

    Sarcoplasmic calcium binding protein (SCP) is a high-affinity calcium buffering protein expressed in muscle of crayfish and other invertebrates. In previous work, we identified three splice variants of Procambarus clarkii SCP (pcSCP1a, pcSCP1b, and pcSCP1c) that differ in a 37 amino acid region that lies mainly between the 2nd and 3ed EF-hand calcium binding domain. To evaluate the function of the proteins encoded by the pcSCP1 transcripts, we produced recombinant pcSCP1 and used tryptophan fluorescence to characterize calcium binding. Tryptophan fluorescence of pcSCP1a decreased in response to increased calcium, while tryptophan fluorescence of the pcSCP1b and pcSCP1c variants increased. We estimated calcium binding constants and Hill coefficients with two different equations: the standard Hill equation and a modified Hill equation that accounts for contributions from two different tryptophans. The approaches gave similar results. Steady-state calcium binding constants (Kd) ranged from 2.7±0.7×10(-8)M to 5.6±0.1×10(-7)M, consistent with previous work. Variants displayed significantly different apparent calcium affinities, which were decreased in the presence of magnesium. Calcium Kd was lowest for pcSCP1a and highest for pcSCP1c. Site-directed mutagenesis of pcSCP1c residues to the amino acids of pcSCP1b decreased the calcium Kd, identifying residues outside the EF-hand domains that contribute to calcium binding in crayfish SCP.

  2. Insulin-Like Growth Factor Binding Proteins: A Structural Perspective

    PubMed Central

    Forbes, Briony E.; McCarthy, Peter; Norton, Raymond S.

    2012-01-01

    Insulin-like growth factor binding proteins (IGFBP-1 to -6) bind insulin-like growth factors-I and -II (IGF-I and IGF-II) with high affinity. These binding proteins maintain IGFs in the circulation and direct them to target tissues, where they promote cell growth, proliferation, differentiation, and survival via the type 1 IGF receptor. IGFBPs also interact with many other molecules, which not only influence their modulation of IGF action but also mediate IGF-independent activities that regulate processes such as cell migration and apoptosis by modulating gene transcription. IGFBPs-1 to -6 are structurally similar proteins consisting of three distinct domains, N-terminal, linker, and C-terminal. There have been major advances in our understanding of IGFBP structure in the last decade and a half. While there is still no structure of an intact IGFBP, several structures of individual N- and C-domains have been solved. The structure of a complex of N-BP-4:IGF-I:C-BP-4 has also been solved, providing a detailed picture of the structural features of the IGF binding site and the mechanism of binding. Structural studies have also identified features important for interaction with extracellular matrix components and integrins. This review summarizes structural studies reported so far and highlights features important for binding not only IGF but also other partners. We also highlight future directions in which structural studies will add to our knowledge of the role played by the IGFBP family in normal growth and development, as well as in disease. PMID:22654863

  3. Neisseria meningitis GNA1030 is a ubiquinone-8 binding protein.

    PubMed

    Donnarumma, Danilo; Golfieri, Giacomo; Brier, Sébastien; Castagnini, Marta; Veggi, Daniele; Bottomley, Matthew James; Delany, Isabel; Norais, Nathalie

    2015-06-01

    Bexsero, a new vaccine against Neisseria meningitidis serogroup B (MenB), is composed of 3 main recombinant proteins and an outer membrane vesicle component. One of the main bactericidal antigens, neisseria heparin binding antigen (NHBA), is present as a fusion protein with the accessory protein genome-derived neisserial antigen (GNA) 1030 to further increase its immunogenicity. The gene encoding for GNA1030 is present and highly conserved in all Neisseria strains, and although orthologs are present in numerous species, its biologic function is unknown. Native mass spectrometry was used to demonstrate that GNA1030 forms a homodimer associated with 2 molecules of ubiquinone-8 (Ub8), a cofactor mainly involved in the electron transport chain and with antioxidant properties. Disc diffusion assays on the wild-type and knockout mutant of GNA1030, in the presence of various compounds, suggested that GNA1030 is not involved in oxidative stress or electron chain transport per se, although it contributes to constitutive refilling of the inner membrane with Ub8. These studies shed light on an accessory protein present in Bexsero and reveal functional insights into the family of related proteins. On the basis of our findings, we propose to name the protein neisseria ubiquinone binding protein (NUbp).

  4. Polyamine binding to proteins in oat and Petunia protoplasts

    NASA Technical Reports Server (NTRS)

    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.

  5. Carotenoid binding to proteins: Modeling pigment transport to lipid membranes.

    PubMed

    Reszczynska, Emilia; Welc, Renata; Grudzinski, Wojciech; Trebacz, Kazimierz; Gruszecki, Wieslaw I

    2015-10-15

    Carotenoid pigments play numerous important physiological functions in human organism. Very special is a role of lutein and zeaxanthin in the retina of an eye and in particular in its central part, the macula lutea. In the retina, carotenoids can be directly present in the lipid phase of the membranes or remain bound to the protein-pigment complexes. In this work we address a problem of binding of carotenoids to proteins and possible role of such structures in pigment transport to lipid membranes. Interaction of three carotenoids, beta-carotene, lutein and zeaxanthin with two proteins: bovine serum albumin and glutathione S-transferase (GST) was investigated with application of molecular spectroscopy techniques: UV-Vis absorption, circular dichroism and Fourier transform infrared spectroscopy (FTIR). Interaction of pigment-protein complexes with model lipid bilayers formed with egg yolk phosphatidylcholine was investigated with application of FTIR, Raman imaging of liposomes and electrophysiological technique, in the planar lipid bilayer models. The results show that in all the cases of protein and pigment studied, carotenoids bind to protein and that the complexes formed can interact with membranes. This means that protein-carotenoid complexes are capable of playing physiological role in pigment transport to biomembranes.

  6. Small Molecule Ligands of Methyl-Lysine Binding Proteins

    PubMed Central

    Herold, J. Martin; Wigle, Tim J.; Norris, Jacqueline L.; Lam, Robert; Korboukh, Victoria K.; Gao, Cen; Ingerman, Lindsey A.; Kireev, Dmitri B.; Senisterra, Guillermo; Vedadi, Masoud; Tripathy, Ashutosh; Brown, Peter J.; Arrowsmith, Cheryl H.; Jin, Jian; Janzen, William P.; Frye, Stephen V.

    2011-01-01

    Proteins which bind methylated lysines (“readers” of the histone code) are important components in the epigenetic regulation of gene expression and can also modulate other proteins that contain methyl-lysine such as p53 and Rb. Recognition of methyl-lysine marks by MBT domains leads to compaction of chromatin and a repressed transcriptional state. Antagonists of MBT domains would serve as probes to interrogate the functional role of these proteins and initiate the chemical biology of methyl-lysine readers as a target class. Small molecule MBT antagonists were designed based on the structure of histone peptide-MBT complexes and their interaction with MBT domains determined using a chemiluminescent assay and ITC. The ligands discovered antagonize native histone peptide binding, exhibiting 5-fold stronger binding affinity to L3MBTL1 than its preferred histone peptide. The first co-crystal structure of a small molecule bound to L3MBTL1 was determined and provides new insights into binding requirements for further ligand design. PMID:21417280

  7. Small-molecule ligands of methyl-lysine binding proteins.

    PubMed

    Herold, J Martin; Wigle, Tim J; Norris, Jacqueline L; Lam, Robert; Korboukh, Victoria K; Gao, Cen; Ingerman, Lindsey A; Kireev, Dmitri B; Senisterra, Guillermo; Vedadi, Masoud; Tripathy, Ashutosh; Brown, Peter J; Arrowsmith, Cheryl H; Jin, Jian; Janzen, William P; Frye, Stephen V

    2011-04-14

    Proteins which bind methylated lysines ("readers" of the histone code) are important components in the epigenetic regulation of gene expression and can also modulate other proteins that contain methyl-lysine such as p53 and Rb. Recognition of methyl-lysine marks by MBT domains leads to compaction of chromatin and a repressed transcriptional state. Antagonists of MBT domains would serve as probes to interrogate the functional role of these proteins and initiate the chemical biology of methyl-lysine readers as a target class. Small-molecule MBT antagonists were designed based on the structure of histone peptide-MBT complexes and their interaction with MBT domains determined using a chemiluminescent assay and ITC. The ligands discovered antagonize native histone peptide binding, exhibiting 5-fold stronger binding affinity to L3MBTL1 than its preferred histone peptide. The first cocrystal structure of a small molecule bound to L3MBTL1 was determined and provides new insights into binding requirements for further ligand design.

  8. Capacitance-modulated transistor detects odorant binding protein chiral interactions

    PubMed Central

    Mulla, Mohammad Yusuf; Tuccori, Elena; Magliulo, Maria; Lattanzi, Gianluca; Palazzo, Gerardo; Persaud, Krishna; Torsi, Luisa

    2015-01-01

    Peripheral events in olfaction involve odorant binding proteins (OBPs) whose role in the recognition of different volatile chemicals is yet unclear. Here we report on the sensitive and quantitative measurement of the weak interactions associated with neutral enantiomers differentially binding to OBPs immobilized through a self-assembled monolayer to the gate of an organic bio-electronic transistor. The transduction is remarkably sensitive as the transistor output current is governed by the small capacitance of the protein layer undergoing minute changes as the ligand–protein complex is formed. Accurate determination of the free-energy balances and of the capacitance changes associated with the binding process allows derivation of the free-energy components as well as of the occurrence of conformational events associated with OBP ligand binding. Capacitance-modulated transistors open a new pathway for the study of ultra-weak molecular interactions in surface-bound protein–ligand complexes through an approach that combines bio-chemical and electronic thermodynamic parameters. PMID:25591754

  9. The Bacillus thuringiensis insecticidal toxin binds biotin-containing proteins.

    PubMed Central

    Du, C; Nickerson, K W

    1996-01-01

    Brush border membrane vesicles from larvae of the tobacco hornworm, Manduca sexta, contain protein bands of 85 and 120 kDa which react directly with streptavidin conjugated to alkaline phosphatase. The binding could be prevented either by including 10 microM biotin in the reaction mixture or by prior incubation of the brush border membrane vesicles with an activated 60- to 65-kDa toxin from Bacillus thuringiensis HD-73. The ability of B. thuringiensis toxins to recognize biotin-containing proteins was confirmed by their binding to pyruvate carboxylase, a biotin-containing enzyme, as well as to biotinylated ovalbumin and biotinylated bovine serum albumin but not to their nonbiotinylated counterparts. Activated HD-73 toxin also inhibited the enzymatic activity of pyruvate carboxylase. The biotin binding site is likely contained in domain III of the toxin. Two highly conserved regions within domain III are similar in sequence to the biotin binding sites of avidin, streptavidin, and a biotin-specific monoclonal antibody. In particular, block 4 of the B. thuringiensis toxin contains the YAS biotin-specific motif. On the basis of its N-terminal amino acid sequence, the 120-kDa biotin-containing protein is totally distinct from the 120-kDa aminopeptidase N reported to be a receptor for Cry1Ac toxin. PMID:8702286

  10. Calcium binding proteins and calcium signaling in prokaryotes.

    PubMed

    Domínguez, Delfina C; Guragain, Manita; Patrauchan, Marianna

    2015-03-01

    With the continued increase of genomic information and computational analyses during the recent years, the number of newly discovered calcium binding proteins (CaBPs) in prokaryotic organisms has increased dramatically. These proteins contain sequences that closely resemble a variety of eukaryotic calcium (Ca(2+)) binding motifs including the canonical and pseudo EF-hand motifs, Ca(2+)-binding β-roll, Greek key motif and a novel putative Ca(2+)-binding domain, called the Big domain. Prokaryotic CaBPs have been implicated in diverse cellular activities such as division, development, motility, homeostasis, stress response, secretion, transport, signaling and host-pathogen interactions. However, the majority of these proteins are hypothetical, and only few of them have been studied functionally. The finding of many diverse CaBPs in prokaryotic genomes opens an exciting area of research to explore and define the role of Ca(2+) in organisms other than eukaryotes. This review presents the most recent developments in the field of CaBPs and novel advancements in the role of Ca(2+) in prokaryotes.

  11. Identification of lipopolysaccharide-binding proteins in porcine milk

    PubMed Central

    Shahriar, Farshid; Gordon, John R.; Simko, Elemir

    2006-01-01

    Septicemia and endotoxemia initiated by bacterial lipopolysaccharide (LPS) are relatively common in suckling and weaned piglets. Maternal milk is a source of both nutrition and immune protection for piglets. Passive transfer of colostral antibodies is necessary for protection of neonatal piglets against diseases, but the concentration of immunoglobulins in milk rapidly declines during the 1st wk of lactation in all mammals. We hypothesized, therefore, that nonimmunoglobulin substances in milk contribute to the innate protection of neonates against septicemia during the suckling period. Using LPS-affinity chromatography for isolation of LPS-binding proteins and liquid chromatography–mass spectrometry for their identification, we identified in porcine milk the following proteins with LPS-binding capacity: lactoferrin, soluble CD14, serum amyloid A, α-S1 casein, β-casein, and κ-casein. For lactoferrin, α-S1 casein, and κ-casein, in vitro pepsin digestion did not inhibit LPS-binding activity, whereas combined digestion with pepsin and pancreatin abolished it. The biologic functions of these LPS-binding proteins and peptides were not determined. PMID:17042375

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

    PubMed

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

    2008-10-23

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

  13. Characterization of flavonoid-protein interactions using fluorescence spectroscopy: Binding of pelargonidin to dairy proteins.

    PubMed

    Arroyo-Maya, Izlia J; Campos-Terán, José; Hernández-Arana, Andrés; McClements, David Julian

    2016-12-15

    In this study, the interaction between the flavonoid pelargonidin and dairy proteins: β-lactoglobulin (β-LG), whey protein (WPI), and caseinate (CAS) was investigated. Fluorescence experiments demonstrated that pelargonidin quenched milk proteins fluorescence strongly. However, the protein secondary structure was not significantly affected by pelargonidin, as judged from far-UV circular dichroism. Analysis of fluorescence data indicated that pelargonidin-induced quenching does not arise from a dynamical mechanism, but instead is due to protein-ligand binding. Therefore, quenching data were analyzed using the model of independent binding sites. Both β-LG and CAS, but not WPI, showed hyperbolic binding isotherms indicating that these proteins firmly bound pelargonidin at both pH 7.0 and 3.0 (binding constants ca. 1.0×10(5) at 25.0°C). To investigate the underlying thermodynamics, binding constants were determined at 25.0, 35.0, and 45.0°C. These results pointed to binding processes that depend on the structural conformation of the milk proteins.

  14. Transport effects on the kinetics of protein-surface binding.

    PubMed Central

    Balgi, G; Leckband, D E; Nitsche, J M

    1995-01-01

    A detailed model is presented for protein binding to active surfaces, with application to the binding of avidin molecules to a biotin-functionalized fiber optic sensor in experiments reported by S. Zhao and W. M. Reichert (American Chemical Society Symposium Series 493, 1992). Kinetic data for binding in solution are used to assign an intrinsic catalytic rate coefficient k to the biotin-avidin pair, deconvoluted from transport and electrostatic factors via application of coagulation theory. This intrinsic chemical constant is built into a reaction-diffusion analysis of surface binding where activity is restricted to localized sites (representing immobilized biotin molecules). The analysis leads to an effective catalytic rate coefficient keff characterizing the active surface. Thereafter, solution of the transport problem describing absorption of avidin molecules by the macroscopic sensor surface leads to predictions of the avidin flux, which are found to be in good agreement with the experimental data. The analysis suggests the following conclusions. 1) Translational diffusion limitations are negligible for avidin-biotin binding in solution owing to the small (kinetically limiting) value k = 0.00045 m/s. 2) The sparse distribution of biotin molecules and the presence of a repulsive hydration force produce an effective surface-average catalytic rate coefficient keff of order 10(-7) m/s, much smaller than k. 3) Avidin binding to the fiber optic sensor occurs in an intermediate regime where the rate is influenced by both kinetics and diffusion. Images FIGURE 1 FIGURE 3 PMID:7647232

  15. Photoaffinity labelling of high affinity dopamine binding proteins

    SciTech Connect

    Ross, G.M.; McCarry, B.E.; Mishra, R.K.

    1986-03-01

    A photoactive analogue of the dopamine agonist 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronapthalene (ADTN) has been synthesized and used to photoaffinity label dopamine binding proteins prepared from bovine caudate nucleus. N-(3-)N'-4-azidobenzamidol)-aminopropyl)-aminopropyl)-ADTN (AzB-AP-ADTN) was incubated with caudate membranes and irradiated with UV light. Membranes were then repeatedly washed by centrifugation to remove excess photolabel. A binding assay, using (/sup 3/H)-SCH 23390 (a D/sub 1/ specific antagonist), was then performed to evaluate the loss of receptor density in the photolyzed preparation. AzB-AP-ADTN irreversibly blocked (/sup 3/H)-SCH 23390 binding in a dose-dependent manner. Scatchard analysis revealed a decrease in the B/sub max/, with no significant change in the K/sub d/, of (/sup 3/H)-SCH 23390 binding. Compounds which compete for D/sub 1/ receptor binding (such as dopamine, SKF 38393 or apomorphine), proteted the SCH 23390 binding site from inactivation. This data would suggest that the novel photoaffinity ligand, AzB-AP-ADTN, can covalently label the D/sub 1/ (adenylate cyclase linked) dopamine receptor.

  16. Predicting protein dynamic binding capacity from batch adsorption tests.

    PubMed

    Carta, Giorgio

    2012-10-01

    The dynamic binding capacity (DBC) and its dependence on residence time influence the design and productivity of adsorption columns used in protein capture applications. This paper offers a very simple approach to predict the DBC of an adsorption column based on a measurement of the equilibrium binding capacity (EBC) and of the time needed to achieve one-half of the EBC in a batch adsorption test. The approach is based on a mass transfer kinetics model that assumes pore diffusion with a rectangular isotherm; however, the same approach is also shown to work for other systems where solute transport inside the particle occurs through other transport mechanisms.

  17. Using protein binding site prediction to improve protein docking.

    PubMed

    Huang, Bingding; Schroeder, Michael

    2008-10-01

    Predicting protein interaction interfaces and protein complexes are two important related problems. For interface prediction, there are a number of tools, such as PPI-Pred, PPISP, PINUP, Promate, and SPPIDER, which predict enzyme-inhibitor interfaces with success rates of 23% to 55% and other interfaces with 10% to 28% on a benchmark dataset of 62 complexes. Here, we develop, metaPPI, a meta server for interface prediction. It significantly improves prediction success rates to 70% for enzyme-inhibitor and 44% for other interfaces. As shown with Promate, predicted interfaces can be used to improve protein docking. Here, we follow this idea using the meta server instead of individual predictions. We confirm that filtering with predicted interfaces significantly improves candidate generation in rigid-body docking based on shape complementarity. Finally, we show that the initial ranking of candidate solutions in rigid-body docking can be further improved for the class of enzyme-inhibitor complexes by a geometrical scoring which rewards deep pockets. A web server of metaPPI is available at scoppi.tu-dresden.de/metappi. The source code of our docking algorithm BDOCK is also available at www.biotec.tu-dresden.de /approximately bhuang/bdock.

  18. Adrenocortical nuclear progesterone-binding protein: Identification by photoaffinity labeling and evidence for deoxyribonucleic acid binding and stimulation by adrenocorticotropin

    SciTech Connect

    Demura, T.; Driscoll, W.J.; Lee, Y.C.; Strott, C.A. )

    1991-01-01

    Nuclei of the guinea pig adrenal cortex contain a protein that specifically binds progesterone and that, biochemically, is clearly distinct from the classical progesterone receptor. The adrenocortical nuclear progesterone-binding protein has now been purified more than 2000-fold by steroid-affinity chromatography with a 75% yield. The purified protein preparation demonstrated three major bands on sodium dodecyl sulfate-polyacrylamide gel of 79K, 74K, and 50K. To determine which of the three might represent the progesterone-binding protein, steroid photoaffinity labeling was performed which resulted in the specific and exclusive labeling of a 50K band. Thus, the adrenocortical nuclear progesterone-binding protein appears to be distinct from the classical progesterone receptor not only biochemically, but also on the basis of molecular size. To test whether the adrenocortical nuclear progesterone-binding protein can be hormonally stimulated, guinea pigs were treated with ACTH. The chronic administration of ACTH caused a 4- to 6-fold increase in the specific progesterone binding capacity without a change in the binding affinity. There appeared to be no significant difference in nuclear progesterone binding between the zona fasciculata and zona reticularis. This finding suggests a mediating role for the progesterone-binding protein in ACTH action. In addition, the nuclear progesterone-binding protein bound to nonspecific DNA sequences, further suggesting a possible transcriptional regulatory role.

  19. Physicochemical characteristics of structurally determined metabolite-protein and drug-protein binding events with respect to binding specificity

    PubMed Central

    Korkuć, Paula; Walther, Dirk

    2015-01-01

    To better understand and ultimately predict both the metabolic activities as well as the signaling functions of metabolites, a detailed understanding of the physical interactions of metabolites with proteins is highly desirable. Focusing in particular on protein binding specificity vs. promiscuity, we performed a comprehensive analysis of the physicochemical properties of compound-protein binding events as reported in the Protein Data Bank (PDB). We compared the molecular and structural characteristics obtained for metabolites to those of the well-studied interactions of drug compounds with proteins. Promiscuously binding metabolites and drugs are characterized by low molecular weight and high structural flexibility. Unlike reported for drug compounds, low rather than high hydrophobicity appears associated, albeit weakly, with promiscuous binding for the metabolite set investigated in this study. Across several physicochemical properties, drug compounds exhibit characteristic binding propensities that are distinguishable from those associated with metabolites. Prediction of target diversity and compound promiscuity using physicochemical properties was possible at modest accuracy levels only, but was consistently better for drugs than for metabolites. Compound properties capturing structural flexibility and hydrogen-bond formation descriptors proved most informative in PLS-based prediction models. With regard to diversity of enzymatic activities of the respective metabolite target enzymes, the metabolites benzylsuccinate, hypoxanthine, trimethylamine N-oxide, oleoylglycerol, and resorcinol showed very narrow process involvement, while glycine, imidazole, tryptophan, succinate, and glutathione were identified to possess broad enzymatic reaction scopes. Promiscuous metabolites were found to mainly serve as general energy currency compounds, but were identified to also be involved in signaling processes and to appear in diverse organismal systems (digestive and nervous

  20. Interaction of Protein Inhibitor of Activated STAT (PIAS) Proteins with the TATA-binding Protein, TBP*

    PubMed Central

    Prigge, Justin R.; Schmidt, Edward E.

    2007-01-01

    Transcription activators often recruit promoter-targeted assembly of a pre-initiation complex; many repressors antagonize recruitment. These activities can involve direct interactions with proteins in the pre-initiation complex. We used an optimized yeast two-hybrid system to screen mouse pregnancy-associated libraries for proteins that interact with TATA-binding protein (TBP). Screens revealed an interaction between TBP and a single member of the zinc finger family of transcription factors, ZFP523. Two members of the protein inhibitor of activated STAT (PIAS) family, PIAS1 and PIAS3, also interacted with TBP in screens. Endogenous PIAS1 and TBP co-immunoprecipitated from nuclear extracts, suggesting the interaction occurred in vivo. In vitro-translated PIAS1 and TBP coimmunopreciptated, which indicated that other nuclear proteins were not required for the interaction. Deletion analysis mapped the PIAS-interacting domain of TBP to the conserved TBPCORE and the TBP-interacting domain on PIAS1 to a 39-amino acid C-terminal region. Mammals issue seven known PIAS proteins from four pias genes, pias1, pias3, piasx, and piasy, each with different cell type-specific expression patterns; the TBP-interacting domain reported here is the only part of the PIAS C-terminal region shared by all seven PIAS proteins. Direct analyses indicated that PIASx and PIASy also interacted with TBP. Our results suggest that all PIAS proteins might mediate situation-specific regulatory signaling at the TBP interface and that previously unknown levels of complexity could exist in the gene regulatory interplay between TBP, PIAS proteins, ZFP523, and other transcription factors. PMID:16522640

  1. Stable Isotope Labeling Strategy for Protein-Ligand Binding Analysis in Multi-Component Protein Mixtures

    NASA Astrophysics Data System (ADS)

    DeArmond, Patrick D.; West, Graham M.; Huang, Hai-Tsang; Fitzgerald, Michael C.

    2011-03-01

    Described here is a stable isotope labeling protocol that can be used with a chemical modification- and mass spectrometry-based protein-ligand binding assay for detecting and quantifying both the direct and indirect binding events that result from protein-ligand binding interactions. The protocol utilizes an H{2/16}O2 and H{2/18}O2 labeling strategy to evaluate the chemical denaturant dependence of methionine oxidation in proteins both in the presence and absence of a target ligand. The differential denaturant dependence to the oxidation reactions performed in the presence and absence of ligand provides a measure of the protein stability changes that occur as a result of direct interactions of proteins with the target ligand and/or as a result of indirect interactions involving other protein-ligand interactions that are either induced or disrupted by the ligand. The described protocol utilizes the 18O/16O ratio in the oxidized protein samples to quantify the ligand-induced protein stability changes. The ratio is determined using the isotopic distributions observed for the methionine-containing peptides used for protein identification in the LC-MS-based proteomics readout. The strategy is applied to a multi-component protein mixture in this proof-of-principle experiment, which was designed to evaluate the technique's ability to detect and quantify the direct binding interaction between cyclosporin A and cyclophilin A and to detect the indirect binding interaction between cyclosporin A and calcineurin (i.e., the protein-protein interaction between cyclophilin A and calcineurin that is induced by cyclosporin A binding to cyclophilin A).

  2. Human erythrocyte dematin and protein 4.2 (pallidin) are ATP binding proteins.

    PubMed

    Azim, A C; Marfatia, S M; Korsgren, C; Dotimas, E; Cohen, C M; Chishti, A H

    1996-03-05

    Dematin and protein 4.2 are peripheral membrane proteins associated with the cytoplasmic surface of the human erythrocyte plasma membrane. Isoforms of dematin and protein 4.2 exist in many nonerythroid cells. In solution, dematin is a trimeric protein containing two subunits of 48 kDa and one subunit of 52 kDa. Recent determination of the primary structure of the 52 kDa subunit of dematin showed that it contains an additional 22-amino acid sequence in the headpiece domain. An alignment of the 22-amino acid insertion sequence revealed that the 52 kDa subunit of dematin shares a novel 11-amino acid motif with protein 4.2. In this communication, we report that the conserved 11-amino acid motif in dematin52 and protein 4.2 contains a nucleotide binding P-loop. Direct binding of ATP is demonstrated to the glutathione S-transferase fusion proteins containing corresponding segments of dematin52 and protein 4.2 as well as to purified protein 4.2. The binding of ATP to the recombinant domains of dematin52 and protein 4.2 is specific, saturable, and of high affinity. The nucleotide specificity of the P-loop is restricted to ATP since no detectable binding was observed with GTP. These results show that the 11-amino acid motif provides an ATP binding site in dematin52 and protein 4.2. Although the functional significance of ATP binding is not yet clear, our findings open new perspectives for the function of dematin and protein 4.2 in vivo.

  3. Identification of pneumococcal surface protein A as a lactoferrin-binding protein of Streptococcus pneumoniae.

    PubMed

    Hammerschmidt, S; Bethe, G; Remane, P H; Chhatwal, G S

    1999-04-01

    Lactoferrin (Lf), an iron-sequestering glycoprotein, predominates in mucosal secretions, where the level of free extracellular iron (10(-18) M) is not sufficient for bacterial growth. This represents a mechanism of resistance to bacterial infections by prevention of colonization of the host by pathogens. In this study we were able to show that Streptococcus pneumoniae specifically recognizes and binds the iron carrier protein human Lf (hLf). Pretreatment of pneumococci with proteases reduced hLf binding significantly, indicating that the hLf receptor is proteinaceous. Binding assays performed with 63 clinical isolates belonging to different serotypes showed that 88% of the tested isolates interacted with hLf. Scatchard analysis showed the existence of two hLf-binding proteins with dissociation constants of 5.7 x 10(-8) and 2.74 x 10(-7) M. The receptors were purified by affinity chromatography, and internal sequence analysis revealed that one of the S. pneumoniae proteins was homologous to pneumococcal surface protein A (PspA). The function of PspA as an hLf-binding protein was confirmed by the ability of purified PspA to bind hLf and to competitively inhibit hLf binding to pneumococci. S. pneumoniae may use the hLf-PspA interaction to overcome the iron limitation at mucosal surfaces, and this might represent a potential virulence mechanism.

  4. Fibronectin-binding protein of Streptococcus pyogenes: sequence of the binding domain involved in adherence of streptococci to epithelial cells.

    PubMed Central

    Talay, S R; Valentin-Weigand, P; Jerlström, P G; Timmis, K N; Chhatwal, G S

    1992-01-01

    The sequence of the fibronectin-binding domain of the fibronectin-binding protein of Streptococcus pyogenes (Sfb protein) was determined, and its role in streptococcal adherence was investigated by use of an Sfb fusion protein in adherence studies. A 1-kb DNA fragment coding for the binding domain of Sfb protein was cloned into the expression vector pEX31 to produce an Sfb fusion protein consisting of the N-terminal part of MS2 polymerase and a C-terminal fragment of the streptococcal protein. Induction of the vector promoter resulted in hyperexpression of fibronectin-binding fusion protein in the cytoplasm of the recombinant Escherichia coli cells. Sequence determination of the cloned 1-kb fragment revealed an in-frame reading frame for a 268-amino-acid peptide composed of a 37-amino-acid sequence which is completely repeated three times and incompletely repeated a fourth time. Cloning of one repeat into pEX31 resulted in expression of small fusion peptides that show fibronectin-binding activity, indicating that one repeat contains at least one binding domain. Each repeat exhibits two charged domains and shows high homology with the 38-amino-acid D3 repeat of the fibronectin-binding protein of Staphylococcus aureus. Sequence comparison with other streptococcal ligand-binding surface proteins, including M protein, failed to reveal significant homology, which suggests that Sfb protein represents a novel type of functional protein in S. pyogenes. The Sfb fusion protein isolated from the cytoplasm of recombinant cells was purified by fast protein liquid chromatography. It showed a strong competitive inhibition of fibronectin binding to S. pyogenes and of the adherence of bacteria to cultured epithelial cells. In contrast, purified streptococcal lipoteichoic acid showed only a weak inhibition of fibronectin binding and streptococcal adherence. These results demonstrate that Sfb protein is directly involved in the fibronectin-mediated adherence of S. pyogenes to

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

    PubMed

    Zhao, Huiying; Wang, Jihua; Zhou, Yaoqi; Yang, Yuedong

    2014-01-01

    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.

  6. A complex of nuclear proteins mediates SR protein binding to a purine-rich splicing enhancer.

    PubMed Central

    Yeakley, J M; Morfin, J P; Rosenfeld, M G; Fu, X D

    1996-01-01

    A purine-rich splicing enhancer from a constitutive exon has been shown to shift the alternative splicing of calcitonin/CGRP pre-mRNA in vivo. Here, we demonstrate that the native repetitive GAA sequence comprises the optimal enhancer element and specifically binds a saturable complex of proteins required for general splicing in vitro. This complex contains a 37-kDa protein that directly binds the repetitive GAA sequence and SRp40, a member of the SR family of non-snRNP splicing factors. While purified SR proteins do not stably bind the repetitive GAA element, exogenous SR proteins become associated with the GAA element in the presence of nuclear extracts and stimulate GAA-dependent splicing. These results suggest that repetitive GAA sequences enhance splicing by binding a protein complex containing a sequence-specific RNA binding protein and a general splicing activator that, in turn, recruit additional SR proteins. This type of mechanism resembles the tra/tra-2-dependent recruitment of SR proteins to the Drosophila doublesex alternative splicing regulatory element. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8755518

  7. Why are hyperactive ice-binding-proteins so active?

    NASA Astrophysics Data System (ADS)

    Braslavsky, Ido; Celik, Yeliz; Pertaya, Natalya; Eun Choi, Young; Bar, Maya; Davies, Peter L.

    2008-03-01

    Ice binding proteins (IBPs), also called `antifreeze proteins' or `ice structuring proteins', are a class of proteins that protect organisms from freezing injury. These proteins have many applications in medicine and agriculture, and as a platform for future biotechnology applications. One of the interesting questions in this field focuses on the hyperactivity of some IBPs. Ice binding proteins can be classified in two groups: moderate ones that can depress the freezing point up to ˜1.0 ^oC and hyperactive ones that can depress the freezing point several-fold further even at lower concentrations. It has been suggested that the hyperactivity of IBPs stem from the fact that they block growth out of specific ice surfaces, more specifically the basal planes of ice. Here we show experimental results based on fluorescence microscopy, highlighting the differences between moderate IBPs and hyperactive IBPs. These include direct evidence for basal plane affinity of hyperactive IBPs, the effects of IBPs on growth-melt behavior of ice and the dynamics of their interaction with ice.

  8. Crystal Structure of Human Retinoblastoma Binding Protein 9

    SciTech Connect

    Vorobiev, S.; Su, M; Seetharaman, J; Huang, Y; Chen, C; Maglaqui, M; Janjua, H; Montelione, G; Tong, L; et. al.

    2009-01-01

    As a step towards better integrating protein three-dimensional (3D) structural information in cancer systems biology, the Northeast Structural Genomics Consortium (NESG) (www.nesg.org) has constructed a Human Cancer Pathway Protein Interaction Network (HCPIN) by analysis of several classical cancer-associated signaling pathways and their physical protein-protein interactions. Many well-known cancer-associated proteins play central roles as hubs or bottlenecks in the HCPIN (http://nmr.cabm.rutgers.edu/hcpin). NESG has selected more than 1000 human proteins and protein domains from the HCPIN for sample production and 3D structure determination. The long-range goal of this effort is to provide a comprehensive 3D structure-function database for human cancer-associated proteins and protein complexes, in the context of their interaction networks. Human retinoblastoma binding protein 9 (RBBP9) is one of the HCPIN proteins targeted by NESG. RBBP9 was initially identified as the product of a new gene, Bog (for B5T over-expressed gene), in several transformed rat liver epithelial cell lines resistant to the growth-inhibitory effect of TGF-1 as well as in primary human liver tumors. RBBP9 contains the retinoblastoma (Rb) binding motif LxCxE in its sequence, and was shown to interact with Rb by yeast two-hybrid and coimmunoprecipitation experiments. Mutation of the Leu residue in this motif to Gln blocked the binding to Rb. RBBP9 can displace E2F1 from E2F1-Rb complexes, and over expression of RBBP9 overcomes TGF-1 induced growth arrest and results in transformation of rat liver epithelial cells leading to hepatoblastoma-like tumors in nude mice. RBBP9 may also play a role in cellular responses to chronic low dose radiation. A close homolog of RBBP9, sharing 93% amino acid sequence identity and also known as RBBP10, interacts with a protein with sua5-yciO-yrdC domains.

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

    PubMed Central

    Chien, Ting-Ying; Lin, Chih-Kang; Lin, Chih-Wei; Weng, Yi-Zhong; Chen, Chien-Yu; Chang, Darby Tien-Hao

    2012-01-01

    By binding to short and highly conserved DNA sequences in genomes, DNA-binding proteins initiate, enhance or repress biological processes. Accurately identifying such binding sites, often represented by position weight matrices (PWMs), is an important step in understanding the control mechanisms of cells. When given coordinates of a DNA-binding domain (DBD) bound with DNA, a potential function can be used to estimate the change of binding affinity after base substitutions, where the changes can be summarized as a PWM. This technique provides an effective alternative when the chromatin immunoprecipitation data are unavailable for PWM inference. To facilitate the procedure of predicting PWMs based on protein–DNA complexes or even structures of the unbound state, the web server, DBD2BS, is presented in this study. The DBD2BS uses an atom-level knowledge-based potential function to predict PWMs characterizing the sequences to which the query DBD structure can bind. For unbound queries, a list of 1066 DBD–DNA complexes (including 1813 protein chains) is compiled for use as templates for synthesizing bound structures. The DBD2BS provides users with an easy-to-use interface for visualizing the PWMs predicted based on different templates and the spatial relationships of the query protein, the DBDs and the DNAs. The DBD2BS is the first attempt to predict PWMs of DBDs from unbound structures rather than from bound ones. This approach increases the number of existing protein structures that can be exploited when analyzing protein–DNA interactions. In a recent study, the authors showed that the kernel adopted by the DBD2BS can generate PWMs consistent with those obtained from the experimental data. The use of DBD2BS to predict PWMs can be incorporated with sequence-based methods to discover binding sites in genome-wide studies. Available at: http://dbd2bs.csie.ntu.edu.tw/, http://dbd2bs.csbb.ntu.edu.tw/, and http://dbd2bs.ee.ncku.edu.tw. PMID:22693214

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

    PubMed Central

    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/. PMID:27907159

  11. Protein-Binding RNA Aptamers Affect Molecular Interactions Distantly from Their Binding Sites

    PubMed Central

    Dupont, Daniel M.; Thuesen, Cathrine K.; Bøtkjær, Kenneth A.; Behrens, Manja A.; Dam, Karen; Sørensen, Hans P.; Pedersen, Jan S.; Ploug, Michael; Jensen, Jan K.; Andreasen, Peter A.

    2015-01-01

    Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126) with therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA). We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A) controlling uPA activities. One of the aptamers (upanap-126) binds to the area 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. PMID:25793507

  12. Protein universe containing a PUA RNA-binding domain.

    PubMed

    Cerrudo, Carolina S; Ghiringhelli, Pablo D; Gomez, Daniel E

    2014-01-01

    Here, we review current knowledge about pseudouridine synthase and archaeosine transglycosylase (PUA)-domain-containing proteins to illustrate progress in this field. A methodological analysis of the literature about the topic was carried out, together with a 'qualitative comparative analysis' to give a more comprehensive review. Bioinformatics methods for whole-protein or protein-domain identification are commonly based on pairwise protein sequence comparisons; we added comparison of structures to detect the whole universe of proteins containing the PUA domain. We present an update of proteins having this domain, focusing on the specific proteins present in Homo sapiens (dyskerin, MCT1, Nip7, eIF2D and Nsun6), and explore the existence of these in other species. We also analyze the phylogenetic distribution of the PUA domain in different species and proteins. Finally, we performed a structural comparison of the PUA domain through data mining of structural databases, determining a conserved structural motif, despite the differences in the sequence, even among eukaryotes, archaea and bacteria. All data discussed in this review, both bibliographic and analytical, corroborate the functional importance of the PUA domain in RNA-binding proteins.

  13. Altering the GTP binding site of the DNA/RNA-binding protein, Translin/TB-RBP, decreases RNA binding and may create a dominant negative phenotype.

    PubMed

    Chennathukuzhi, V M; Kurihara, Y; Bray, J D; Yang, J; Hecht, N B

    2001-11-01

    The DNA/RNA-binding protein, Translin/Testis Brain RNA-binding protein (Translin/TB-RBP), contains a putative GTP binding site in its C-terminus which is highly conserved. To determine if guanine nucleotide binding to this site functionally alters nucleic acid binding, electrophoretic mobility shift assays were performed with RNA and DNA binding probes. GTP, but not GDP, reduces RNA binding by approximately 50% and the poorly hydrolyzed GTP analog, GTPgammaS, reduces binding by >90% in gel shift and immunoprecipitation assays. No similar reduction of DNA binding is seen. When the putative GTP binding site of TB-RBP, amino acid sequence VTAGD, is altered to VTNSD by site directed mutagenesis, GTP will no longer bind to TB-RBP(GTP) and TB-RBP(GTP) no longer binds to RNA, although DNA binding is not affected. Yeast two-hybrid assays reveal that like wild-type TB-RBP, TB-RBP(GTP) will interact with itself, with wild-type TB-RBP and with Translin associated factor X (Trax). Transfection of TB-RBP(GTP) into NIH 3T3 cells leads to a marked increase in cell death suggesting a dominant negative function for TB-RBP(GTP) in cells. These data suggest TB-RBP is an RNA-binding protein whose activity is allosterically controlled by nucleotide binding.

  14. Difference in DNA-binding abilities of Fur-homolog DNA binding protein from Neisseria gonorrhoeae.

    PubMed

    Bagchi, Angshuman

    2016-10-01

    Gonorrhea is a severe disease infecting both men and women worldwide. The causative agent of the disease is Neisseria gonorrhoeae. The organism mostly affects human beings in iron restricted environments. In such an environment the organism produces a set of proteins which are mostly absent in iron rich environments. The expressions of the genes for the proteins are regulated by the transcription factor (TF) belonging to the Fur family. Interestingly, the same TF acts as the activator and repressor of genes. In this present work, an attempt has been made to analyze the molecular details of the differential DNA-binding activities of the TF from Neisseria gonorrhoeae to come up with a plausible molecular reason behind the difference DNA binding activities of the same TF. Computational modelling technique was used to build the three dimensional structure of the TF. Molecular docking and molecular dynamics simulations were employed to determine the binding interactions between the TF and the promoter DNA. With the help of the computational techniques, the biochemical reason behind the different modes of DNA binding by the TF was analyzed. Results from this analysis may be useful to future drug development endeavours to curtail the spread of Gonorrhea.

  15. Expression patterns and binding properties of three pheromone binding proteins in the diamondback moth, Plutella xyllotella.

    PubMed

    Sun, Mengjing; Liu, Yang; Wang, Guirong

    2013-01-01

    Pheromone binding proteins (PBPs) play a key role in transporting hydrophobic sex pheromone components emitted by con-specific female across aqueous sensillar lymph to the surface of olfactory receptor neurons. A number of PBPs have been cloned, however, details of their function are still largely unknown. Here three pheromone binding protein genes in the diamondback moth, Plutella xyllotella were cloned. The three PxylPBP genes are not only expressed in chemosensory tissues but also expressed in female reproductive organs and male legs. To better understand the functions of PxylPBPs in the initial steps of pheromone recognition, three PxylPBPs were expressed in Escherichia coli and the ligand-binding specificities of purified recombinant PBPs were investigated. Fluorescence binding assays indicate that three PxylPBPs not only robustly bound all four sex pheromone components but also significantly bound pheromone analogs with at least one double bond, while weakly bound tested plant volatiles. Although pheromone analogs bound PBPs, they could not elicit the moth's electrophysiological response. These experiments provide evidence that PxylPBPs have limited selectivity of pheromone components and analogs and some downstream components such as odor receptors might be involved in selectivity and specificity of pheromone perception in P. xyllotella.

  16. Pyruvate kinase M2 is a phosphotyrosine-binding protein

    SciTech Connect

    Christofk, H.R.; Vander Heiden, M.G.; Wu, N.; Asara, J.M.; Cantley, L.C.

    2008-06-03

    Growth factors stimulate cells to take up excess nutrients and to use them for anabolic processes. The biochemical mechanism by which this is accomplished is not fully understood but it is initiated by phosphorylation of signalling proteins on tyrosine residues. Using a novel proteomic screen for phosphotyrosine-binding proteins, we have made the observation that an enzyme involved in glycolysis, the human M2 (fetal) isoform of pyruvate kinase (PKM2), binds directly and selectively to tyrosine-phosphorylated peptides. We show that binding of phosphotyrosine peptides to PKM2 results in release of the allosteric activator fructose-1,6-bisphosphate, leading to inhibition of PKM2 enzymatic activity. We also provide evidence that this regulation of PKM2 by phosphotyrosine signalling diverts glucose metabolites from energy production to anabolic processes when cells are stimulated by certain growth factors. Collectively, our results indicate that expression of this phosphotyrosine-binding form of pyruvate kinase is critical for rapid growth in cancer cells.

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

    PubMed

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

    2015-03-01

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

  18. The binding of immunoglobulin Fc to cationic proteins.

    PubMed Central

    Pambakian, S; Poston, R N

    1987-01-01

    The interaction of cationic proteins with IgG, IgA and IgM were investigated by solid phase radioimmunoassay. All these immunoglobulins showed avid binding, IgM giving the strongest reaction, followed by IgA and then IgG. Fc fragments of IgG gave binding, but F(ab')2 fragments from the three main Ig classes did not, showing that the Fc region is the active part of the molecule. The effects of changes of ionic strength and pH are compatible with the interaction being ionic, and are similar to those seen between immunoglobulins and both Clq and cationic ion exchange gels. The addition of other serum proteins resulted in marked inhibition of the interaction. These phenomena are likely to have fundamental significance for the understanding of interactions of immunoglobulins in vivo and in vitro. Images Fig. 6 PMID:3652520

  19. Immunochemical similarity of GTP-binding proteins from different systems

    SciTech Connect

    Kalinina, S.N.

    1986-06-20

    It was found that antibodies against the GTP-binding proteins of bovine retinal photoreceptor membranes blocked the inhibitory effect of estradiol on phosphodiesterase from rat and human uterine cytosol and prevented the cumulative effect of catecholamines and guanylyl-5'-imidodiphosphate on rat skeletal muscle adenylate cyclase. It was established by means of double radial immunodiffusion that these antibodies form a precipitating complex with purified bovine brain tubulin as well as with retinal preparations obtained from eyes of the bull, pig, rat, frog, some species of fish, and one reptile species. Bands of precipitation were not observed with these antibodies when retinal preparations from invertebrates (squid and octopus) were used as the antigens. The antibodies obtained interacted with the ..cap alpha..- and ..beta..-subunits of GTP-binding proteins from bovine retinal photoreceptor membranes.

  20. Isolation and Characterization of the DNA and Protein Binding Activities of Adenovirus Core Protein V

    PubMed Central

    Pérez-Vargas, Jimena; Vaughan, Robert C.; Houser, Carolyn; Hastie, Kathryn M.; Kao, C. Cheng

    2014-01-01

    ABSTRACT The structure of adenovirus outer capsid was revealed recently at 3- to 4-Å resolution (V. Reddy, S. Natchiar, P. Stewart, and G. Nemerow, Science 329:1071–1075, 2010, http://dx.doi.org/10.1126/science.1187292); however, precise details on the function and biochemical and structural features for the inner core still are lacking. Protein V is one the most important components of the adenovirus core, as it links the outer capsid via association with protein VI with the inner DNA core. Protein V is a highly basic protein that strongly binds to DNA in a nonspecific manner. We report the expression of a soluble protein V that exists in monomer-dimer equilibrium. Using reversible cross-linking affinity purification in combination with mass spectrometry, we found that protein V contains multiple DNA binding sites. The binding sites from protein V mediate heat-stable nucleic acid associations, with some of the binding sites possibly masked in the virus by other core proteins. We also demonstrate direct interaction between soluble proteins V and VI, thereby revealing the bridging of the inner DNA core with the outer capsid proteins. These findings are consistent with a model of nucleosome-like structures proposed for the adenovirus core and encapsidated DNA. They also suggest an additional role for protein V in linking the inner nucleic acid core with protein VI on the inner capsid shell. IMPORTANCE Scant knowledge exists of how the inner core of adenovirus containing its double-stranded DNA (dsDNA) genome and associated proteins is organized. Here, we report a purification scheme for a recombinant form of protein V that allowed analysis of its interactions with the nucleic acid core region. We demonstrate that protein V exhibits stable associations with dsDNA due to the presence of multiple nucleic acid binding sites identified both in the isolated recombinant protein and in virus particles. As protein V also binds to the membrane lytic protein VI molecules

  1. DNA binding protein identification by combining pseudo amino acid composition and profile-based protein representation

    NASA Astrophysics Data System (ADS)

    Liu, Bin; Wang, Shanyi; Wang, Xiaolong

    2015-10-01

    DNA-binding proteins play an important role in most cellular processes. Therefore, it is necessary to develop an efficient predictor for identifying DNA-binding proteins only based on the sequence information of proteins. The bottleneck for constructing a useful predictor is to find suitable features capturing the characteristics of DNA binding proteins. We applied PseAAC to DNA binding protein identification, and PseAAC was further improved by incorporating the evolutionary information by using profile-based protein representation. Finally, Combined with Support Vector Machines (SVMs), a predictor called iDNAPro-PseAAC was proposed. Experimental results on an updated benchmark dataset showed that iDNAPro-PseAAC outperformed some state-of-the-art approaches, and it can achieve stable performance on an independent dataset. By using an ensemble learning approach to incorporate more negative samples (non-DNA binding proteins) in the training process, the performance of iDNAPro-PseAAC was further improved. The web server of iDNAPro-PseAAC is available at http://bioinformatics.hitsz.edu.cn/iDNAPro-PseAAC/.

  2. Identification of albumin-binding proteins in capillary endothelial cells

    PubMed Central

    1988-01-01

    Isolated fat tissue microvessels and lung, whose capillary endothelia express in situ specific binding sites for albumin, were homogenized and subjected to SDS-gel electrophoresis and electroblotting. The nitrocellulose strips were incubated with either albumin-gold (Alb-Au) and directly visualized, or with [125I]albumin (monomeric or polymeric) and autoradiographed. The extracts of both microvascular endothelium and the lung express albumin-binding proteins (ABPs) represented by two pairs of polypeptides with major components of molecular mass 31 and 18 kD. The ABP peptides have pIs 8.05 to 8.75. Rabbit aortic endothelium, used as control, does not express detectable amounts of ABPs. The ABPs subjected to electrophoresis bind specifically and with high affinity (Kd = approximately 60 X 10(-9)M) both monomeric and polymeric albumin: the binding is saturable at approximately 80 nM concentration and 50% inhibition is reached at 5.5 micrograms/ml albumin concentration. Sulfhydryl-reducing agents beta-mercaptoethanol and dithiothreitol do not markedly affect the ABPs electrophoretic mobility and binding properties. As indicated by cell surface iodination of isolated capillary endothelium followed by electroblotting, autoradiography, and incubation with Alb-Au, the bands specifically stained by this ligand are also labeled with radioiodine. PMID:2839518

  3. Anchored Clathrate Waters Bind Antifreeze Proteins to Ice

    SciTech Connect

    C Garnham; R Campbell; P Davies

    2011-12-31

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

  4. Identifying Interactions that Determine Fragment Binding at Protein Hotspots.

    PubMed

    Radoux, Chris J; Olsson, Tjelvar S G; Pitt, Will R; Groom, Colin R; Blundell, Tom L

    2016-05-12

    Locating a ligand-binding site is an important first step in structure-guided drug discovery, but current methods do little to suggest which interactions within a pocket are the most important for binding. Here we illustrate a method that samples atomic hotspots with simple molecular probes to produce fragment hotspot maps. These maps specifically highlight fragment-binding sites and their corresponding pharmacophores. For ligand-bound structures, they provide an intuitive visual guide within the binding site, directing medicinal chemists where to grow the molecule and alerting them to suboptimal interactions within the original hit. The fragment hotspot map calculation is validated using experimental binding positions of 21 fragments and subsequent lead molecules. The ligands are found in high scoring areas of the fragment hotspot maps, with fragment atoms having a median percentage rank of 97%. Protein kinase B and pantothenate synthetase are examined in detail. In each case, the fragment hotspot maps are able to rationalize a Free-Wilson analysis of SAR data from a fragment-based drug design project.

  5. Anchored clathrate waters bind antifreeze proteins to ice.

    PubMed

    Garnham, Christopher P; Campbell, Robert L; Davies, Peter L

    2011-05-03

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

  6. Analysis of zinc binding sites in protein crystal structures.

    PubMed Central

    Alberts, I. L.; Nadassy, K.; Wodak, S. J.

    1998-01-01

    The geometrical properties of zinc binding sites in a dataset of high quality protein crystal structures deposited in the Protein Data Bank have been examined to identify important differences between zinc sites that are directly involved in catalysis and those that play a structural role. Coordination angles in the zinc primary coordination sphere are compared with ideal values for each coordination geometry, and zinc coordination distances are compared with those in small zinc complexes from the Cambridge Structural Database as a guide of expected trends. We find that distances and angles in the primary coordination sphere are in general close to the expected (or ideal) values. Deviations occur primarily for oxygen coordinating atoms and are found to be mainly due to H-bonding of the oxygen coordinating ligand to protein residues, bidentate binding arrangements, and multi-zinc sites. We find that H-bonding of oxygen containing residues (or water) to zinc bound histidines is almost universal in our dataset and defines the elec-His-Zn motif. Analysis of the stereochemistry shows that carboxyl elec-His-Zn motifs are geometrically rigid, while water elec-His-Zn motifs show the most geometrical variation. As catalytic motifs have a higher proportion of carboxyl elec atoms than structural motifs, they provide a more rigid framework for zinc binding. This is understood biologically, as a small distortion in the zinc position in an enzyme can have serious consequences on the enzymatic reaction. We also analyze the sequence pattern of the zinc ligands and residues that provide elecs, and identify conserved hydrophobic residues in the endopeptidases that also appear to contribute to stabilizing the catalytic zinc site. A zinc binding template in protein crystal structures is derived from these observations. PMID:10082367

  7. Structure of human guanylate-binding protein 1 representing a unique class of GTP-binding proteins.

    PubMed

    Prakash, B; Praefcke, G J; Renault, L; Wittinghofer, A; Herrmann, C

    2000-02-03

    Interferon-gamma is an immunomodulatory substance that induces the expression of many genes to orchestrate a cellular response and establish the antiviral state of the cell. Among the most abundant antiviral proteins induced by interferon-gamma are guanylate-binding proteins such as GBP1 and GBP2. These are large GTP-binding proteins of relative molecular mass 67,000 with a high-turnover GTPase activity and an antiviral effect. Here we have determined the crystal structure of full-length human GBP1 to 1.8 A resolution. The amino-terminal 278 residues constitute a modified G domain with a number of insertions compared to the canonical Ras structure, and the carboxy-terminal part is an extended helical domain with unique features. From the structure and biochemical experiments reported here, GBP1 appears to belong to the group of large GTP-binding proteins that includes Mx and dynamin, the common property of which is the ability to undergo oligomerization with a high concentration-dependent GTPase activity.

  8. Drug Discovery Toward Antagonists of Methyl-Lysine Binding Proteins

    PubMed Central

    Herold, J. Martin; Ingerman, Lindsey A; Gao, Cen; Frye, Stephen V

    2011-01-01

    The recognition of methyl-lysine and -arginine residues on both histone and other proteins by specific “reader” elements is important for chromatin regulation, gene expression, and control of cell-cycle progression. Recently the crucial role of these reader proteins in cancer development and dedifferentiation has emerged, owing to the increased interest among the scientific community. The methyl-lysine and -arginine readers are a large and very diverse set of effector proteins and targeting them with small molecule probes in drug discovery will inevitably require a detailed understanding of their structural biology and mechanism of binding. In the following review, the critical elements of methyl-lysine and -arginine recognition will be summarized with respect to each protein family and initial results in assay development, probe design, and drug discovery will be highlighted. PMID:22145013

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

    NASA Astrophysics Data System (ADS)

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

    2008-12-01

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

  10. Binding of S100 proteins to RAGE: an update.

    PubMed

    Leclerc, Estelle; Fritz, Günter; Vetter, Stefan W; Heizmann, Claus W

    2009-06-01

    The Receptor for Advanced Glycation Endproducts (RAGE) is a multi-ligand receptor of the immunoglobulin family. RAGE interacts with structurally different ligands probably through the oligomerization of the receptor on the cell surface. However, the exact mechanism is unknown. Among RAGE ligands are members of the S100 protein family. S100 proteins are small calcium binding proteins with high structural homology. Several members of the family have been shown to interact with RAGE in vitro or in cell-based assays. Interestingly, many RAGE ligands appear to interact with distinct domains of the extracellular portion of RAGE and to trigger various cellular effects. In this review, we summarize the modes of S100 protein-RAGE interaction with regard to their cellular functions.

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

    NASA Technical Reports Server (NTRS)

    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

  12. Maltose-Binding Protein (MBP), a Secretion-Enhancing Tag for Mammalian Protein Expression Systems.

    PubMed

    Reuten, Raphael; Nikodemus, Denise; Oliveira, Maria B; Patel, Trushar R; Brachvogel, Bent; Breloy, Isabelle; Stetefeld, Jörg; Koch, Manuel

    2016-01-01

    Recombinant proteins are commonly expressed in eukaryotic expression systems to ensure the formation of disulfide bridges and proper glycosylation. Although many proteins can be expressed easily, some proteins, sub-domains, and mutant protein versions can cause problems. Here, we investigated expression levels of recombinant extracellular, intracellular as well as transmembrane proteins tethered to different polypeptides in mammalian cell lines. Strikingly, fusion of proteins to the prokaryotic maltose-binding protein (MBP) generally enhanced protein production. MBP fusion proteins consistently exhibited the most robust increase in protein production in comparison to commonly used tags, e.g., the Fc, Glutathione S-transferase (GST), SlyD, and serum albumin (ser alb) tag. Moreover, proteins tethered to MBP revealed reduced numbers of dying cells upon transient transfection. In contrast to the Fc tag, MBP is a stable monomer and does not promote protein aggregation. Therefore, the MBP tag does not induce artificial dimerization of tethered proteins and provides a beneficial fusion tag for binding as well as cell adhesion studies. Using MBP we were able to secret a disease causing laminin β2 mutant protein (congenital nephrotic syndrome), which is normally retained in the endoplasmic reticulum. In summary, this study establishes MBP as a versatile expression tag for protein production in eukaryotic expression systems.

  13. A novel LZAP-binding protein, NLBP, inhibits cell invasion.

    PubMed

    Kwon, Junhye; Cho, Hyun Jung; Han, Seung Hun; No, Jin Gu; Kwon, Jae Young; Kim, Hongtae

    2010-04-16

    LXXLL/leucine zipper-containing alternative reading frame (ARF)-binding protein (LZAP) was recently shown to function as a tumor suppressor through inhibition of the NF-kappaB signaling pathway. LZAP is also known as a negative regulator of cell invasion, and its expression was demonstrated to be reduced in several tumor tissues. However, the molecular mechanism of the negative effect of LZAP on cell invasion is unclear. In this study, we identify NLBP as a novel LZAP-binding protein using tandem affinity purification. We demonstrate the negative effects of NLBP on cell invasion and the NF-kappaB signaling pathway. NLBP expression was not detected in hepatocellular carcinoma cells with strong invasive activity, whereas its expression was detected in a hepatocellular carcinoma cell line with no invasive activity. We also demonstrate that these two proteins mutually affect the stability of each other by inhibiting ubiquitination of the other protein. Based on these results, we suggest that NLBP may act as a novel tumor suppressor by inhibiting cell invasion, blocking NF-kappaB signaling, and increasing stability of the LZAP protein.

  14. A Novel LZAP-binding Protein, NLBP, Inhibits Cell Invasion*

    PubMed Central

    Kwon, Junhye; Cho, Hyun Jung; Han, Seung Hun; No, Jin Gu; Kwon, Jae Young; Kim, Hongtae

    2010-01-01

    LXXLL/leucine zipper-containing alternative reading frame (ARF)-binding protein (LZAP) was recently shown to function as a tumor suppressor through inhibition of the NF-κB signaling pathway. LZAP is also known as a negative regulator of cell invasion, and its expression was demonstrated to be reduced in several tumor tissues. However, the molecular mechanism of the negative effect of LZAP on cell invasion is unclear. In this study, we identify NLBP as a novel LZAP-binding protein using tandem affinity purification. We demonstrate the negative effects of NLBP on cell invasion and the NF-κB signaling pathway. NLBP expression was not detected in hepatocellular carcinoma cells with strong invasive activity, whereas its expression was detected in a hepatocellular carcinoma cell line with no invasive activity. We also demonstrate that these two proteins mutually affect the stability of each other by inhibiting ubiquitination of the other protein. Based on these results, we suggest that NLBP may act as a novel tumor suppressor by inhibiting cell invasion, blocking NF-κB signaling, and increasing stability of the LZAP protein. PMID:20164180

  15. Binding of tobamovirus replication protein with small RNA duplexes.

    PubMed

    Kurihara, Yukio; Inaba, Naoko; Kutsuna, Natsumaro; Takeda, Atsushi; Tagami, Yuko; Watanabe, Yuichiro

    2007-08-01

    The sequence profiles of small interfering RNAs (siRNAs) in Arabidopsis infected with the crucifer tobamovirus tobacco mosaic virus (TMV)-Cg were determined by using a small RNA cloning technique. The majority of TMV-derived siRNAs were 21 nt in length. The size of the most abundant endogenous small RNAs in TMV-infected plants was 21 nt, whilst in mock-inoculated plants, it was 24 nt. Northern blot analysis revealed that some microRNAs (miRNAs) accumulated more in TMV-infected plants than in mock-inoculated plants. The question of whether the TMV-Cg-encoded 126K replication protein, an RNA-silencing suppressor, caused small RNA enrichment was examined. Transient expression of the replication protein did not change the pattern of miRNA processing. However, miRNA, miRNA* (the opposite strand of the miRNA duplex) and hairpin-derived siRNA all co-immunoprecipitated with the replication protein. Gel mobility-shift assays indicated that the replication protein binds small RNA duplexes. These results suggest that the tobamovirus replication protein functions as a silencing suppressor by binding small RNA duplexes, changing the small RNA profile in infected plants.

  16. Mycobacteriophage cell binding proteins for the capture of mycobacteria

    PubMed Central

    Arutyunov, Denis; Singh, Upasana; El-Hawiet, Amr; Seckler, Henrique dos Santos; Nikjah, Sanaz; Joe, Maju; Bai, Yu; Lowary, Todd L; Klassen, John S; Evoy, Stephane; Szymanski, Christine M

    2014-01-01

    Slow growing Mycobacterium avium subsp. paratuberculosis (MAP) causes a deadly condition in cattle known as Johne's disease where asymptomatic carriers are the major source of disease transmission. MAP was also shown to be associated with chronic Crohn's disease in humans. Mycobacterium smegmatis is a model mycobacterium that can cause opportunistic infections in a number of human tissues and, rarely, a respiratory disease. Currently, there are no rapid, culture-independent, reliable and inexpensive tests for the diagnostics of MAP or M. smegmatis infections. Bacteriophages are viruses producing a number of proteins that effectively and specifically recognize the cell envelopes of their bacterial hosts. We demonstrate that the mycobacterial phage L5 minor tail protein Gp6 and lysin Gp10 are useful tools for the rapid capture of mycobacteria. Immobilized Gp10 was able to bind both MAP and M. smegmatis cells whereas Gp6 was M. smegmatis specific. Neither of the 2 proteins was able to capture E. coli, salmonella, campylobacter or Mycobacterium marinum cells. Gp6 was detected previously as a component of the phage particle and shows no homology to proteins with known function. Therefore, electrospray ionization mass spectrometry was used to determine whether recombinant Gp6 could bind to a number of chemically synthesized fragments of mycobacterial surface glycans. These findings demonstrate that mycobacteriophage proteins could be used as a pathogen capturing platform that can potentially improve the effectiveness of existing diagnostic methods. PMID:26713219

  17. Spatial Analysis and Quantification of the Thermodynamic Driving Forces in Protein-Ligand Binding: Binding Site Variability

    PubMed Central

    Raman, E. Prabhu; MacKerell, Alexander D.

    2015-01-01

    The thermodynamic driving forces behind small molecule-protein binding are still not well understood, including the variability of those forces associated with different types of ligands in different binding pockets. To better understand these phenomena we calculate spatially resolved thermodynamic contributions of the different molecular degrees of freedom for the binding of propane and methanol to multiple pockets on the proteins Factor Xa and p38 MAP kinase. Binding thermodynamics are computed using a statistical thermodynamics based end-point method applied on a canonical ensemble comprising the protein-ligand complexes and the corresponding free states in an explicit solvent environment. Energetic and entropic contributions of water and ligand degrees of freedom computed from the configurational ensemble provides an unprecedented level of detail into the mechanisms of binding. Direct protein-ligand interaction energies play a significant role in both non-polar and polar binding, which is comparable to water reorganization energy. Loss of interactions with water upon binding strongly compensates these contributions leading to relatively small binding enthalpies. For both solutes, the entropy of water reorganization is found to favor binding in agreement with the classical view of the “hydrophobic effect”. Depending on the specifics of the binding pocket, both energy-entropy compensation and reinforcement mechanisms are observed. Notable is the ability to visualize the spatial distribution of the thermodynamic contributions to binding at atomic resolution showing significant differences in the thermodynamic contributions of water to the binding of propane versus methanol. PMID:25625202

  18. Tight binding of proteins to membranes from older human cells.

    PubMed

    Truscott, Roger J W; Comte-Walters, Susana; Ablonczy, Zsolt; Schwacke, John H; Berry, Yoke; Korlimbinis, Anastasia; Friedrich, Michael G; Schey, Kevin L

    2011-12-01

    The lens is an ideal model system for the study of macromolecular aging and its consequences for cellular function, since there is no turnover of lens fibre cells. To examine biochemical processes that take place in the lens and that may also occur in other long-lived cells, membranes were isolated from defined regions of human lenses that are synthesised at different times during life, and assayed for the presence of tightly bound cytosolic proteins using quantitative iTRAQ proteomics technology. A majority of lens beta crystallins and all gamma crystallins became increasingly membrane bound with age, however, the chaperone proteins alpha A and alpha B crystallin, as well as the thermally-stable protein, βB2 crystallin, did not. Other proteins such as brain-associated signal protein 1 and paralemmin 1 became less tightly bound in the older regions of the lens. It is evident that protein-membrane interactions change significantly with age. Selected proteins that were formerly cytosolic become increasingly tightly bound to cell membranes with age and are not removed even by treatment with 7 M urea. It is likely that such processes reflect polypeptide denaturation over time and the untoward binding of proteins to membranes may alter membrane properties and contribute to impairment of communication between older cells.

  19. Characterization of microtubule-binding and dimerization activity of Giardia lamblia end-binding 1 protein.

    PubMed

    Kim, Juri; Nagami, Sara; Lee, Kyu-Ho; Park, Soon-Jung

    2014-01-01

    End-binding 1 (EB1) proteins are evolutionarily conserved components of microtubule (MT) plus-end tracking protein that regulate MT dynamics. Giardia lamblia, with two nuclei and cytoskeletal structures, requires accurate MT distribution for division. In this study, we show that a single EB1 homolog gene of G. lamblia regulates MT dynamics in mitosis. The haemagglutinin-tagged G. lamblia EB1 (GlEB1) localizes to the nuclear envelopes and median bodies, and is transiently present in mitotic spindles of dividing cells. Knockdown of GlEB1 expression using the morpholinos-based anti-EB1 oligonucleotides, resulted in a significant defect in mitosis of Giardia trophozoites. The MT-binding assays using recombinant GlEB1 (rGlEB1) proteins demonstrated that rGlEB1102-238, but not rGlEB11-184, maintains an MT-binding ability comparable with that of the full length protein, rGlEB11-238. Size exclusion chromatography showed that rGlEB1 is present as a dimer formed by its C-terminal domain and a disulfide bond. In vitro-mutagenesis of GlEB1 indicated that an intermolecular disulfide bond is made between cysteine #13 of the two monomers. Complementation assay using the BIM1 knockout mutant yeast, the yeast homolog of mammalian EB1, indicated that expression of the C13S mutant GlEB1 protein cannot rescue the mitotic defect of the BIM1 mutant yeast. These results suggest that dimerization of GlEB1 via the 13th cysteine residues plays a role during mitosis in Giardia.

  20. Identification of trichoplein, a novel keratin filament-binding protein.

    PubMed

    Nishizawa, Miwako; Izawa, Ichiro; Inoko, Akihito; Hayashi, Yuko; Nagata, Koh-ichi; Yokoyama, Tomoya; Usukura, Jiro; Inagaki, Masaki

    2005-03-01

    Keratins 8 and 18 (K8/18) are major components of the intermediate filaments (IFs) of simple epithelia. We report here the identification of a novel protein termed trichoplein. This protein shows a low degree of sequence similarity to trichohyalin, plectin and myosin heavy chain, and is a K8/18-binding protein. Among interactions between trichoplein and various IF proteins that we tested using two-hybrid methods, trichoplein interacted significantly with K16 and K18, and to some extent with K5, K6a, K8 and K14. In in vitro co-sedimentation assays, trichoplein directly binds to K8/18, but not with vimentin, desmin, actin filaments or microtubules. An antibody raised against trichoplein specifically recognized a polypeptide with a relative molecular mass of 61 kDa in cell lysates. Trichoplein was immunoprecipitated using this antibody in a complex with K8/18 and immunostaining revealed that trichoplein colocalized with K8/18 filaments in HeLa cells. In polarized Caco-2 cells, trichoplein colocalized not only with K8/18 filaments in the apical region but also with desmoplakin, a constituent of desmosomes. In the absorptive cells of the small intestine, trichoplein colocalized with K8/18 filaments at the apical cortical region, and was also concentrated at desmosomes. Taken together, these results suggest that trichoplein is a keratin-binding protein that may be involved in the organization of the apical network of keratin filaments and desmosomes in simple epithelial cells.

  1. Prenylation of an interferon-gamma-induced GTP-binding protein: the human guanylate binding protein, huGBP1.

    PubMed

    Nantais, D E; Schwemmle, M; Stickney, J T; Vestal, D J; Buss, J E

    1996-09-01

    Interferons (IFN) and lipopolysaccharide (LPS) cause multiple changes in isoprenoid-modified proteins in murine macrophages, the most dramatic being the expression of a prenyl protein of 65 kDa. The guanylate binding proteins (GBPs) are IFN-inducible GTP-binding proteins of approximately 65 kDa that possess a CaaX motif at their C-terminus, indicating that they might be substrates for prenyltransferases. The human GBP1 protein, when expressed in transfected COS-1 cells, incorporates radioactivity from the isoprenoid precursor [3H]mevalonate. In addition, huGBPs expressed from the endogenous genes in IFN-gamma-treated human fibroblasts or monocytic cells were also found to be isoprenoid modified. IFN-gamma-induced huGBPs in HL-60 cells were not labeled by the specific C20 isoprenoid, [3H]geranylgeraniol, but did show decreased isoprenoid incorporation in cells treated with the farnesyl transferase inhibitor BZA-5B, indicating that huGBPs in HL-60 cells are probably modified by a C15 farnesyl rather than the more common C20 lipid. Differentiated HL-60 cells treated with IFN-gamma/LPS showed no change in the profile of constitutive isoprenylated proteins and the IFN-gamma/LPS-induced huGBPs remained prenylated. Despite being prenylated, huGBP1 in COS cells and endogenous huGBPs in HL-60 cells were primarily (approximately 85%) cytosolic. Human GBPs are thus among the select group of prenyl proteins whose synthesis is tightly regulated by a cytokine. HuGBP1 is an abundant protein whose prenylation may be vulnerable to farnesyl transferase inhibitors that are designed to prevent farnesylation of Ras proteins.

  2. Prediction of nucleic acid binding probability in proteins: a neighboring residue network based score.

    PubMed

    Miao, Zhichao; Westhof, Eric

    2015-06-23

    We describe a general binding score for predicting the nucleic acid binding probability in proteins. The score is directly derived from physicochemical and evolutionary features and integrates a residue neighboring network approach. Our process achieves stable and high accuracies on both DNA- and RNA-binding proteins and illustrates how the main driving forces for nucleic acid binding are common. Because of the effective integration of the synergetic effects of the network of neighboring residues and the fact that the prediction yields a hierarchical scoring on the protein surface, energy funnels for nucleic acid binding appear on protein surfaces, pointing to the dynamic process occurring in the binding of nucleic acids to proteins.

  3. THE ROLE OF PROTEIN BINDING OF TRIVALENT ARSENICALS IN ARSENIC CARCINOGENESIS AND TOXICITY

    EPA Science Inventory

    Three of the most plausible biological theories of arsenic carcinogenesis are protein binding, oxidative stress and altered DNA methylation. This review presents the role of trivalent arsenicals binding to proteins in arsenic carcinogenesis. Using vacuum filtration based receptor...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  5. Expression profile and ligand-binding characterization of odorant-binding protein 2 in Batocera horsfieldi (Hope)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Odorant-binding proteins (OBPs) are important components in insect olfactory systems that transport semiochemicals through the aqueous sensillum lymph to surface of olfactory receptor neurons. In this study, we cloned the cDNA of odorant-binding protein 2 (BhorOBP2) in Batocera horsfieldi (Hope) and...

  6. Ligand binding and thermodynamic stability of a multidomain protein, calmodulin.

    PubMed Central

    Masino, L.; Martin, S. R.; Bayley, P. M.

    2000-01-01

    Chemical and thermal denaturation of calmodulin has been monitored spectroscopically to determine the stability for the intact protein and its two isolated domains as a function of binding of Ca2+ or Mg2+. The reversible urea unfolding of either isolated apo-domain follows a two-state mechanism with relatively low deltaG(o)20 values of approximately 2.7 (N-domain) and approximately 1.9 kcal/mol (C-domain). The apo-C-domain is significantly unfolded at normal temperatures (20-25 degrees C). The greater affinity of the C-domain for Ca2+ causes it to be more stable than the N-domain at [Ca2+] > or = 0.3 mM. By contrast, Mg2+ causes a greater stabilization of the N- rather than the C-domain, consistent with measured Mg2+ affinities. For the intact protein (+/-Ca2+), the bimodal denaturation profiles can be analyzed to give two deltaG(o)20 values, which differ significantly from those of the isolated domains, with one domain being less stable and one domain more stable. The observed stability of the domains is strongly dependent on solution conditions such as ionic strength, as well as specific effects due to metal ion binding. In the intact protein, different folding intermediates are observed, depending on the ionic composition. The results illustrate that a protein of low intrinsic stability is liable to major perturbation of its unfolding properties by environmental conditions and liganding processes and, by extension, mutation. Hence, the observed stability of an isolated domain may differ significantly from the stability of the same structure in a multidomain protein. These results address questions involved in manipulating the stability of a protein or its domains by site directed mutagenesis and protein engineering. PMID:10975573

  7. Interactome map uncovers phosphatidylserine transport by oxysterol-binding proteins.

    PubMed

    Maeda, Kenji; Anand, Kanchan; Chiapparino, Antonella; Kumar, Arun; Poletto, Mattia; Kaksonen, Marko; Gavin, Anne-Claude

    2013-09-12

    The internal organization of eukaryotic cells into functionally specialized, membrane-delimited organelles of unique composition implies a need for active, regulated lipid transport. Phosphatidylserine (PS), for example, is synthesized in the endoplasmic reticulum and then preferentially associates--through mechanisms not fully elucidated--with the inner leaflet of the plasma membrane. Lipids can travel via transport vesicles. Alternatively, several protein families known as lipid-transfer proteins (LTPs) can extract a variety of specific lipids from biological membranes and transport them, within a hydrophobic pocket, through aqueous phases. Here we report the development of an integrated approach that combines protein fractionation and lipidomics to characterize the LTP-lipid complexes formed in vivo. We applied the procedure to 13 LTPs in the yeast Saccharomyces cerevisiae: the six Sec14 homology (Sfh) proteins and the seven oxysterol-binding homology (Osh) proteins. We found that Osh6 and Osh7 have an unexpected specificity for PS. In vivo, they participate in PS homeostasis and the transport of this lipid to the plasma membrane. The structure of Osh6 bound to PS reveals unique features that are conserved among other metazoan oxysterol-binding proteins (OSBPs) and are required for PS recognition. Our findings represent the first direct evidence, to our knowledge, for the non-vesicular transfer of PS from its site of biosynthesis (the endoplasmic reticulum) to its site of biological activity (the plasma membrane). We describe a new subfamily of OSBPs, including human ORP5 and ORP10, that transfer PS and propose new mechanisms of action for a protein family that is involved in several human pathologies such as cancer, dyslipidaemia and metabolic syndrome.

  8. Protein Affinity Chromatography with Purified Yeast DNA Polymerase α Detects Proteins that Bind to DNA Polymerase

    NASA Astrophysics Data System (ADS)

    Miles, Jeff; Formosa, Tim

    1992-02-01

    We have overexpressed the POL1 gene of the yeast Saccharomyces cerevisiae and purified the resulting DNA polymerase α polypeptide in an apparently intact form. We attached the purified DNA polymerase covalently to an agarose matrix and used this matrix to chromatograph extracts prepared from yeast cells. At least six proteins bound to the yeast DNA polymerase α matrix that did not bind to a control matrix. We speculate that these proteins might be DNA polymerase α accessory proteins. Consistent with this interpretation, one of the binding proteins, which we have named POB1 (polymerase one binding), is required for normal chromosome transmission. Mutations in this gene cause increased chromosome loss and an abnormal cell morphology, phenotypes that also occur in the presence of mutations in the yeast α or δ polymerase genes. These results suggest that the interactions detected by polymerase affinity chromatography are biologically relevant and may help to illuminate the architecture of the eukaryotic DNA replication machinery.

  9. Preferential binding of an unfolded protein to DsbA.

    PubMed Central

    Frech, C; Wunderlich, M; Glockshuber, R; Schmid, F X

    1996-01-01

    The oxidoreductase DsbA from the periplasm of escherichia coli introduces disulfide bonds into proteins at an extremely high rate. During oxidation, a mixed disulfide is formed between DsbA and the folding protein chain, and this covalent intermediate reacts very rapidly either to form the oxidized protein or to revert back to oxidized DsbA. To investigate its properties, a stable form of the intermediate was produced by reacting the C33A variant of DsbA with a variant of RNase T1. We find that in this stable mixed disulfide the conformational stability of the substrate protein is decreased by 5 kJ/mol, whereas the conformational stability of DsbA is increased by 5 kJ/mol. This reciprocal effect suggests strongly that DsbA interacts with the unfolded substrate protein not only by the covalent disulfide bond, but also by preferential non-covalent interactions. The existence of a polypeptide binding site explains why DsbA oxidizes protein substrates much more rapidly than small thiol compounds. Such a very fast reaction is probably important for protein folding in the periplasm, because the accessibility of the thiol groups for DsbA can decrease rapidly when newly exported polypeptide chains begin to fold. PMID:8617214

  10. A conserved NAD(+) binding pocket that regulates protein-protein interactions during aging.

    PubMed

    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.

  11. Cytoskeletal protein binding kinetics at planar phospholipid membranes.

    PubMed Central

    Mc Kiernan, A E; MacDonald, R I; MacDonald, R C; Axelrod, D

    1997-01-01

    It has been hypothesized that nonspecific reversible binding of cytoskeletal proteins to lipids in cells may guide their binding to integral membrane anchor proteins. In a model system, we measured desorption rates k(off) (off-rates) of the erythrocyte cytoskeletal proteins spectrin and protein 4.1 labeled with carboxyfluorescein (CF), at two different compositions of planar phospholipid membranes (supported on glass), using the total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP) technique. The lipid membranes consisted of either pure phosphatidylcholine (PC) or a 3:1 mixture of PC with phosphatidylserine (PS). In general, the off-rates were not single exponentials and were fit to a combination of fast, slow, and irreversible fractions, reported both separately and as a weighted average. By a variation of TIR/FRAP, we also measured equilibrium affinities (the ratio of surface-bound to bulk protein concentration) and thereby calculated on-rates, k(on). The average off-rate of CF-4.1 from PC/PS (approximately 0.008/s) is much slower than that from pure PC (approximately 1.7/s). Despite the consequent increase in equilibrium affinity at PC/PS, the on-rate at PC/PS is also substantially decreased (by a factor of 40) relative to that at pure PC. The simultaneous presence of (unlabeled) spectrin tends to substantially decrease the on-rate (and the affinity) of CF-4.1 at both membrane types. Similar experiments for CF-spectrin alone showed much less sensitivity to membrane type and generally faster off-rates than those exhibited by CF-4.1. However, when mixed with (unlabeled) 4.1, both the on-rate and off-rate of CF-spectrin decreased drastically at PC/PS (but not PC), leading to a somewhat increased affinity. Clearly, changes in affinity often involve countervailing changes in both on-rates and off-rates. In many of these studies, the effect of varying ionic strength and bulk concentrations was examined; it appears that the binding is an

  12. Insights into cellulase-lignin non-specific binding revealed by computational redesign of the surface of green fluorescent protein: Protein Redesign to Lower Protein-lignin Binding

    SciTech Connect

    Haarmeyer, Carolyn N.; Smith, Matthew D.; Chundawat, Shishir P. S.; Sammond, Deanne; Whitehead, Timothy A.

    2016-11-07

    Biological-mediated conversion of pretreated lignocellulosic biomass to biofuels and biochemicals is a promising avenue towards energy sustainability. However, a critical impediment to the commercialization of cellulosic biofuel production is the high cost of cellulase enzymes needed to deconstruct biomass into fermentable sugars. One major factor driving cost is cellulase adsorption and inactivation in the presence of lignin, yet we currently have a poor understanding of the protein structure-function relationships driving this adsorption. In this work, we have systematically investigated the role of protein surface potential on lignin adsorption using a model monomeric fluorescent protein. We have designed and experimentally characterized 16 model protein variants spanning the physiological range of net charge (-24 to +16 total charges) and total charge density (0.28 to 0.40 charges per sequence length) typical for natural proteins. Protein designs were expressed, purified, and subjected to in silico and in vitro biophysical measurements to evaluate the relationship between protein surface potential and lignin adsorption properties. The designs were comparable to model fluorescent protein in terms of thermostability and heterologous expression yield, although the majority of the designs unexpectedly formed homodimers. Protein adsorption to lignin was studied at two different temperatures using Quartz Crystal Microbalance with Dissipation Monitoring and a subtractive mass balance assay. We found a weak correlation between protein net charge and protein-binding capacity to lignin. No other single characteristic, including apparent melting temperature and 2nd virial coefficient, showed correlation with lignin binding. Analysis of an unrelated cellulase dataset with mutations localized to a family I carbohydrate-binding module showed a similar correlation between net charge and lignin binding capacity. Overall, our study provides strategies to identify highly active

  13. Membrane Binding of HIV-1 Matrix Protein: Dependence on Bilayer Composition and Protein Lipidation

    PubMed Central

    Barros, Marilia; Nanda, Hirsh

    2016-01-01

    ABSTRACT By assembling in a protein lattice on the host's plasma membrane, the retroviral Gag polyprotein triggers formation of the viral protein/membrane shell. The MA domain of Gag employs multiple signals—electrostatic, hydrophobic, and lipid-specific—to bring the protein to the plasma membrane, thereby complementing protein-protein interactions, located in full-length Gag, in lattice formation. We report the interaction of myristoylated and unmyristoylated HIV-1 Gag MA domains with bilayers composed of purified lipid components to dissect these complex membrane signals and quantify their contributions to the overall interaction. Surface plasmon resonance on well-defined planar membrane models is used to quantify binding affinities and amounts of protein and yields free binding energy contributions, ΔG, of the various signals. Charge-charge interactions in the absence of the phosphatidylinositide PI(4,5)P2 attract the protein to acidic membrane surfaces, and myristoylation increases the affinity by a factor of 10; thus, our data do not provide evidence for a PI(4,5)P2 trigger of myristate exposure. Lipid-specific interactions with PI(4,5)P2, the major signal lipid in the inner plasma membrane, increase membrane attraction at a level similar to that of protein lipidation. While cholesterol does not directly engage in interactions, it augments protein affinity strongly by facilitating efficient myristate insertion and PI(4,5)P2 binding. We thus observe that the isolated MA protein, in the absence of protein-protein interaction conferred by the full-length Gag, binds the membrane with submicromolar affinities. IMPORTANCE Like other retroviral species, the Gag polyprotein of HIV-1 contains three major domains: the N-terminal, myristoylated MA domain that targets the protein to the plasma membrane of the host; a central capsid-forming domain; and the C-terminal, genome-binding nucleocapsid domain. These domains act in concert to condense Gag into a membrane

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

    NASA Astrophysics Data System (ADS)

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

    2016-11-01

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

  15. Targeting Human Cancer by a Glycosaminoglycan Binding Malaria Protein.

    PubMed

    Salanti, Ali; Clausen, Thomas M; Agerbæk, Mette Ø; Al Nakouzi, Nader; Dahlbäck, Madeleine; Oo, Htoo Z; Lee, Sherry; Gustavsson, Tobias; Rich, Jamie R; Hedberg, Bradley J; Mao, Yang; Barington, Line; Pereira, Marina A; LoBello, Janine; Endo, Makoto; Fazli, Ladan; Soden, Jo; Wang, Chris K; Sander, Adam F; Dagil, Robert; Thrane, Susan; Holst, Peter J; Meng, Le; Favero, Francesco; Weiss, Glen J; Nielsen, Morten A; Freeth, Jim; Nielsen, Torsten O; Zaia, Joseph; Tran, Nhan L; Trent, Jeff; Babcook, John S; Theander, Thor G; Sorensen, Poul H; Daugaard, Mads

    2015-10-12

    Plasmodium falciparum engineer infected erythrocytes to present the malarial protein, VAR2CSA, which binds a distinct type chondroitin sulfate (CS) exclusively expressed in the placenta. Here, we show that the same CS modification is present on a high proportion of malignant cells and that it can be specifically targeted by recombinant VAR2CSA (rVAR2). In tumors, placental-like CS chains are linked to a limited repertoire of cancer-associated proteoglycans including CD44 and CSPG4. The rVAR2 protein localizes to tumors in vivo and rVAR2 fused to diphtheria toxin or conjugated to hemiasterlin compounds strongly inhibits in vivo tumor cell growth and metastasis. Our data demonstrate how an evolutionarily refined parasite-derived protein can be exploited to target a common, but complex, malignancy-associated glycosaminoglycan modification.

  16. Isolation of chlorophyll-binding proteins of green plants

    SciTech Connect

    Williams, R.S.; Shaw, E.K.; Sieburth, L.E.; Bennett, J.

    1986-01-01

    This chapter describes procedures for isolating most of the known chlorophyll-binding proteins of the photosynthetic membranes of green plant chloroplasts. The procedures yield purified apoproteins which may be used for protein chemistry (amino acid analysis, N-terminal sequencing, etc.) and for the preparation of antibodies, but not directly for functional studies. Monoclonal and polyclonal antibodies raised against the apoproteins may be employed in a variety of studies on chlorophyll-protein complexes, including analysis of their exposure at either surface of the thylakoid membrane, the assay of their mRNAs, the localization of their genes, the study of the photoregulation of their biosynthesis, and, in the case of apoproteins synthesized in the cytoplasm, the uptake of precursor forms into chloroplasts. 2 figures, 1 table.

  17. Dynamics of nucleosome invasion by DNA binding proteins.

    PubMed

    Tims, Hannah S; Gurunathan, Kaushik; Levitus, Marcia; Widom, Jonathan

    2011-08-12

    Nucleosomes sterically occlude their wrapped DNA from interacting with many large protein complexes. How proteins gain access to nucleosomal DNA target sites in vivo is not known. Outer stretches of nucleosomal DNA spontaneously unwrap and rewrap with high frequency, providing rapid and efficient access to regulatory DNA target sites located there; however, rates for access to the nucleosome interior have not been measured. Here we show that for a selected high-affinity nucleosome positioning sequence, the spontaneous DNA unwrapping rate decreases dramatically with distance inside the nucleosome. The rewrapping rate also decreases, but only slightly. Our results explain the previously known strong position dependence on the equilibrium accessibility of nucleosomal DNA, which is characteristic of both selected and natural sequences. Our results point to slow nucleosome conformational fluctuations as a potential source of cell-cell variability in gene activation dynamics, and they reveal the dominant kinetic path by which multiple DNA binding proteins cooperatively invade a nucleosome.

  18. Crystal structure of the Locusta migratoria odorant binding protein.

    PubMed

    Zheng, Jiangge; Li, Junru; Han, Lei; Wang, Yang; Wu, Wei; Qi, Xiaoxuan; Tao, Ye; Zhang, Long; Zhang, Ziding; Chen, Zhongzhou

    2015-01-16

    Locusta migratoria (Lmig) causes enormous losses to agricultural products, especially because it often infests the world with great swarms as locust plagues. Locusts find their plant hosts on which they feed through their olfactory system, in which odorant binding proteins (OBPs) play an important role. Previous study indicated that the amino acid sequences of LmigOBP showed low similarity to OBPs from other insect orders and we speculated that it might perform unique binding behavior. Here, we solved the first LmigOBP1 structure at 1.65Å, which is a monomer in solution and disulfide bonds play a key role in maintaining its function. We show that LmigOBP1 possesses a unique seventh α-helix, which is located at the surface with strong interactions with the LmigOBP1 scaffold consisting of other six α-helices. Moreover, the seventh α-helix forms a wall of an "L" shaped internal hydrophobic cavity to accommodate linear ligands, which is consistent with the binding experiments. We also demonstrate that the ligand-binding pocket in LmigOBP1 is greatly different from that in the closest homologs mosquito OBPs. Taken together, this study provides a structural basis for designing small inhibitors to control locust.

  19. Arabidopsis chloroplast chaperonin 10 is a calmodulin-binding protein

    NASA Technical Reports Server (NTRS)

    Yang, T.; Poovaiah, B. W.

    2000-01-01

    Calcium regulates diverse cellular activities in plants through the action of calmodulin (CaM). By using (35)S-labeled CaM to screen an Arabidopsis seedling cDNA expression library, a cDNA designated as AtCh-CPN10 (Arabidopsis thaliana chloroplast chaperonin 10) was cloned. Chloroplast CPN10, a nuclear-encoded protein, is a functional homolog of E. coli GroES. It is believed that CPN60 and CPN10 are involved in the assembly of Rubisco, a key enzyme involved in the photosynthetic pathway. Northern analysis revealed that AtCh-CPN10 is highly expressed in green tissues. The recombinant AtCh-CPN10 binds to CaM in a calcium-dependent manner. Deletion mutants revealed that there is only one CaM-binding site in the last 31 amino acids of the AtCh-CPN10 at the C-terminal end. The CaM-binding region in AtCh-CPN10 has higher homology to other chloroplast CPN10s in comparison to GroES and mitochondrial CPN10s, suggesting that CaM may only bind to chloroplast CPN10s. Furthermore, the results also suggest that the calcium/CaM messenger system is involved in regulating Rubisco assembly in the chloroplast, thereby influencing photosynthesis. Copyright 2000 Academic Press.

  20. In vitro binding of selenium by rat liver mitochondrial selenium-binding protein

    SciTech Connect

    Brian, W.R.; Hoekstra, W.G.

    1986-03-01

    Last year the authors reported that upon freezing and thawing mitochondria from rats injected with (/sup 75/Se)Na/sub 2/SeO/sub 3/ (/sup 75/Se-selenite), a /sup 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 /sup 75/Se-selenite but not when matrix material alone was incubated with /sup 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 /sup 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 /sup 75/Se-selenite. Labelling did not occur when matrix alone and /sup 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.

  1. A supramolecular bioactive surface for specific binding of protein.

    PubMed

    Hu, Changming; Qu, Yangcui; Zhan, Wenjun; Wei, Ting; Cao, Limin; Yu, Qian; Chen, Hong

    2017-04-01

    Bioactive surfaces with immobilized bioactive molecules aimed specifically at promoting or supporting particular interactions are of great interest for application of biosensors and biological detection. In this work, we fabricated a supramolecular bioactive surface with specific protein binding capability using two noncovalent interactions as the driving forces. The substrates were first layer-by-layer (LbL) deposited with a multilayered polyelectrolyte film containing "guest" adamantane groups via electrostatic interactions, followed by incorporation of "host" β-cyclodextrin derivatives bearing seven biotin units (CD-B) into the films via host-guest interactions. The results of fluorescence microscopy and quartz crystal microbalance measurement demonstrated that these surfaces exhibited high binding capacity and high selectivity for avidin due to the high density of biotin residues. Moreover, since host-guest interactions are inherently reversible, the avidin-CD-B complex is easily released by treatment with the sodium dodecyl sulfate, and the "regenerated" surfaces, after re-introducing fresh CD-B, can be used repeatedly for avidin binding. Given the generality and versatility of this approach, it may pave a way for development of re-usable biosensors for the detection and measurement of specific proteins.

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

    PubMed

    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.

  3. Two mechanisms of ion selectivity in protein binding sites.

    PubMed

    Yu, Haibo; Noskov, Sergei Yu; Roux, Benoît

    2010-11-23

    A theoretical framework is presented to clarify the molecular determinants of ion selectivity in protein binding sites. The relative free energy of a bound ion is expressed in terms of the main coordinating ligands coupled to an effective potential of mean force representing the influence of the rest of the protein. The latter is separated into two main contributions. The first includes all the forces keeping the ion and the coordinating ligands confined to a microscopic subvolume but does not prevent the ligands from adapting to a smaller or larger ion. The second regroups all the remaining forces that control the precise geometry of the coordinating ligands best adapted to a given ion. The theoretical framework makes it possible to delineate two important limiting cases. In the limit where the geometric forces are dominant (rigid binding site), ion selectivity is controlled by the ion-ligand interactions within the matching cavity size according to the familiar "snug-fit" mechanism of host-guest chemistry. In the limit where the geometric forces are negligible, the ion and ligands behave as a "confined microdroplet" that is free to fluctuate and adapt to ions of different sizes. In this case, ion selectivity is set by the interplay between ion-ligand and ligand-ligand interactions and is controlled by the number and the chemical type of ion-coordinating ligands. The framework is illustrated by considering the ion-selective binding sites in the KcsA channel and the LeuT transporter.

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

    PubMed

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

    2017-04-01

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

  5. Flies expand the repertoire of protein structures that bind ice

    PubMed Central

    Basu, Koli; Graham, Laurie A.; Campbell, Robert L.; Davies, Peter L.

    2015-01-01

    An antifreeze protein (AFP) with no known homologs has been identified in Lake Ontario midges (Chironomidae). The midge AFP is expressed as a family of isoforms at low levels in adults, which emerge from fresh water in spring before the threat of freezing temperatures has passed. The 9.1-kDa major isoform derived from a preproprotein precursor is glycosylated and has a 10-residue tandem repeating sequence xxCxGxYCxG, with regularly spaced cysteines, glycines, and tyrosines comprising one-half its 79 residues. Modeling and molecular dynamics predict a tightly wound left-handed solenoid fold in which the cysteines form a disulfide core to brace each of the eight 10-residue coils. The solenoid is reinforced by intrachain hydrogen bonds, side-chain salt bridges, and a row of seven stacked tyrosines on the hydrophobic side that forms the putative ice-binding site. A disulfide core is also a feature of the similar-sized beetle AFP that is a β-helix with seven 12-residue coils and a comparable circular dichroism spectrum. The midge and beetle AFPs are not homologous and their ice-binding sites are radically different, with the latter comprising two parallel arrays of outward-pointing threonines. However, their structural similarities is an amazing example of convergent evolution in different orders of insects to cope with change to a colder climate and provide confirmation about the physical features needed for a protein to bind ice. PMID:25561557

  6. Retinoic acid binding protein in normal and neopolastic rat prostate.

    PubMed

    Gesell, M S; Brandes, M J; Arnold, E A; Isaacs, J T; Ueda, H; Millan, J C; Brandes, D

    1982-01-01

    Sucrose density gradient analysis of cytosol from normal and neoplastic rat prostatic tissues exhibited a peak of (3H) retinoic acid binding in the 2S region, corresponding to the cytoplasmic retinoic acid binding protein (cRABP). In the Fisher-Copenhagen F1 rat, cRABP was present in the lateral lobe, but could not be detected in the ventral nor in the dorsal prostatic lobes. Four sublines of the R-3327 rat prostatic tumor contained similar levels of this binding protein. The absence of cRABP in the normal tissue of origin of the R-3327 tumor, the rat dorsal prostate, and reappearance in the neoplastic tissues follows a pattern described in other human and animal tumors. The occurrence of cRABP in the well-differentiated as well as in the anaplastic R-3327 tumors in which markers which reflect a state of differentiation and hormonal regulation, such as androgen receptor, 5 alpha reductase, and secretory acid phosphatase are either markedly reduced or absent, points to cRABP as a marker of malignant transformation.

  7. RNA-binding protein Lin28 in cancer and immunity.

    PubMed

    Jiang, Shuai; Baltimore, David

    2016-05-28

    The highly conserved RNA-binding protein, Lin28, is involved in many biological processes, including development, reprogramming, pluripotency, and metabolism. Importantly, Lin28 functions as an oncogene, promoting tumor progression and metastasis in various human cancers. Lin28 can regulate gene expression either by directly binding to mRNAs or by blocking microRNA biogenesis, and the underlying mechanisms include Let-7-dependent and Let-7-independent modes of action. Recent evidence shows that Lin28 also plays a fundamental role in immunity. The roles of Lin28 in disease are complex and require characterization of its physiological functions in cancer and immunological contexts. Here we review emerging information on the role of Lin28 in cancer and immunity and the molecular mechanisms it uses. We discuss our present knowledge of the system and highlight remaining mysteries related to the functions of this small RNA-binding protein. This knowledge may lead to Lin28 becoming a diagnostic marker for cancer or immune-related diseases and a possible therapeutic target.

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

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

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

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

    PubMed

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

    2002-03-22

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

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

    PubMed Central

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

    2013-01-01

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

  11. Protein-protein interface-binding peptides inhibit the cancer therapy target human thymidylate synthase.

    PubMed

    Cardinale, Daniela; Guaitoli, Giambattista; Tondi, Donatella; Luciani, Rosaria; Henrich, Stefan; Salo-Ahen, Outi M H; Ferrari, Stefania; Marverti, Gaetano; Guerrieri, Davide; Ligabue, Alessio; Frassineti, Chiara; Pozzi, Cecilia; Mangani, Stefano; Fessas, Dimitrios; Guerrini, Remo; Ponterini, Glauco; Wade, Rebecca C; Costi, M Paola

    2011-08-23

    Human thymidylate synthase is a homodimeric enzyme that plays a key role in DNA synthesis and is a target for several clinically important anticancer drugs that bind to its active site. We have designed peptides to specifically target its dimer interface. Here we show through X-ray diffraction, spectroscopic, kinetic, and calorimetric evidence that the peptides do indeed bind at the interface of the dimeric protein and stabilize its di-inactive form. The "LR" peptide binds at a previously unknown binding site and shows a previously undescribed mechanism for the allosteric inhibition of a homodimeric enzyme. It inhibits the intracellular enzyme in ovarian cancer cells and reduces cellular growth at low micromolar concentrations in both cisplatin-sensitive and -resistant cells without causing protein overexpression. This peptide demonstrates the potential of allosteric inhibition of hTS for overcoming platinum drug resistance in ovarian cancer.

  12. Leptospiral Outer Membrane Protein Microarray, a Novel Approach to Identification of Host Ligand-Binding Proteins

    PubMed Central

    Matsunaga, James; Haake, David A.

    2012-01-01

    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. PMID:22961849

  13. Protein binding onto surfactant-based synthetic vesicles.

    PubMed

    Letizia, Caterina; Andreozzi, Patrizia; Scipioni, Anita; La Mesa, Camillo; Bonincontro, Adalberto; Spigone, Elisabetta

    2007-02-01

    Synthetic vesicles were prepared by mixing anionic and cationic surfactants, aqueous sodium dodecylsulfate with didodecyltrimethylammonium or cetyltrimethylammonium bromide. The overall surfactant content and the (anionic/cationic) mole ratios allow one to obtain negatively charged vesicles. In the phase diagram, the vesicular region is located between a solution phase, a lamellar liquid crystalline dispersion, and a precipitate area. Characterization of the vesicles was performed by electrophoretic mobility, NMR, TEM, and DLS and we determined their uni-lamellar character, size, stability, and charge density. Negatively charged vesicular dispersions, made of sodium dodecylsulfate/didodecyltrimethylammonium bromide or sodium dodecylsulfate/cetyltrimethylammonium bromide, were mixed with lysozyme, to form lipoplexes. Depending on the protein/vesicle charge ratio, binding, surface saturation, and lipoplexes flocculation, or precipitation, occurs. The free protein in excess remains in solution, after binding saturation. The systems were investigated by thermodynamic (surface tension and solution calorimetry), DLS, CD, TEM, 1H NMR, transport properties, electrophoretic mobility, and dielectric relaxation. The latter two methods give information on the vesicle charge neutralization by adsorbed protein. Binding is concomitant to modifications in the double layer thickness of vesicles and in the surface charge density of the resulting lipoplexes. This is also confirmed by developing the electrophoretic mobility results in terms of a Langmuir-like adsorption isotherm. Charges in excess with respect to the amount required to neutralize the vesicle surface promote lipoplexes clustering and/or flocculation. Protein-vesicle interactions were observed by DLS, indicating changes in particle size (and in their distribution functions) upon addition of LYSO. According to CD, the bound protein retains its native conformation, at least in the SDS/CTAB vesicular system. In fact

  14. Calmodulin-binding proteins are developmentally regulated in gametes and embryos of fucoid algae

    SciTech Connect

    Brawley, S.H.; Roberts, D.M.

    1989-02-01

    Calcium-binding proteins and calmodulin-binding proteins were identified in gametes and zygotes of the marine brown algae Fucus vesiculosus, Fucus distichus, and Pelvetia fastigiata using gel (SDS-PAGE) overlay techniques. A calcium current appears to be important during cell polarization in fucoid zygotes, but there are no biochemical data on calcium-binding proteins in these algae. By using a sensitive 45Ca2+ overlay method designed to detect high-affinity calcium-binding proteins, at least 9-11 polypeptides were detected in extracts of fucoid gametes and zygotes. All samples had calcium-binding proteins with apparent molecular weights of about 17 and 30 kDa. A 17-kDa calcium-binding protein was purified by calcium-dependent hydrophobic chromatography and was identified as calmodulin by immunological and enzyme activator criteria. A 125I-calmodulin overlay assay was used to identify potential targets of calmodulin action. Sperm contained one major calmodulin-binding protein of about 45 kDa. Eggs lacked major calmodulin-binding activity. A 72-kDa calmodulin-binding protein was prominent in zygotes from 1-65 hr postfertilization. Both calmodulin-binding proteins showed calcium-dependent binding activity. Overall, the data suggest that the appearance and distribution of certain calcium-binding and calmodulin-binding proteins are under developmental regulation, and may reflect the different roles of calcium during fertilization and early embryogenesis.

  15. Specific mutagenesis of a chlorophyll-binding protein. Progress report.

    SciTech Connect

    Eaton-Rye, Dr., Julian; Shen, Gaozhong

    1990-01-01

    During the first phase of the project regarding specific mutagenesis of the chlorophyll-binding protein CP47 in photosystem II (PS II) most of the time has been devoted to (1) establishment of an optimal procedure for the reintroduction of psbB (the gene encoding CP47) carrying a site-directed mutation into the experimental organism, the cyanobacterium Synechocystis sp. PCC 6803, (2) preparations for site-directed mutagenesis, and (3) creation and analysis of chimaeric spinach/cyanobacterial CP47 mutants of Synechocystis. In the coming year, psbB constructs with site-directed mutations in potential chlorophyll-binding regions of CP47 will be introduced into the Synechocystis genome, and site-directed mutants will be characterized according to procedures described in the original project description. In addition, analysis of chimaeric CP47 mutants will be continued.

  16. Insulin-Like Growth Factor Binding Proteins--an Update.

    PubMed

    Bach, Leon A

    2015-12-01

    The insulin-like growth factor (IGF) system is essential for normal growth and development, and its perturbation is implicated in a number of diseases. IGF activity is finely regulated by a family of six high-affinity IGF binding proteins (IGFBPs). 1GFBPs usually inhibit IGF actions but may enhance them under certain conditions. Additionally, IGFBPs bind non-IGF ligands in the extracellular space, cell membrane, cytoplasm and nucleus, thereby modulating cell proliferation, survival and migration in an IGF-independent manner. IGFBP activity is regulated by transcriptional mechanisms as well as by post-translational modifications and proteolysis. Understanding the balance between the various actions of IGFBPs in vivo may lead to novel insights into disease processes and possible IGFBP-based therapeutics.

  17. Insulin-like growth factor binding proteins 4-6.

    PubMed

    Bach, Leon A

    2015-10-01

    Insulin-like growth factor binding proteins (IGFBPs) 4-6 have important roles as modulators of IGF actions. IGFBP-4 and IGFBP-6 predominantly inhibit IGF actions, whereas IGFBP-5 may enhance these actions under some circumstances. IGFBP-6 is unique among the IGFBPs for its marked IGF-II binding preference. IGFBPs 4-6 are found in the circulation as binary complexes with IGFs that can enter tissues. Additionally, about half of the circulating IGFBP-5 is found in ternary complexes with IGFs and an acid labile subunit; this high molecular complex cannot leave the circulation and acts as an IGF reservoir. IGFBPs 4-6 also have IGF-independent actions. These IGFBPs are regulated in a cell-specific manner and their dysregulation may play a role in a range of diseases including cancer. However, there is no clear clinical indication for measuring serum levels of these IGFBPs at present.

  18. RPI-Bind: a structure-based method for accurate identification of RNA-protein binding sites.

    PubMed

    Luo, Jiesi; Liu, Liang; Venkateswaran, Suresh; Song, Qianqian; Zhou, Xiaobo

    2017-04-04

    RNA and protein interactions play crucial roles in multiple biological processes, while these interactions are significantly influenced by the structures and sequences of protein and RNA molecules. In this study, we first performed an analysis of RNA-protein interacting complexes, and identified interface properties of sequences and structures, which reveal the diverse nature of the binding sites. With the observations, we built a three-step prediction model, namely RPI-Bind, for the identification of RNA-protein binding regions using the sequences and structures of both proteins and RNAs. The three steps include 1) the prediction of RNA binding regions on protein, 2) the prediction of protein binding regions on RNA, and 3) the prediction of interacting regions on both RNA and protein simultaneously, with the results from steps 1) and 2). Compared with existing methods, most of which employ only sequences, our model significantly improves the prediction accuracy at each of the three steps. Especially, our model outperforms the catRAPID by >20% at the 3(rd) step. All of these results indicate the importance of structures in RNA-protein interactions, and suggest that the RPI-Bind model is a powerful theoretical framework for studying RNA-protein interactions.

  19. Characterization of the DNA binding protein encoded by the N-specific filamentous Escherichia coli phage IKe. Binding properties of the protein and nucleotide sequence of the gene.

    PubMed

    Peeters, B P; Konings, R N; Schoenmakers, J G

    1983-09-05

    A DNA binding protein encoded by the filamentous single-stranded DNA phage IKe has been isolated from IKe-infected Escherichia coli cells. Fluorescence and in vitro binding studies have shown that the protein binds co-operatively and with a high specificity to single-stranded but not to double-stranded DNA. From titration of the protein to poly(dA) it has been calculated that approximately four bases of the DNA are covered by one monomer of protein. These binding characteristics closely resemble those of gene V protein encoded by the F-specific filamentous phages M13 and fd. The nucleotide sequence of the gene specifying the IKe DNA binding protein has been established. When compared to the nucleotide sequence of gene V of phage M13 it shows an homology of 58%, indicating that these two phages are evolutionarily related. The IKe DNA binding protein is 88 amino acids long which is one amino acid residue larger than the gene V protein sequence. When the IKe DNA binding protein sequence is compared with that of gene V protein it was found that 39 amino acid residues have identical positions in both proteins. The positions of all five tyrosine residues, a number of which are known to be involved in DNA binding, are conserved. Secondary structure predictions indicate that the two proteins contain similar structural domains. It is proposed that the tyrosine residues which are involved in DNA binding are the ones in or next to a beta-turn, at positions 26, 41 and 56 in gene V protein and at positions 27, 42 and 57 in the IKe DNA binding protein.

  20. Identification of novel PTEN-binding partners: PTEN interaction with fatty acid binding protein FABP4.

    PubMed

    Gorbenko, O; Panayotou, G; Zhyvoloup, A; Volkova, D; Gout, I; Filonenko, V

    2010-04-01

    PTEN is a tumor suppressor with dual protein and lipid-phosphatase activity, which is frequently deleted or mutated in many human advanced cancers. Recent studies have also demonstrated that PTEN is a promising target in type II diabetes and obesity treatment. Using C-terminal PTEN sequence in pEG202-NLS as bait, yeast two-hybrid screening on Mouse Embryo, Colon Cancer, and HeLa cDNA libraries was carried out. Isolated positive clones were validated by mating assay and identified through automated DNA sequencing and BLAST database searches. Sequence analysis revealed a number of PTEN-binding proteins linking this phosphatase to a number of different signaling cascades, suggesting that PTEN may perform other functions besides tumor-suppressing activity in different cell types. In particular, the interplay between PTEN function and adipocyte-specific fatty-acid-binding protein FABP4 is of notable interest. The demonstrable tautology of PTEN to FABP4 suggested a role for this phosphatase in the regulation of lipid metabolism and adipocyte differentiation. This interaction was further studied using coimmunoprecipitation and gel-filtration assays. Finally, based on Biacore assay, we have calculated the K(D) of PTEN-FABP4 complex, which is around 2.8 microM.

  1. Control of nuclear organization by F-actin binding proteins.

    PubMed

    Pfisterer, Karin; Jayo, Asier; Parsons, Maddy

    2017-03-04

    The regulation of nuclear shape and deformability is a key factor in controlling diverse events from embryonic development to cancer cell metastasis, but the mechanisms governing this process are still unclear. Our recent study demonstrated an unexpected role for the F-actin bundling protein fascin in controlling nuclear plasticity through a direct interaction with Nesprin-2. Nesprin-2 is a component of the LINC complex that is known to couple the F-actin cytoskeleton to the nuclear envelope. We demonstrated that fascin, which is predominantly associated with peripheral F-actin rich filopodia, binds directly to Nesprin-2 at the nuclear envelope in a range of cell types. Depleting fascin or specifically blocking the fascin-Nesprin-2 complex leads to defects in nuclear polarization, movement and cell invasion. These studies reveal a novel role for an F-actin bundling protein in control of nuclear plasticity and underline the importance of defining nuclear-associated roles for F-actin binding proteins in future.

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

    SciTech Connect

    Rothe, Francoise; Gueydan, Cyril; Bellefroid, Eric; Huez, Georges; Kruys, Veronique . E-mail: vkruys@ulb.ac.be

    2006-04-28

    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.

  3. Coactosin-like protein, a human F-actin-binding protein: critical role of lysine-75.

    PubMed Central

    Provost, P; Doucet, J; Stock, A; Gerisch, G; Samuelsson, B; Rådmark, O

    2001-01-01

    Coactosin-like protein (CLP) was recently identified in a yeast two-hybrid screen using 5-lipoxygenase as bait. In the present study, we report the functional characterization of CLP as a human filamentous actin (F-actin)-binding protein. CLP mRNA shows a wide tissue distribution and is predominantly expressed in placenta, lung, kidney and peripheral-blood leucocytes. Endogenous CLP is localized in the cytosol of myeloid cells. Using a two-hybrid approach, actin was identified as a CLP-interacting protein. Binding experiments indicated that CLP associates with F-actin, but does not form a stable complex with globular actin. In transfected mammalian cells, CLP co-localized with actin stress fibres. CLP bound to actin filaments with a stoichiometry of 1:2 (CLP: actin subunits), but could be cross-linked to only one subunit of actin. Site-directed mutagenesis revealed the involvement of Lys(75) of CLP in actin binding, a residue highly conserved in related proteins and supposed to be exposed on the surface of the CLP protein. Our results identify CLP as a new human protein that binds F-actin in vitro and in vivo, and indicate that Lys(75) is essential for this interaction. PMID:11583571

  4. DNA-Binding Proteins Essential for Protein-Primed Bacteriophage Φ29 DNA Replication

    PubMed Central

    Salas, Margarita; Holguera, Isabel; Redrejo-Rodríguez, Modesto; de Vega, Miguel

    2016-01-01

    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 and

  5. Eukaryotic damaged DNA-binding proteins: DNA repair proteins or transcription factors?

    SciTech Connect

    Protic, M.

    1994-12-31

    Recognition and removal of structural defects in the genome, caused by diverse physical and chemical agents, are among the most important cell functions. Proteins that recognize and bind to modified DNA, and thereby initiate damage-induced recovery processes, have been identified in prokaryotic and eukaryotic cells. Damaged DNA-binding (DDB) proteins from prokaryotes are either DNA repair enzymes or noncatalytic subunits of larger DNA repair complexes that participate in excision repair, or in recombinational repair and SOS-mutagenesis. Although the methods employed may not have allowed detection of all eukaryotic DDB proteins and identification of their functions, it appears that during evolution cells have developed a wide array of DDB proteins that can discriminate among the diversity of DNA conformations found in the eukaryotic nucleus, as well as a gene-sharing feature found in DDB proteins that also act as transcription factors.

  6. Biologically active protein fragments containing specific binding regions of serum albumin or related proteins

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C. (Inventor)

    1998-01-01

    In accordance with the present invention, biologically active protein fragments can be constructed which contain only those specific portions of the serum albumin family of proteins such as regions known as subdomains IIA and IIIA which are primarily responsible for the binding properties of the serum albumins. The artificial serums that can be prepared from these biologically active protein fragments are advantageous in that they can be produced much more easily than serums containing the whole albumin, yet still retain all or most of the original binding potential of the full albumin proteins. In addition, since the protein fragment serums of the present invention can be made from non-natural sources using conventional recombinant DNA techniques, they are far safer than serums containing natural albumin because they do not carry the potentially harmful viruses and other contaminants that will be found in the natural substances.

  7. Protein-protein and protein-DNA interaction regions within the DNA end-binding protein Ku70-Ku86.

    PubMed Central

    Wu, X; Lieber, M R

    1996-01-01

    DNA ends are generated during double-strand-break repair and recombination. A p70-p86 heterodimer, Ku, accounts for the DNA end binding activity in eukaryotic cell extracts. When one or both subunits of Ku are missing, mammalian cells are deficient in double-strand-break repair and in specialized recombination, such as V(D)J recombination. Little is known of which regions of Ku70 and Ku86 bind to each other to form the heterodimeric complex or of which regions are important for DNA end binding. We have done genetic and biochemical studies to examine the domains within the two subunits important for protein assembly and for DNA end binding. We found that the C-terminal 20-kDa region of Ku70 and the C-terminal 32-kDa region of Ku86 are important for subunit-subunit interaction. For DNA binding, full-length individual subunits are inactive, indicating that heterodimer assembly precedes DNA binding. DNA end binding activity by the heterodimer requires the C-terminal 40-kDa region of Ku70 and the C-terminal 45-kDa region of Ku86. Leucine zipper-like motifs in both subunits that have been suggested as the Ku70-Ku86 interaction domains do not appear to be the sites of such interaction because these are dispensable for both assembly and DNA end binding. On the basis of these studies, we have organized Ku70 into nine sequence regions conserved between Saccharomyces cerevisiae, Drosophila melanogaster, mice, and humans; only the C-terminal three regions are essential for assembly (amino acids [aa] 439 to 609), and the C-terminal four regions appear to be essential for DNA end binding (aa 254 to 609). Within the minimal active fragment of Ku86 necessary for subunit interaction (aa 449 to 732) and DNA binding (aa 334 to 732), a proline-rich region is the only defined motif. PMID:8756676

  8. Integrating computational methods and experimental data for understanding the recognition mechanism and binding affinity of protein-protein complexes.

    PubMed

    Gromiha, M Michael; Yugandhar, K

    2017-01-07

    Protein-protein interactions perform several functions inside the cell. Understanding the recognition mechanism and binding affinity of protein-protein complexes is a challenging problem in experimental and computational biology. In this review, we focus on two aspects (i) understanding the recognition mechanism and (ii) predicting the binding affinity. The first part deals with computational techniques for identifying the binding site residues and the contribution of important interactions for understanding the recognition mechanism of protein-protein complexes in comparison with experimental observations. The second part is devoted to the methods developed for discriminating high and low affinity complexes, and predicting the binding affinity of protein-protein complexes using three-dimensional structural information and just from the amino acid sequence. The overall view enhances our understanding of the integration of experimental data and computational methods, recognition mechanism of protein-protein complexes and the binding affinity.

  9. Proteins and Their Interacting Partners: An Introduction to Protein-Ligand Binding Site Prediction Methods.

    PubMed

    Roche, Daniel Barry; Brackenridge, Danielle Allison; McGuffin, Liam James

    2015-12-15

    Elucidating the biological and biochemical roles of proteins, and subsequently determining their interacting partners, can be difficult and time consuming using in vitro and/or in vivo methods, and consequently the majority of newly sequenced proteins will have unknown structures and functions. However, in silico methods for predicting protein-ligand binding sites and protein biochemical functions offer an alternative practical solution. The characterisation of protein-ligand binding sites is essential for investigating new functional roles, which can impact the major biological research spheres of health, food, and energy security. In this review we discuss the role in silico methods play in 3D modelling of protein-ligand binding sites, along with their role in predicting biochemical functionality. In addition, we describe in detail some of the key alternative in silico prediction approaches that are available, as well as discussing the Critical Assessment of Techniques for Protein Structure Prediction (CASP) and the Continuous Automated Model EvaluatiOn (CAMEO) projects, and their impact on developments in the field. Furthermore, we discuss the importance of protein function prediction methods for tackling 21st century problems.

  10. Developmentally Regulated RNA-binding Protein 1 (Drb1)/RNA-binding Motif Protein 45 (RBM45), a Nuclear-Cytoplasmic Trafficking Protein, Forms TAR DNA-binding Protein 43 (TDP-43)-mediated Cytoplasmic Aggregates.

    PubMed

    Mashiko, Takafumi; Sakashita, Eiji; Kasashima, Katsumi; Tominaga, Kaoru; Kuroiwa, Kenji; Nozaki, Yasuyuki; Matsuura, Tohru; Hamamoto, Toshiro; Endo, Hitoshi

    2016-07-15

    Cytoplasmic protein aggregates are one of the pathological hallmarks of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Several RNA-binding proteins have been identified as components of inclusion bodies. Developmentally regulated RNA-binding protein 1 (Drb1)/RNA-binding motif protein 45 is an RNA-binding protein that was recently described as a component in ALS- and FTLD-related inclusion bodies. However, the molecular mechanism underlying cytoplasmic Drb1 aggregation remains unclear. Here, using an in vitro cellular model, we demonstrated that Drb1 co-localizes with cytoplasmic aggregates mediated by TAR DNA-binding protein 43, a major component of ALS and FTLD-related inclusion bodies. We also defined the domains involved in the subcellular localization of Drb1 to clarify the role of Drb1 in the formation of cytoplasmic aggregates in ALS and FTLD. Drb1 predominantly localized in the nucleus via a classical nuclear localization signal in its carboxyl terminus and is a shuttling protein between the nucleus and cytoplasm. Furthermore, we identify a double leucine motif serving as a nuclear export signal. The Drb1 mutant, presenting mutations in both nuclear localization signal and nuclear export signal, is prone to aggregate in the cytoplasm. The mutant Drb1-induced cytoplasmic aggregates not only recruit TAR DNA-binding protein 43 but also decrease the mitochondrial membrane potential. Taken together, these results indicate that perturbation of Drb1 nuclear-cytoplasmic trafficking induces toxic cytoplasmic aggregates, suggesting that mislocalization of Drb1 is involved in the cause of cytotoxicity in neuronal cells.

  11. Interacting protein partners of Arabidopsis RNA-binding protein AtRBP45b.

    PubMed

    Muthuramalingam, M; Wang, Y; Li, Y; Mahalingam, R

    2017-05-01

    RNA binding proteins, important players in post-transcriptional gene regulation, usually exist in ribonuclear complexes. However, even in model systems like Arabidopsis characterisation of RBP associated proteins is limited. In this study, we investigated the interacting proteins of the Arabidopsis AtRBP45b, which is involved in stress signalling. In vivo localisation of AtRBP45b was conducted using 35S-GFP. FLAG-tagged AtRBP45b under control of the 35S promoter in the Atrbp45b-1 mutant background was used to pull down AtRBP45b interacting proteins. Yeast two-hybrid analysis, fluorescence energy resonance transfer assays were used to confirm the veracity of the AtRBP45b interacting proteins. In planta GFP-tagging indicated AtRBP45b is localised to the nucleus and the cytosol. AtRBP45b protein has a N-terminal proline-rich region and a C-terminal glutamine-rich domain that are usually involved in protein-protein interactions. Co-immunoprecipitation followed by mass spectrometry-based protein sequencing led to identification of 30 proteins that interacted with AtRBP45b. Using information from interactome databases (BIOGRID, INTACT and STRING), pull-down assays and localisation data, 12 putative interacting proteins were selected for yeast two-hybrid analysis. Cap-binding protein (CBP20, At5g44200) and polyA-binding protein (PAB8, At1g49760) were shown to interact with AtRBP45b. Based on its interacting partners we speculate that AtRBP45b may play an important role in RNA metabolism, especially in aspects related to mRNA stability and translation initiation during stress conditions in plants.

  12. Dynamic profiling of double-stranded RNA binding proteins

    PubMed Central

    Wang, Xinlei; Vukovic, Lela; Koh, Hye Ran; Schulten, Klaus; Myong, Sua

    2015-01-01

    Double-stranded (ds) RNA is a key player in numerous biological activities in cells, including RNA interference, anti-viral immunity and mRNA transport. The class of proteins responsible for recognizing dsRNA is termed double-stranded RNA binding proteins (dsRBP). However, little is known about the molecular mechanisms underlying the interaction between dsRBPs and dsRNA. Here we examined four human dsRBPs, ADAD2, TRBP, Staufen 1 and ADAR1 on six dsRNA substrates that vary in length and secondary structure. We combined single molecule pull-down (SiMPull), single molecule protein-induced fluorescence enhancement (smPIFE) and molecular dynamics (MD) simulations to investigate the dsRNA-dsRBP interactions. Our results demonstrate that despite the highly conserved dsRNA binding domains, the dsRBPs exhibit diverse substrate specificities and dynamic properties when in contact with different RNA substrates. While TRBP and ADAR1 have a preference for binding simple duplex RNA, ADAD2 and Staufen1 display higher affinity to highly structured RNA substrates. Upon interaction with RNA substrates, TRBP and Staufen1 exhibit dynamic sliding whereas two deaminases ADAR1 and ADAD2 mostly remain immobile when bound. MD simulations provide a detailed atomic interaction map that is largely consistent with the affinity differences observed experimentally. Collectively, our study highlights the diverse nature of substrate specificity and mobility exhibited by dsRBPs that may be critical for their cellular function. PMID:26184879

  13. Polyclonal and monoclonal IgG binding on Protein A resins - Evidence of competitive binding effects.

    PubMed

    Weinberg, Justin; Zhang, Shaojie; Crews, Gillian; Healy, Edward; Carta, Giorgio; Przybycien, Todd

    2017-03-14

    Protein A (ProA) chromatography is used extensively in the biopharmaceutical industry for the selective capture of both polyclonal and monoclonal antibodies (mAbs). This work provides a comparison of the adsorptive behavior of a highly heterogeneous polyclonal hIgG versus that of a mAb as well as the behavior of their mixtures on representative ProA resins. Both pH gradient elution and frontal loading experiments using human polyclonal IgG (hIgG) reveal a distribution of IgG-ProA binding strengths likely associated with multiple IgG subclasses and the heterogeneity of the variable region. pH gradient analysis of fractions collected along the breakthrough curve demonstrate a clear progression from weaker binding (higher pH eluting) to stronger binding (lower pH eluting) IgG species leaving the column suggesting the possibility of stronger binding species displacing the weaker binding ones. Displacement is directly observed by visualizing the adsorption of fluorescently labeled mAb and hIgG using confocal laser scanning microscopy (CLSM). Here, the displacement of hIgG results in a broad adsorption front compared to the sharp, 'shrinking core' behavior typically observed with mAbs. Sequential CLSM adsorption experiments with a mAb and hIgG confirm that stronger or equivalent-binding hIgG species are able to displace and desorb bound mAb molecules. These phenomena are examined using a variety of ProA resins including CaptivA PriMAB, MabSelect, and MabSelect SuRe to understand the effect of different ligand properties on binding strength and competition among different IgG species. The results of these comparisons suggest that the competition kinetics are slower with ligands that have a single-point covalent attachment to the base matrix compared to a multi-point attachment. This article is protected by copyright. All rights reserved.

  14. Bacterial chitinases and chitin-binding proteins as virulence factors.

    PubMed

    Frederiksen, Rikki F; Paspaliari, Dafni K; Larsen, Tanja; Storgaard, Birgit G; Larsen, Marianne H; Ingmer, Hanne; Palcic, Monica M; Leisner, Jørgen J

    2013-05-01

    Bacterial chitinases (EC 3.2.1.14) and chitin-binding proteins (CBPs) play a fundamental role in the degradation of the ubiquitous biopolymer chitin, and the degradation products serve as an important nutrient source for marine- and soil-dwelling bacteria. However, it has recently become clear that representatives of both Gram-positive and Gram-negative bacterial pathogens encode chitinases and CBPs that support infection of non-chitinous mammalian hosts. This review addresses this biological role of bacterial chitinases and CBPs in terms of substrate specificities, regulation, secretion and involvement in cellular and animal infection.

  15. Vitamin D binding protein polymorphism protects against development of blastomycosis.

    PubMed

    Sainsbury, J P; Trajtman, A; Stalker, A T; Embil, J M; Keynan, Y

    2014-12-01

    Blastomycosis is an uncommon endemic fungal infection. It is presumed that in the endemic regions, the number of exposed individuals is significantly greater than those in whom clinical manifestations develop. We conducted a case-control study of individuals with clinical blastomycosis and controls with similar exposure but who did not develop disease. A genetic association was observed between the Gc-2 allele of vitamin D binding protein and reduced susceptibility to blastomycosis in a Canadian cohort. The Gc-2 allele can affect increased antimicrobial activity of macrophages. It may be possible to mimic this mechanism of protection by vitamin D supplementation.

  16. Thermostability and reversibility of silver nanoparticle-protein binding.

    PubMed

    Wang, Bo; Seabrook, Shane A; Nedumpully-Govindan, Praveen; Chen, Pengyu; Yin, Hong; Waddington, Lynne; Epa, V Chandana; Winkler, David A; Kirby, Jason K; Ding, Feng; Ke, Pu Chun

    2015-01-21

    The interactions between nanoparticles (NPs) and proteins in living systems are a precursor to the formation of a NP-protein "corona" that underlies cellular and organism responses to nanomaterials. However, the thermodynamic properties and reversibility of NP-protein interactions have rarely been examined. Using an automated, high-throughput and temperature-controlled dynamic light scattering (DLS) technique we observed a distinct hysteresis in the hydrodynamic radius of branched polyethyleneimine (BPEI) coated-silver nanoparticles (bAgNPs) exposed to like-charged lysozyme during the processes of heating and cooling, in contrast to the irreversible interactions between bAgNPs and oppositely charged alpha lactalbumin (ALact). Our discrete molecular dynamics (DMD) simulations offered a new molecular insight into the differential structure, dynamics and thermodynamics of bAgNPs binding with the two protein homologs and further revealed the different roles of the capping agents of citrate and BPEI in NP-protein interactions. This study facilitates our understanding of the transformation of nanomaterials in living systems, whose implications range from the field study of nanotoxicology to nanomaterials synthesis, nanobiotechnology and nanomedicine.

  17. Revisiting Apoplastic Auxin Signaling Mediated by AUXIN BINDING PROTEIN 1.

    PubMed

    Feng, Mingxiao; Kim, Jae-Yean

    2015-10-01

    It has been suggested that AUXIN BINDING PROTEIN 1 (ABP1) functions as an apoplastic auxin receptor, and is known to be involved in the post-transcriptional process, and largely independent of the already well-known SKP-cullin-F-box-transport inhibitor response (TIR1) /auxin signaling F-box (AFB) (SCF(TIR1/AFB)) pathway. In the past 10 years, several key components downstream of ABP1 have been reported. After perceiving the auxin signal, ABP1 interacts, directly or indirectly, with plasma membrane (PM)-localized transmembrane proteins, transmembrane kinase (TMK) or SPIKE1 (SPK1), or other unidentified proteins, which transfer the signal into the cell to the Rho of plants (ROP). ROPs interact with their effectors, such as the ROP interactive CRIB motif-containing protein (RIC), to regulate the endocytosis/exocytosis of the auxin efflux carrier PIN-FORMED (PIN) proteins to mediate polar auxin transport across the PM. Additionally, ABP1 is a negative regulator of the traditional SCF(TIR1/AFB) auxin signaling pathway. However, Gao et al. (2015) very recently reported that ABP1 is not a key component in auxin signaling, and the famous abp1-1 and abp1-5 mutant Arabidopsis lines are being called into question because of possible additional mutantion sites, making it necessary to reevaluate ABP1. In this review, we will provide a brief overview of the history of ABP1 research.

  18. HTLV-I Tax protein stimulation of DNA binding of bZIP proteins by enhancing dimerization.

    PubMed

    Wagner, S; Green, M R

    1993-10-15

    The Tax protein of human T cell leukemia virus type-1 (HTLV-I) transcriptionally activates the HTLV-I promoter. This activation requires binding sites for activating transcription factor (ATF) proteins, a family of cellular proteins that contain basic region-leucine zipper (bZIP) DNA binding domains. Data are presented showing that Tax increases the in vitro DNA binding activity of multiple ATF proteins. Tax also stimulated DNA binding by other bZIP proteins, but did not affect DNA binding proteins that lack a bZIP domain. The increase in DNA binding occurred because Tax promotes dimerization of the bZIP domain in the absence of DNA, and the elevated concentration of the bZIP homodimer then facilitates the DNA binding reaction. These results help explain how Tax activates viral transcription and transforms cells.

  19. A vicilin-like seed protein of cycads: similarity to sucrose-binding proteins.

    PubMed

    Braun, H; Czihal, A; Shutov, A D; Bäumlein, H

    1996-04-01

    Seed storage globulins of the 7S and 11S type are synthesized in the seeds of angiosperms and gymnosperms. We have isolated and characterized a vicilin-like gene expressed in the cycad Zamia furfuraceae. Sequence comparisons reveal clear similarities to a sucrose-binding protein isolated from soybean. We suggest the existence of a superfamily of related genes including both vicilin-like and legumin-like seed globulin genes as well as genes coding for spherulins, germins and sucrose-binding-proteins.

  20. Computational characterization of TTHA0379: A potential glycerophosphocholine binding protein of Ugp ATP-binding cassette transporter.

    PubMed

    Chandravanshi, Monika; Gogoi, Prerana; Kanaujia, Shankar Prasad

    2016-11-05

    For the de novo biosynthesis of phospholipids, byproducts such as sn-glycerol-3-phosphate (G3P) and glycerophosphocholine (GPC) of glycerophospholipid metabolic pathway are imported inside the cell by an ATP-binding cassette (ABC) transporter known as UgpABCE. Of which, UgpA and UgpE constitutes the transmembrane domains (TMDs), UgpC forms the dimer of ATP-hydrolyzing component and UgpB is the periplasmic substrate binding protein. Structurally, UgpABCE transporter displays similarity to the maltose ABC transporter of Escherichia coli; thus, has been grouped into the CUT1 (Carbohydrate Uptake Transporter-1) family of bacterial ABC transporters. Being a member of CUT1 family, several Ugp (Uptake glycerol phosphate) protein sequences in biological database(s) exhibit sequence and structure similarity to sugar ABC transporters and have been annotated as sugar binding proteins; one of such proteins is TTHA0379 from Thermus thermophilus HB8. Here, in this study, we used computational method(s) to distinguish UgpB and sugar binding proteins based on their primary and tertiary structure features. A comprehensive analysis of these proteins indicates that they are evolutionarily related to each other having common conserved features at their primary and tertiary structure levels. However, they display differences at their active sites owing to the dissimilarity in their ligand preferences. In addition, phylogenetic analysis of TTHA0379 along with UgpB and sugar binding proteins reveals that both the groups of proteins forms two distinct clades and TTHA0379 groups with UgpB proteins. Furthermore, analysis of the ligand binding pocket shows that all the essential features of glycerophosphocholine binding protein i.e. UgpB, are conserved in TTHA0379 as well. Combining these features, here, we designate TTHA0379 to be a GPC binding protein.

  1. Identification of AKAP79 as a Protein Phosphatase 1 catalytic binding protein

    PubMed Central

    Le, Andrew. V.; Tavalin, Steven. J.

    2011-01-01

    The ubiquitously expressed and highly promiscuous Protein Phosphatase 1 (PP1) regulates many cellular processes. Targeting PP1 to specific locations within the cell allows for the regulation of PP1 by conferring substrate specificity. In the present study, we identified AKAP79 as a novel PP1 regulatory subunit. Immunoprecipitaiton of the AKAP from rat brain extract found that the PP1 catalytic subunit co-purified with the anchoring protein. This is a direct interaction, demonstrated by pulldown experiments using purified proteins. Interestingly, the addition of AKAP79 to purified PP1 catalytic subunit decreased phosphatase activity with an IC50 of 811±0.56 nM of the anchoring protein. Analysis of AKAP79 identified a PP1 binding site that conformed to a consensus PP1 binding motif (FxxR/KxR/K) in the first 44 amino acids of the anchoring protein. This was confirmed when a peptide mimicking this region of AKAP79 was able to bind PP1 by both pulldown assay and Surface Plasmon Resonance. However, PP1 was still able to bind to AKAP79 upon deletion of this region, suggestion additional sites of contact between the anchoring protein and the phosphatase. Importantly, this consensus PP1 binding motif was found not to be responsible for PP1 inhibition, but rather enhanced phosphatase activity, as deletion of this domain resulted in an increased inhibition of PP1 activity. Instead, a second interaction domain localized to residues 150–250 of AKAP79 was required for the inhibition of PP1. However, the inhibitory actions of AKAP79 on PP1 are substrate dependent, as the anchoring protein did not inhibit PP1 dephosphorylation of phospho-PSD-95, a substrate found in AKAP79 complexes in the brain. These combined observations suggest that AKAP79 acts as a PP1 regulatory subunit that can direct PP1 activity towards specific targets in the AKAP79 complex. PMID:21561082

  2. Light-activated DNA binding in a designed allosteric protein

    SciTech Connect

    Strickland, Devin; Moffat, Keith; Sosnick, Tobin R.

    2008-09-03

    An understanding of how allostery, the conformational coupling of distant functional sites, arises in highly evolvable systems is of considerable interest in areas ranging from cell biology to protein design and signaling networks. We reasoned that the rigidity and defined geometry of an {alpha}-helical domain linker would make it effective as a conduit for allosteric signals. To test this idea, we rationally designed 12 fusions between the naturally photoactive LOV2 domain from Avena sativa phototropin 1 and the Escherichia coli trp repressor. When illuminated, one of the fusions selectively binds operator DNA and protects it from nuclease digestion. The ready success of our rational design strategy suggests that the helical 'allosteric lever arm' is a general scheme for coupling the function of two proteins.

  3. Oxysterol-related-binding-protein related Protein-2 (ORP2) regulates cortisol biosynthesis and cholesterol homeostasis.

    PubMed

    Escajadillo, Tamara; Wang, Hongxia; Li, Linda; Li, Donghui; Sewer, Marion B

    2016-05-15

    Oxysterol binding protein-related protein 2 (ORP2) is a lipid binding protein that has been implicated in various cellular processes, including lipid sensing, cholesterol efflux, and endocytosis. We recently identified ORP2 as a member of a protein complex that regulates glucocorticoid biosynthesis. Herein, we examine the effect of silencing ORP2 on adrenocortical function and show that the ORP2 knockdown cells exhibit reduced amounts of multiple steroid metabolites, including progesterone, 11-deoxycortisol, and cortisol, but have increased concentrations of androgens, and estrogens. Moreover, silencing ORP2 suppresses the expression of most proteins required for cortisol production and reduces the expression of steroidogenic factor 1 (SF1). ORP2 silencing also increases cellular cholesterol, concomitant with decreased amounts of 22-hydroxycholesterol and 7-ketocholesterol, two molecules that have been shown to bind to ORP2. Further, we show that ORP2 binds to liver X receptor (LXR) and is required for nuclear LXR expression. LXR and ORP2 are recruited to the CYP11B1 promoter in response to cAMP signaling. Additionally, ORP2 is required for the expression of other LXR target genes, including ABCA1 and the LDL receptor (LDLR). In summary, we establish a novel role for ORP2 in regulating steroidogenic capacity and cholesterol homeostasis in the adrenal cortex.

  4. Protein-protein binding before and after photo-modification of albumin

    NASA Astrophysics Data System (ADS)

    Rozinek, Sarah C.; Glickman, Randolph D.; Thomas, Robert J.; Brancaleon, Lorenzo

    2016-03-01

    Bioeffects of directed-optical-energy encompass a wide range of applications. One aspect of photochemical interactions involves irradiating a photosensitizer with visible light in order to induce protein unfolding and consequent changes in function. In the past, irradiation of several dye-protein combinations has revealed effects on protein structure. Beta lactoglobulin, human serum albumin (HSA) and tubulin have all been photo-modified with meso-tetrakis(4- sulfonatophenyl)porphyrin (TSPP) bound, but only in the case of tubulin has binding caused a verified loss of biological function (loss of ability to form microtubules) as a result of this light-induced structural change. The current work questions if the photo-induced structural changes that occur to HSA, are sufficient to disable its biological function of binding to osteonectin. The albumin-binding protein, osteonectin, is about half the molecular weight of HSA, so the two proteins and their bound product can be separated and quantified by size exclusion high performance liquid chromatography. TSPP was first bound to HSA and irradiated, photo-modifying the structure of HSA. Then native HSA or photo-modified HSA (both with TSPP bound) were compared, to assess loss in HSA's innate binding ability as a result of light-induced structure modification.

  5. Competitive Binding to Cuprous Ions of Protein and BCA in the Bicinchoninic Acid Protein Assay

    PubMed Central

    Huang, Tao; Long, Mian; Huo, Bo

    2010-01-01

    Although Bicinchoninic acid (BCA) has been widely used to determine protein concentration, the mechanism of interaction between protein, copper ion and BCA in this assay is still not well known. Using the Micro BCA protein assay kit (Pierce Company), we measured the absorbance at 562 nm of BSA solutions with different concentrations of protein, and also varied the BCA concentration. When the concentration of protein was increased, the absorbance exhibited the known linear and nonlinear increase, and then reached an unexpected plateau followed by a gradual decrease. We introduced a model in which peptide chains competed with BCA for binding to cuprous ions. Formation of the well-known chromogenic complex of BCA-Cu1+-BCA was competed with the binding of two peptide bonds (NTPB) to cuprous ion, and there is the possibility of the existence of two new complexes. A simple equilibrium equation was established to describe the correlations between the substances in solution at equilibrium, and an empirical exponential function was introduced to describe the reduction reaction. Theoretical predictions of absorbance from the model were in good agreement with the measurements, which not only validated the competitive binding model, but also predicted a new complex of BCA-Cu1+-NTPB that might exist in the final solution. This work provides a new insight into understanding the chemical bases of the BCA protein assay and might extend the assay to higher protein concentration. PMID:21625379

  6. Competitive Binding to Cuprous Ions of Protein and BCA in the Bicinchoninic Acid Protein Assay.

    PubMed

    Huang, Tao; Long, Mian; Huo, Bo

    2010-01-01

    Although Bicinchoninic acid (BCA) has been widely used to determine protein concentration, the mechanism of interaction between protein, copper ion and BCA in this assay is still not well known. Using the Micro BCA protein assay kit (Pierce Company), we measured the absorbance at 562 nm of BSA solutions with different concentrations of protein, and also varied the BCA concentration. When the concentration of protein was increased, the absorbance exhibited the known linear and nonlinear increase, and then reached an unexpected plateau followed by a gradual decrease. We introduced a model in which peptide chains competed with BCA for binding to cuprous ions. Formation of the well-known chromogenic complex of BCA-Cu(1+)-BCA was competed with the binding of two peptide bonds (NTPB) to cuprous ion, and there is the possibility of the existence of two new complexes. A simple equilibrium equation was established to describe the correlations between the substances in solution at equilibrium, and an empirical exponential function was introduced to describe the reduction reaction. Theoretical predictions of absorbance from the model were in good agreement with the measurements, which not only validated the competitive binding model, but also predicted a new complex of BCA-Cu(1+)-NTPB that might exist in the final solution. This work provides a new insight into understanding the chemical bases of the BCA protein assay and might extend the assay to higher protein concentration.

  7. Gc protein (vitamin D-binding protein): Gc genotyping and GcMAF precursor activity.

    PubMed

    Nagasawa, Hideko; Uto, Yoshihiro; Sasaki, Hideyuki; Okamura, Natsuko; Murakami, Aya; Kubo, Shinichi; Kirk, Kenneth L; Hori, Hitoshi

    2005-01-01

    The Gc protein (human group-specific component (Gc), a vitamin D-binding protein or Gc globulin), has important physiological functions that include involvement in vitamin D transport and storage, scavenging of extracellular G-actin, enhancement of the chemotactic activity of C5a for neutrophils in inflammation and macrophage activation (mediated by a GalNAc-modified Gc protein (GcMAF)). In this review, the structure and function of the Gc protein is focused on especially with regard to Gc genotyping and GcMAF precursor activity. A discussion of the research strategy "GcMAF as a target for drug discovery" is included, based on our own research.

  8. Specific binding of the methyl binding domain protein 2 at the BRCA1-NBR2 locus.

    PubMed

    Auriol, Emilie; Billard, Lise-Marie; Magdinier, Frédérique; Dante, Robert

    2005-01-01

    The methyl-CpG binding domain (MBD) proteins are key molecules in the interpretation of DNA methylation signals leading to gene silencing. We investigated their binding specificity at the constitutively methylated region of a CpG island containing the bidirectional promoter of the Breast cancer predisposition gene 1, BRCA1, and the Near BRCA1 2 (NBR2) gene. In HeLa cells, quantitative chromatin immunoprecipitation assays indicated that MBD2 is associated with the methylated region, while MeCP2 and MBD1 were not detected at this locus. MBD2 depletion (approximately 90%), mediated by a transgene expressing a small interfering RNA (siRNA), did not induce MeCP2 or MBD1 binding at the methylated area. Furthermore, the lack of MBD2 at the BRCA1-NBR2 CpG island is associated with an elevated level of NBR2 transcripts and with a significant reduction of induced-DNA-hypomethylation response. In MBD2 knockdown cells, transient expression of a Mbd2 cDNA, refractory to siRNA-mediated decay, shifted down the NBR2 mRNA level to that observed in unmodified HeLa cells. Variations in MBD2 levels did not affect BRCA1 expression despite its stimulation by DNA hypomethylation. Collectively, our data indicate that MBD2 has specific targets and its presence at these targets is indispensable for gene repression.

  9. Paracetamol and cytarabine binding competition in high affinity binding sites of transporting protein

    NASA Astrophysics Data System (ADS)

    Sułkowska, A.; Bojko, B.; Równicka, J.; Sułkowski, W. W.

    2006-07-01

    Paracetamol (acetaminophen, AA) the most popular analgesic drug is commonly used in the treatment of pain in patients suffering from cancer. In our studies, we evaluated the competition in binding with serum albumin between paracetamol (AA) and cytarabine, antyleukemic drug (araC). The presence of one drug can alter the binding affinity of albumin towards the second one. Such interaction can result in changing of the free fraction of the one of these drugs in blood. Two spectroscopic methods were used to determine high affinity binding sites and the competition of the drugs. Basing on the change of the serum albumin fluorescence in the presence of either of the drugs the quenching ( KQ) constants for the araC-BSA and AA-BSA systems were calculated. Analysis of UV difference spectra allowed us to describe the changes in drug-protein complexes (araC-albumin and AA-albumin) induced by the presence of the second drug (AA and araC, respectively). The mechanism of competition between araC and AA has been proposed.

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

    PubMed

    Narla, Satya Nandana; Sun, Xue-Long

    2012-05-14

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

  11. Identification of proteins binding coding and non-coding human RNAs using protein microarrays

    PubMed Central

    2012-01-01

    Background The regulation and function of mammalian RNAs has been increasingly appreciated to operate via RNA-protein interactions. With the recent discovery of thousands of novel human RNA molecules by high-throughput RNA sequencing, efficient methods to uncover RNA-protein interactions are urgently required. Existing methods to study proteins associated with a given RNA are laborious and require substantial amounts of cell-derived starting material. To overcome these limitations, we have developed a rapid and large-scale approach to characterize binding of in vitro transcribed labeled RNA to ~9,400 human recombinant proteins spotted on protein microarrays. Results We have optimized methodology to probe human protein microarrays with full-length RNA molecules and have identified 137 RNA-protein interactions specific for 10 coding and non-coding RNAs. Those proteins showed strong enrichment for common human RNA binding domains such as RRM, RBD, as well as K homology and CCCH type zinc finger motifs. Previously unknown RNA-protein interactions were discovered using this technique, and these interactions were biochemically verified between TP53 mRNA and Staufen1 protein as well as between HRAS mRNA and CNBP protein. Functional characterization of the interaction between Staufen 1 protein and TP53 mRNA revealed a novel role for Staufen 1 in preserving TP53 RNA stability. Conclusions Our approach demonstrates a scalable methodology, allowing rapid and efficient identification of novel human RNA-protein interactions using RNA hybridization to human protein microarrays. Biochemical validation of newly identified interactions between TP53-Stau1 and HRAS-CNBP using reciprocal pull-down experiments, both in vitro and in vivo, demonstrates the utility of this approach to study uncharacterized RNA-protein interactions. PMID:23157412

  12. Identification of Actin-Binding Proteins from Maize Pollen

    SciTech Connect

    Staiger, C.J.

    2004-01-13

    Specific Aims--The goal of this project was to gain an understanding of how actin filament organization and dynamics are controlled in flowering plants. Specifically, we proposed to identify unique proteins with novel functions by investigating biochemical strategies for the isolation and characterization of actin-binding proteins (ABPs). In particular, our hunt was designed to identify capping proteins and nucleation factors. The specific aims included: (1) to use F-actin affinity chromatography (FAAC) as a general strategy to isolate pollen ABPs (2) to produce polyclonal antisera and perform subcellular localization in pollen tubes (3) to isolate cDNA clones for the most promising ABPs (4) to further purify and characterize ABP interactions with actin in vitro. Summary of Progress By employing affinity chromatography on F-actin or DNase I columns, we have identified at least two novel ABPs from pollen, PrABP80 (gelsolin-like) and ZmABP30, We have also cloned and expressed recombinant protein, as well as generated polyclonal antisera, for 6 interesting ABPs from Arabidopsis (fimbrin AtFIM1, capping protein a/b (AtCP), adenylyl cyclase-associated protein (AtCAP), AtCapG & AtVLN1). We performed quantitative analyses of the biochemical properties for two of these previously uncharacterized ABPs (fimbrin and capping protein). Our studies provide the first evidence for fimbrin activity in plants, demonstrate the existence of barbed-end capping factors and a gelsolin-like severing activity, and provide the quantitative data necessary to establish and test models of F-actin organization and dynamics in plant cells.

  13. Crystal structure of the adenovirus DNA binding protein reveals a hook-on model for cooperative DNA binding.

    PubMed Central

    Tucker, P A; Tsernoglou, D; Tucker, A D; Coenjaerts, F E; Leenders, H; van der Vliet, P C

    1994-01-01

    The adenovirus single-stranded DNA binding protein (Ad DBP) is a multifunctional protein required, amongst other things, for DNA replication and transcription control. It binds to single- and double-stranded DNA, as well as to RNA, in a sequence-independent manner. Like other single-stranded DNA binding proteins, it binds ssDNA, cooperatively. We report the crystal structure, at 2.6 A resolution, of the nucleic acid binding domain. This domain is active in DNA replication. The protein contains two zinc atoms in different, novel coordinations. The zinc atoms appear to be required for the stability of the protein fold rather than being involved in direct contacts with the DNA. The crystal structure shows that the protein contains a 17 amino acid C-terminal extension which hooks onto a second molecule, thereby forming a protein chain. Deletion of this C-terminal arm reduces cooperativity in DNA binding, suggesting a hook-on model for cooperativity. Based on this structural work and mutant studies, we propose that DBP forms a protein core around which the single-stranded DNA winds. Images PMID:8039495

  14. Cadmium-binding proteins in the mussel, Mytilus edulis.

    PubMed Central

    Frazier, J M

    1986-01-01

    Inducible cadmium-binding proteins (Cd-BP) in the mussel, Mytilus edulis, were resolved into two molecular weight components, designated Cd-BP10 and Cd-BP20, by gel-permeation chromatography on Sephadex G-75. Each of these two molecular weight components were further resolved into four subcomponents by DEAE-ion-exchange chromatography. All eight subcomponents bound cadmium and exhibited significant UV absorption at 254 nm and little absorption at 280 nm. Each subcomponent was purified and subjected to amino acid composition analysis. Two classes were identified, one having higher cysteine (23.9-26.6 mole-%) and lower glutamic acid contents compared to the other class (11.6-18.2 mole-% cysteine). All subcomponents have a relatively high glycine content (approximately 15 mole-%) relative to mammalian metallothioneins (approximately 8 mole-%). Although the Cd-BP20 have apparent molecular weights almost twice the Cd-BP10, the exact molecular relationship between these binding proteins is not known. PMID:3709463

  15. Slob, a Slowpoke channel–binding protein, modulates synaptic transmission

    PubMed Central

    Zhang, Jiaming

    2011-01-01

    Modulation of ion channels by regulatory proteins within the same macromolecular complex is a well-accepted concept, but the physiological consequences of such modulation are not fully understood. Slowpoke (Slo), a potassium channel critical for action potential repolarization and transmitter release, is regulated by Slo channel–binding protein (Slob), a Drosophila melanogaster Slo (dSlo) binding partner. Slob modulates the voltage dependence of dSlo channel activation in vitro and exerts similar effects on the dSlo channel in Drosophila central nervous system neurons in vivo. In addition, Slob modulates action potential duration in these neurons. Here, we investigate further the functional consequences of the modulation of the dSlo channel by Slob in vivo, by examining larval neuromuscular synaptic transmission in flies in which Slob levels have been altered. In Slob-null flies generated through P-element mutagenesis, as well as in Slob knockdown flies generated by RNA interference (RNAi), we find an enhancement of synaptic transmission but no change in the properties of the postsynaptic muscle cell. Using targeted transgenic rescue and targeted expression of Slob-RNAi, we find that Slob expression in neurons (but not in the postsynaptic muscle cell) is critical for its effects on synaptic transmission. Furthermore, inhibition of dSlo channel activity abolishes these effects of Slob. These results suggest that presynaptic Slob, by regulating dSlo channel function, participates in the modulation of synaptic transmission. PMID:21282401

  16. Purification of Capping Protein Using the Capping Protein Binding Site of CARMIL as an Affinity Matrix

    PubMed Central

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

    2009-01-01

    Capping Protein (CP) is a ubiquitously expressed, heterodimeric actin binding protein that is essential for normal actin dynamics in cells. The existing methods for purifying native CP from tissues and recombinant CP from bacteria are time-consuming processes that involve numerous conventional chromatographic steps and functional assays to achieve a homogeneous preparation of the protein. Here we report the rapid purification of Acanthamoeba CP from amoeba extracts and recombinant mouse CP from E. coli extracts using as an affinity matrix GST fusion proteins containing the CP binding site from Acanthamoeba CARMIL and mouse CARMIL-1, respectively. This improved method for CP purification should facilitate the in vitro analysis of CP structure, function and regulation. PMID:19427903

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

    PubMed

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

    2009-10-01

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

  18. Transient Protein-Protein Interaction of the SH3-Peptide Complex via Closely Located Multiple Binding Sites

    PubMed Central

    Hahn, Seungsoo; Kim, Dongsup

    2012-01-01

    Protein-protein interactions play an essential role in cellular processes. Certain proteins form stable complexes with their partner proteins, whereas others function by forming transient complexes. The conventional protein-protein interaction model describes an interaction between two proteins under the assumption that a protein binds to its partner protein through a single binding site. In this study, we improved the conventional interaction model by developing a Multiple-Site (MS) model in which a protein binds to its partner protein through closely located multiple binding sites on a surface of the partner protein by transiently docking at each binding site with individual binding free energies. To test this model, we used the protein-protein interaction mediated by Src homology 3 (SH3) domains. SH3 domains recognize their partners via a weak, transient interaction and are therefore promiscuous in nature. Because the MS model requires large amounts of data compared with the conventional interaction model, we used experimental data from the positionally addressable syntheses of peptides on cellulose membranes (SPOT-synthesis) technique. From the analysis of the experimental data, individual binding free energies for each binding site of peptides were extracted. A comparison of the individual binding free energies from the analysis with those from atomistic force fields gave a correlation coefficient of 0.66. Furthermore, application of the MS model to 10 SH3 domains lowers the prediction error by up to 9% compared with the conventional interaction model. This improvement in prediction originates from a more realistic description of complex formation than the conventional interaction model. The results suggested that, in many cases, SH3 domains increased the protein complex population through multiple binding sites of their partner proteins. Our study indicates that the consideration of general complex formation is important for the accurate description of

  19. ATPase and ubiquitin-binding proteins of the yeast proteasome.

    PubMed

    Rubin, D M; van Nocker, S; Glickman, M; Coux, O; Wefes, I; Sadis, S; Fu, H; Goldberg, A; Vierstra, R; Finley, D

    1997-03-01

    The 26S proteasome is a 2-Megadalton proteolytic complex with over 30 distinct subunits. The 19S particle, a subcomplex of the 26S proteasome, is thought to confer ATP-dependence and ubiquitin-dependence on the proteolytic core particle of the proteasome. Given the complexity of the 19S particle, genetic approaches are likely to play an important role in its analysis. We have initiated biochemical and genetic studies of the 19S particle in Saccharomyces cerevisiae. Here we describe the localization to the proteasome of several ATPases that were previously proposed to be involved in transcription. Independent studies indicate that the mammalian 26S proteasome contains closely related ATPases. We have also found that the multiubiquitin chain binding protein Mcb1, a homolog of the mammalian S5a protein, is a subunit of the yeast proteasome. However, contrary to expectation, MCB1 is not an essential gene in yeast. The mcb1 mutant grows at a nearly wild-type rate, and the breakdown of most ubiquitin-protein conjugates is unaffected in this strain. One substrate, Ub-Proline-beta gal, was found to require MCB1 for its breakdown, but it remains unclear whether Mcb1 serves as a ubiquitin receptor in this process. Our data suggest that the recognition of ubiquitin conjugates by the proteasome is a complex process which must involve proteins other than Mcb1.

  20. TAR DNA-binding protein 43 in neurodegenerative disease

    PubMed Central

    Chen-Plotkin, Alice S.; Lee, Virginia M.-Y.; Trojanowski, John Q.

    2010-01-01

    In 2006, TAR DNA-binding protein 43 (TDP-43), a highly conserved nuclear protein, was identified as the major disease protein in amyotrophic lateral sclerosis (ALS) and in the most common variant of frontotemporal lobar degeneration (FTLD), FTLD-U, which is characterized by cytoplasmic inclusions that stain positive for ubiquitin but negative for tau and α-synuclein. Since then, rapid advances have been made in our understanding of the physiological function of TDP-43 and the role of this protein in neurodegeneration. These advances link ALS and FTLD-U (now designated FTLD-TDP) to a shared mechanism of disease. In this Review, we summarize the current evidence regarding the normal function of TDP-43 and the TDP-43 pathology observed in FTLD-TDP, ALS, and other neurodegenerative diseases wherein TDP-43 pathology co-occurs with other disease-specific lesions (for example, with amyloid plaques and neurofibrillary tangles in Alzheimer disease). Moreover, we discuss the accumulating data that support our view that FTLD-TDP and ALS represent two ends of a spectrum of primary TDP-43 proteinopathies. Finally, we comment on the importance of recent advances in TDP-43-related research to neurological practice, including the new opportunities to develop better diagnostics and disease-modifying therapies for ALS, FTLD-TDP, and related disorders exhibiting TDP-43 pathology. PMID:20234357

  1. BET Acetyl-Lysine Binding Proteins Control Pathological Cardiac Hypertrophy

    PubMed Central

    Spiltoir, Jessica I.; Stratton, Matthew S.; Cavasin, Maria A.; Demos-Davies, Kim; Reid, Brian G.; Qi, Jun; Bradner, James E.; McKinsey, Timothy A.

    2014-01-01

    Cardiac hypertrophy is an independent predictor of adverse outcomes in patients with heart failure, and thus represents an attractive target for novel therapeutic intervention. JQ1, a small molecule inhibitor of bromodomain and extraterminal (BET) acetyl-lysine reader proteins, was identified in a high throughput screen designed to discover novel small molecule regulators of cardiomyocyte hypertrophy. JQ1 dose-dependently blocked agonist-dependent hypertrophy of cultured neonatal rat ventricular myocytes (NRVMs) and reversed the prototypical gene program associated with pathological cardiac hypertrophy. JQ1 also blocked left ventricular hypertrophy (LVH) and improved cardiac function in adult mice subjected to transverse aortic constriction (TAC). The BET family consists of BRD2, BRD3, BRD4 and BRDT. BRD4 protein expression was increased during cardiac hypertrophy, and hypertrophic stimuli promoted recruitment of BRD4 to the transcriptional start site (TSS) of the gene encoding atrial natriuretic factor (ANF). Binding of BRD4 to the ANF TSS was associated with increased phosphorylation of local RNA polymerase II. These findings define a novel function for BET proteins as signal-responsive regulators of cardiac hypertrophy, and suggest that small molecule inhibitors of these epigenetic reader proteins have potential as therapeutics for heart failure. PMID:23939492

  2. Grizzly bear corticosteroid binding globulin: Cloning and serum protein expression.

    PubMed

    Chow, Brian A; Hamilton, Jason; Alsop, Derek; Cattet, Marc R L; Stenhouse, Gordon; Vijayan, Mathilakath M

    2010-06-01

    Serum corticosteroid levels are routinely measured as markers of stress in wild animals. However, corticosteroid levels rise rapidly in response to the acute stress of capture and restraint for sampling, limiting its use as an indicator of chronic stress. We hypothesized that serum corticosteroid binding globulin (CBG), the primary transport protein for corticosteroids in circulation, may be a better marker of the stress status prior to capture in grizzly bears (Ursus arctos). To test this, a full-length CBG cDNA was cloned and sequenced from grizzly bear testis and polyclonal antibodies were generated for detection of this protein in bear sera. The deduced nucleotide and protein sequences were 1218 bp and 405 amino acids, respectively. Multiple sequence alignments showed that grizzly bear CBG (gbCBG) was 90% and 83% identical to the dog CBG nucleotide and amino acid sequences, respectively. The affinity purified rabbit gbCBG antiserum detected grizzly bear but not human CBG. There were no sex differences in serum total cortisol concentration, while CBG expression was significantly higher in adult females compared to males. Serum cortisol levels were significantly higher in bears captured by leg-hold snare compared to those captured by remote drug delivery from helicopter. However, serum CBG expression between these two groups did not differ significantly. Overall, serum CBG levels may be a better marker of chronic stress, especially because this protein is not modulated by the stress of capture and restraint in grizzly bears.

  3. Metals and Neuronal Metal Binding Proteins Implicated in Alzheimer's Disease.

    PubMed

    Cristóvão, Joana S; Santos, Renata; Gomes, Cláudio M

    2016-01-01

    Alzheimer's disease (AD) is the most prevalent age-related dementia affecting millions of people worldwide. Its main pathological hallmark feature is the formation of insoluble protein deposits of amyloid-β and hyperphosphorylated tau protein into extracellular plaques and intracellular neurofibrillary tangles, respectively. Many of the mechanistic details of this process remain unknown, but a well-established consequence of protein aggregation is synapse dysfunction and neuronal loss in the AD brain. Different pathways including mitochondrial dysfunction, oxidative stress, inflammation, and metal metabolism have been suggested to be implicated in this process. In particular, a body of evidence suggests that neuronal metal ions such as copper, zinc, and iron play important roles in brain function in health and disease states and altered homeostasis and distribution as a common feature across different neurodegenerative diseases and aging. In this focused review, we overview neuronal proteins that are involved in AD and whose metal binding properties may underlie important biochemical and regulatory processes occurring in the brain during the AD pathophysiological process.

  4. Centromeric binding and activity of Protein Phosphatase 4

    PubMed Central

    Lipinszki, Zoltan; Lefevre, Stephane; Savoian, Matthew S.; Singleton, Martin R.; Glover, David M.; Przewloka, Marcin R.

    2015-01-01

    The cell division cycle requires tight coupling between protein phosphorylation and dephosphorylation. However, understanding the cell cycle roles of multimeric protein phosphatases has been limited by the lack of knowledge of how their diverse regulatory subunits target highly conserved catalytic subunits to their sites of action. Phosphoprotein phosphatase 4 (PP4) has been recently shown to participate in the regulation of cell cycle progression. We now find that the EVH1 domain of the regulatory subunit 3 of Drosophila PP4, Falafel (Flfl), directly interacts with the centromeric protein C (CENP-C). Unlike other EVH1 domains that interact with proline-rich ligands, the crystal structure of the Flfl amino-terminal EVH1 domain bound to a CENP-C peptide reveals a new target-recognition mode for the phosphatase subunit. We also show that binding of Flfl to CENP-C is required to bring PP4 activity to centromeres to maintain CENP-C and attached core kinetochore proteins at chromosomes during mitosis. PMID:25562660

  5. Localization of Cellular Retinol-Binding Protein and Retinol-Binding Protein in Cells Comprising the Blood-Brain Barrier of Rat and Human

    NASA Astrophysics Data System (ADS)

    MacDonald, Paul N.; Bok, Dean; Ong, David E.

    1990-06-01

    Brain is not generally recognized as an organ that requiries 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 endothelial cells of the brain microvasculature and 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.

  6. Novel approach for selecting the best predictor for identifying the binding sites in DNA binding proteins.

    PubMed

    Nagarajan, R; Ahmad, Shandar; Gromiha, M Michael

    2013-09-01

    Protein-DNA complexes play vital roles in many cellular processes by the interactions of amino acids with DNA. Several computational methods have been developed for predicting the interacting residues in DNA-binding proteins using sequence and/or structural information. These methods showed different levels of accuracies, which may depend on the choice of data sets used in training, the feature sets selected for developing a predictive model, the ability of the models to capture information useful for prediction or a combination of these factors. In many cases, different methods are likely to produce similar results, whereas in others, the predictors may return contradictory predictions. In this situation, a priori estimates of prediction performance applicable to the system being investigated would be helpful for biologists to choose the best method for designing their experiments. In this work, we have constructed unbiased, stringent and diverse data sets for DNA-binding proteins based on various biologically relevant considerations: (i) seven structural classes, (ii) 86 folds, (iii) 106 superfamilies, (iv) 194 families, (v) 15 binding motifs, (vi) single/double-stranded DNA, (vii) DNA conformation (A, B, Z, etc.), (viii) three functions and (ix) disordered regions. These data sets were culled as non-redundant with sequence identities of 25 and 40% and used to evaluate the performance of 11 different methods in which online services or standalone programs are available. We observed that the best performing methods for each of the data sets showed significant biases toward the data sets selected for their benchmark. Our analysis revealed important data set features, which could be used to estimate these context-specific biases and hence suggest the best method to be used for a given problem. We have developed a web server, which considers these features on demand and displays the best method that the investigator should use. The web server is freely available at

  7. A putative GTP binding protein homologous to interferon-inducible Mx proteins performs an essential function in yeast protein sorting.

    PubMed

    Rothman, J H; Raymond, C K; Gilbert, T; O'Hara, P J; Stevens, T H

    1990-06-15

    Members of the Mx protein family promote interferon-inducible resistance to viral infection in mammals and act by unknown mechanisms. We identified an Mx-like protein in yeast and present genetic evidence for its cellular function. This protein, the VPS1 product, is essential for vacuolar protein sorting, normal organization of intracellular membranes, and growth at high temperature, implying that Mx-like proteins are engaged in fundamental cellular processes in eukaryotes. Vps1p contains a tripartite GTP binding motif, which suggests that binding to GTP is essential to its role in protein sorting. Vps1p-specific antibody labels punctate cytoplasmic structures that condense to larger structures in a Golgi-accumulating sec7 mutant; thus, Vps1p may associate with an intermediate organelle of the secretory pathway.

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

    PubMed

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

    1996-05-28

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

  9. NMR studies reveal the role of biomembranes in modulating ligand binding and release by intracellular bile acid binding proteins.

    PubMed

    Pedò, Massimo; Löhr, Frank; D'Onofrio, Mariapina; Assfalg, Michael; Dötsch, Volker; Molinari, Henriette

    2009-12-18

    Bile acid molecules are transferred vectorially between basolateral and apical membranes of hepatocytes and enterocytes in the context of the enterohepatic circulation, a process regulating whole body lipid homeostasis. This work addresses the role of the cytosolic lipid binding proteins in the intracellular transfer of bile acids between different membrane compartments. We present nuclear magnetic resonance (NMR) data describing the ternary system composed of the bile acid binding protein, bile acids, and membrane mimetic systems, such as anionic liposomes. This work provides evidence that the investigated liver bile acid binding protein undergoes association with the anionic membrane and binding-induced partial unfolding. The addition of the physiological ligand to the protein-liposome mixture is capable of modulating this interaction, shifting the equilibrium towards the free folded holo protein. An ensemble of NMR titration experiments, based on nitrogen-15 protein and ligand observation, confirm that the membrane and the ligand establish competing binding equilibria, modulating the cytoplasmic permeability of bile acids. These results support a mechanism of ligand binding and release controlled by the onset of a bile salt concentration gradient within the polarized cell. The location of a specific protein region interacting with liposomes is highlighted.

  10. Interacting protein partners of Arabidopsis RNA binding protein AtRBP45b

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA binding proteins (RBPs) are important players in post-transcriptional gene regulation and shown to play an important role in normal development and in response to environmental perturbations. Arabidopsis RBP, AtRBP45b with triple RNA recognition motifs (RRMs) have are closely related to the yeas...

  11. Molecular cloning and expression of chicken carbohydrate response element binding protein and Max-like protein X gene homologues

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Carbohydrate response element binding protein (ChREBP) and sterol regulatory element binding protein-1c (SREBP-1c) are transcription factors that are known to be key regulators of glucose metabolism and lipid synthesis in mammals. Since ChREBP and its co-activator Max-like protein X (Mlx) have not ...

  12. Blastomyces Virulence Adhesin-1 Protein Binding to Glycosaminoglycans Is Enhanced by Protein Disulfide Isomerase

    PubMed Central

    Beaussart, Audrey; Brandhorst, Tristan

    2015-01-01

    ABSTRACT Blastomyces adhesin-1 (BAD-1) protein mediates the virulence of the yeast Blastomyces dermatitidis, in part by binding host lung tissue, the extracellular matrix, and cellular receptors via glycosaminoglycans (GAGs), such as heparan sulfate. The tandem repeats that make up over 90% of BAD-1 appear in their native state to be tightly folded into an inactive conformation, but recent work has shown that they become activated and adhesive upon reduction of a disulfide linkage. Here, atomic force microscopy (AFM) of a single BAD-1 molecule interacting with immobilized heparin revealed that binding is enhanced upon treatment with protein disulfide isomerase and dithiothreitol (PDI/DTT). PDI/DTT treatment of BAD-1 induced a plateau effect in atomic force signatures that was consistent with sequential rupture of tandem binding domains. Inhibition of PDI in murine macrophages blunted BAD-1 binding to heparin in vitro. Based on AFM, we found that a short Cardin-Weintraub sequence paired with a WxxWxxW sequence in the first, degenerate repeat at the N terminus of BAD-1 was sufficient to initiate heparin binding. Removal of half of the 41 BAD-1 tandem repeats led to weaker adhesion, illustrating their role in enhanced binding. Mass spectroscopy of the tandem repeat revealed that the PDI-induced interaction with heparin is characterized by ruptured disulfide bonds and that cysteine thiols remain reduced. Further binding studies showed direct involvement of thiols in heparin ligation. Thus, we propose that the N-terminal domain of BAD-1 governs the initial association with host GAGs and that proximity to GAG-associated host PDI catalyzes activation of additional binding motifs conserved within the tandem repeats, leading to enhanced avidity and availability of reduced thiols. PMID:26396244

  13. Properties of the cadmium binding proteins of Euglena gracilis

    SciTech Connect

    Weber, D.; Petering, D.H.; Shaw, C.F. III

    1986-05-01

    The two Euglena cadmium binding proteins (Cd-BPI and II) resolved by HPLC-DEAE, are smaller and more negatively charged than mammalian metallothiones (MT). Pyridylazoresorcinol (PAR) reacts with Cd/sup +2/ at pH 7.5 to form Cd(PAR). Cd-BPII reacts biphasically, each step involving both a PAR dependent and independent term. Cd-BPI appears to fit a saturation curve suggesting a long-lived BP-Cd-PAR complex which reacts to form Cd-PAR+apoBP. These differences reflect substantial differences in the preliminary amino acid analyses for BPI and BPII. Dithionitrobenzoic acid (DTNB) reacts with protein sulfhydryls. Both proteins show biphasic reaction, although DTNB dependence in the slow step is slight. Rates for both BPs are an order of magnitude greater than for horse kidney MT. This may indicate either greater accessibility of the metal-bound thiolates or weaker Cd-S bonds. DTNB reactions indicate a 2-3 thiol:1 Cd.pH titrations of Cd-BPI show 2 1/2 titration values (pH 8.5 +/- 0.5 and pH 5.9 +/- 0.3) and a 1/2 titration value of 5.5 +/- 0.5 for Cd-BPII. Mammalian MT has a value between pH 3 and 4. These differences are consistent with low levels of cellular Cd always being bound to mammalian MT but not to Euglena BP which only bind Cd after exposure to high levels of Cd/sup +2/.

  14. THE INFLUENCE OF SERUM BINDING PROTEINS AND CLEARANCE ON THE COMPARATIVE RECEPTOR BINDING POTENCY OF ENDOCRINE ACTIVE COMPOUNDS

    EPA Science Inventory

    THE INFLUENCE OF SERUM BINDING PROTEINS AND CLEARANCE ON THE COMPARATIVE RECEPTOR BINDING POTENCY OF ENDOCRINE ACTIVE COMPOUNDS. JG Teeguarden1 and HA Barton2. 1ENVIRON International, Ruston LA; 2US EPA, ORD, NHEERL, ETD, Pharmacokinetics Branch, RTP, NC.

    One measure of th...

  15. The yeast telomere-binding protein RAP1 binds to and promotes the formation of DNA quadruplexes in telomeric DNA.

    PubMed Central

    Giraldo, R; Rhodes, D

    1994-01-01

    The protein RAP1 is essential for the maintenance of the telomeres of Saccharomyces cerevisiae and binds in vitro to multiple sites found within the TG1-3 telomeric repeats. We show here that, in addition to its known binding activity for double-stranded DNA, RAP1 binds sequence-specifically to the GT-strands. This indicates that RAP1 is the protein that binds to the telomeric terminal GT-tails. Furthermore, we have found that RAP1 binds to and promotes the formation of G-tetrads, i.e. DNA quadruplexes, in GT-strand oligonucleotides at nanomolar concentrations. The formation of DNA quadruplexes appears to involve the intermolecular association of GT-strands. The minimal DNA-binding domain of RAP1 (DBD) binds only to double-stranded DNA, so that the novel DNA-binding activity we have found involves regions of the protein located outside of the DBD. The finding that a telomeric protein promotes the formation of G-tetrads argues for the use of DNA quadruplexes in telomere association. Images PMID:8194531

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

    NASA Astrophysics Data System (ADS)

    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. Characterization of a 100-kilodalton binding protein for the six serotypes of coxsackie B viruses.

    PubMed Central

    de Verdugo, U R; Selinka, H C; Huber, M; Kramer, B; Kellermann, J; Hofschneider, P H; Kandolf, R

    1995-01-01

    Viral infection of host cells primarily depends on binding of the virus to a specific cell surface protein. In order to characterize the binding protein for group B coxsackieviruses (CVB), detergent-solubilized membrane proteins of different cell lines were tested in virus overlay protein-binding assays. A prominent virus-binding protein with a molecular mass of 100 kDa was detected in various CVB-permissive human and monkey cell lines but was not detected in nonpermissive cell lines. The specificity of CVB binding to the 100-kDa protein on permissive human cells was substantiated by binding of all six serotypes of CVB and by competition experiments. In contrast, poliovirus and Sendai virus did not bind to the 100-kDa CVB-specific protein. A fraction of HeLa membrane proteins enriched in the range of 100 kDa showed functional activity by transforming infectious CVB (160S) into A-particles (135S). In order to purify this CVB-binding protein, solubilized membrane proteins from HeLa cells were separated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by elution of the 100-kDa protein. Amino acid sequence analysis of tryptic fragments of the CVB-binding protein indicated that this 100-kDa CVB-specific protein is a cell surface protein related to nucleolin. These results were confirmed by immunoprecipitations of the CVB-binding protein with nucleolin-specific antibodies, suggesting that a nucleolin-related membrane protein acts as a specific binding protein for the six serotypes of CVB. PMID:7474086

  18. Reagentless, Electrochemical Approach for the Specific Detection of Double- and Single-Stranded DNA Binding Proteins

    PubMed Central

    Ricci, Francesco; Bonham, Andrew J.; Mason, Aaron C.; Reich, Norbert O.; Plaxco, Kevin W.

    2009-01-01

    Here we demonstrate a reagentless, electrochemical platform for the specific detection of proteins that bind to single- or double-stranded DNA. The sensor is composed of a double- or single-stranded, redox-tagged DNA probe which is covalently attached to an interrogating electrode. Upon protein binding the current arising from the redox tag is suppressed, indicating the presence of the target. Using this approach we have fabricated sensors against the double-stranded DNA binding proteins TATA-box binding protein and M.HhaI methyltransferase, and against the single-strand binding proteins Escherichia coli SSBP and replication protein A. All four targets are detected at nanomolar concentrations, in minutes, and in a convenient, general, readily reusable, electrochemical format. The approach is specific; we observed no significant cross-reactivity between the sensors. Likewise the approach is selective; it supports, for example, the detection of single strand binding protein directly in crude nuclear extracts. The generality of our approach (including its ability to detect both double- and single-strand binding proteins) and a strong, non-monotonic dependence of signal gain on probe density support a collisional signaling mechanism in which binding alters the collision efficiency, and thus electron transfer efficiency, of the attached redox tag. Given the ubiquity with which protein binding will alter the collisional dynamics of an oligonucleotide, we believe this approach may prove of general utility in the detection of DNA and RNA binding proteins. PMID:19199570

  19. Inactivation of rice bran thiamine-binding protein by N,N'-dicyclohexylcarbodiimide.

    PubMed

    Nishimura, H; Sempuku, K; Nosaka, K; Iwashima, A

    1984-10-01

    The addition of a carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide (DCCD) to thiamine-binding protein isolated from rice bran resulted in a remarkable loss of its binding activity with [14C]thiamine. Thiamine and chloroethylthiamine substantially protected the protein against inactivation by DCCD, whereas thiamine phosphates did not. Another carboxyl reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) also inactivated rice bran thiamine-binding protein. Inactivation of the thiamine-binding protein was accompanied by covalent binding of DCCD to the protein as shown by the use of [14C]DCCD. The binding of [14C]DCCD to the thiamine-binding protein was specific, and significantly inhibited by the addition of thiamine. The loss of thiamine-binding activity was proportional to the specific binding of [14C]DCCD. For complete inactivation of the thiamine-binding activity, the binding of 2.46 mol of [14C]DCCD per mol of thiamine-binding protein was required. Furthermore, limited proteolysis of the binding protein by trypsin yielded two polypeptides with molecular weights of 35,000 (large polypeptide) and 12,500 (small polypeptide) which were separated by SDS-polyacrylamide gel electrophoresis. The binding sites of [14C]DCCD were found to be located on the large polypeptide. These results suggest that a specific carboxyl residue in the large polypeptide releasable from rice bran thiamine-binding protein by trypsin digestion when modified by DCCD is involved in the binding of thiamine.

  20. Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins

    PubMed Central

    Bokhove, Marcel; Sadat Al Hosseini, Hamed; Saito, Takako; Dioguardi, Elisa; Gegenschatz-Schmid, Katharina; Nishimura, Kaoru; Raj, Isha; de Sanctis, Daniele; Han, Ling; Jovine, Luca

    2016-01-01

    We present a strategy to obtain milligrams of highly post-translationally modified eukaryotic proteins, transiently expressed in mammalian cells as rigid or cleavable fusions with a mammalianized version of bacterial maltose-binding protein (mMBP). This variant was engineered to combine mutations that enhance MBP solubility and affinity purification, as well as provide crystal-packing interactions for increased crystallizability. Using this cell type-independent approach, we could increase the expression of secreted and intracellular human proteins up to 200-fold. By molecular replacement with MBP, we readily determined five novel high-resolution structures of rigid fusions of targets that otherwise defied crystallization. PMID:26850170

  1. Structure, sarcomeric organization, and thin filament binding of cardiac myosin-binding protein-C.

    PubMed

    Craig, Roger; Lee, Kyoung Hwan; Mun, Ji Young; Torre, Iratxe; Luther, Pradeep K

    2014-03-01

    Myosin-binding protein-C (MyBP-C) is an accessory protein of the myosin filaments of vertebrate striated muscle. In the heart, it plays a key role in modulating contractility in response to β-adrenergic stimulation. Mutations in the cardiac isoform (cMyBP-C) are a leading cause of inherited hypertrophic cardiomyopathy. Understanding cMyBP-C function and its role in disease requires knowledge of the structure of the molecule, its organization in the sarcomere, and its interactions with other sarcomeric proteins. Here we review the main structural features of this modular, elongated molecule and the properties of some of its key domains. We describe observations suggesting that the bulk of the molecule extends perpendicular to the thick filament, enabling it to reach neighboring thin filaments in the sarcomere. We review structural and functional evidence for interaction of its N-terminal domains with actin and how this may modulate thin filament activation. We also discuss the effects that phosphorylation of cMyBP-C has on some of these structural features and how this might relate to cMyBP-C function in the beating heart.

  2. Accumulation of guanylate binding proteins in patients treated with interferons.

    PubMed

    Cheng, Y S; Becker-Manley, M F; Rucker, R G; Borden, E C

    1988-06-01

    We have previously described an interferon (IFN)-induced protein with a molecular weight of 67,000. This protein has an affinity to guanylates and is thus called guanylate binding protein (GBP). The synthesis of GBP is inducible by IFNs in all human diploid fibroblast cell lines that we studied. To determine whether or not the GBP synthesis is IFN-inducible in humans as well as in cultured cells, we have studied the levels of GBP in the peripheral blood leukocytes (PBL) of patients treated with either type I or type II IFN. An increased GBP level was found the day immediately after treatment with either type of IFN, and the elevated GBP levels were maintained for at least 8 days. Among the patients studied, we found a higher level GBP accumulation (2.3x) in patients treated with IFN-beta than in those treated with IFN-gamma (1.6x). The increase of GBP in patients receiving IFN-gamma correlated with increases in class II histocompatibility antigens, HLA-DR and HLA-DQ in monocytes. Thus, the levels of GBP in peripheral blood leukocytes may be used as a parameter for the study of IFN responses in patients.

  3. Interferon induction of fibroblast proteins with guanylate binding activity.

    PubMed

    Cheng, Y S; Colonno, R J; Yin, F H

    1983-06-25

    Treatment of human diploid fibroblastic cells with interferon induces the synthesis of two guanylate binding proteins (GBP) with molecular weights of 67,000 and 56,000. The Mr = 67,000 protein (67K GBP) is synthesized upon treatment with either alpha-, beta-, or gamma-interferon. Among these interferons, gamma-interferon induces a higher level of 67K GBP synthesis. The 67K GBP synthesized in either beta- or gamma-interferon-treated cells has two charge forms with isoelectric points of 6.0 and 5.8, respectively. The synthesis of the Mr = 56,000 protein is induced by the treatment using either alpha- or beta-interferon, but its synthesis in gamma-interferon-treated cells is undetectable. The amounts of the radioactive GBPs synthesized in human fibroblasts are proportional to the amounts of the purified beta-interferon used for the inductions. Syntheses of GBPs require the transcription of cellular genes because their syntheses are completely blocked by actinomycin D treatments. The mRNA for the 67K GBP is found in fibroblasts that are treated by either alpha-, beta-, or gamma-interferon, but it is not detected in untreated cells. More 67K GBP mRNA is accumulated in the gamma-interferon-treated than in alpha- or beta-interferon-treated fibroblasts. This is consistent with more 67K GBP synthesis found in gamma-interferon-treated fibroblasts.

  4. Growth hormone receptor/binding protein: Physiology and function

    SciTech Connect

    Herington, A.C.; Ymer, S.I.; Stevenson, J.L.; Roupas, P.

    1994-12-31

    Soluble truncated forms of the growth hormone receptor (GHR) are present in the circulation of many species and are also produced by many tissues/cell types. The major high-affinity forms of these GH-binding proteins (GHBP) are derived by alternative splicing of GHR mRNA in rodents, but probably by proteolytic cleavage in other species. Questions still remain with respect to the origins, native molecular forms(s), physiology, and function of the GHBPs, however. The observation that GH induces dimerization of the soluble GHBP and a membrane GHR, and that dimerization of GHR appears to be critical for GH bioactivity suggests that the presentation of GH to target cells, in an unbound form or as a monomeric or dimeric complex with GHBP, may have significant implications for the ability of GH to activate specific postreceptor signaling pathways (tyrosine kinase, protein kinase C, G-protein pathways) known to be utilized by GH for its diverse biological effects. This minireview addresses some of these aspects and highlights several new questions which have arisen as a result of recent advances in our understanding of the structure, function, and signaling mechanisms of the membrane bound GHR. 43 refs.

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

    PubMed

    Zhu, Yu; Ye, Xin-Hai; Liu, Yang; Yan, Zhi-Chao; Stanley, David; Ye, Gong-Yin; Fang, Qi

    2015-11-30

    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.

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

    PubMed

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

    2016-05-01

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

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

    PubMed Central

    Zhu, Yu; Ye, Xin-Hai; Liu, Yang; Yan, Zhi-Chao; Stanley, David; Ye, Gong-Yin; Fang, Qi

    2015-01-01

    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. PMID:26633500

  8. RNA-binding proteins in plants: the tip of an iceberg?

    NASA Technical Reports Server (NTRS)

    Fedoroff, Nina V.; Federoff, N. V. (Principal Investigator)

    2002-01-01

    RNA-binding proteins, which are involved in the synthesis, processing, transport, translation, and degradation of RNA, are emerging as important, often multifunctional, cellular regulatory proteins. Although relatively few RNA-binding proteins have been studied in plants, they are being identified with increasing frequency, both genetically and biochemically. RNA-binding proteins that regulate chloroplast mRNA stability and translation in response to light and that have been elegantly analyzed in Clamydomonas reinhardtii have counterparts with similar functions in higher plants. Several recent reports describe mutations in genes encoding RNA-binding proteins that affect plant development and hormone signaling.

  9. Adenovirus DNA-binding protein forms a multimeric protein complex with double-stranded DNA and enhances binding of nuclear factor I.

    PubMed Central

    Stuiver, M H; van der Vliet, P C

    1990-01-01

    The 72-kilodalton adenovirus DNA-binding protein (DBP) binds to single-stranded DNA as well as to RNA and double-stranded DNA and is essential for the replication of viral DNA. We investigated the binding of DBP to double-stranded DNA by gel retardation analysis. By using a 114-base-pair DNA fragment, five or six different complexes were observed by gel retardation. The mobility of these complexes is dependent on the DBP concentration, suggesting that the complexes arise by sequential binding of DBP molecules to the DNA. In contrast to binding to single-stranded DNA, the binding of DBP to double-stranded DNA appears to be noncooperative. DBP binds to linear DNA as well as to circular DNA, while linear DNA containing the adenovirus terminal protein was also recognized. No specificity for adenovirus origin sequences was observed. To study whether the binding of DBP could influence initiation of DNA replication, we analyzed the effect of DBP on the binding of nuclear factor I (NFI) and NFIII, two sequence-specific origin-recognizing proteins that enhance initiation. At subsaturating levels of NFI, DBP increases the rate of binding of NFI considerably, while no effect was seen on NFIII. This stimulation of NFI binding is specific for DBP and was not observed with another protein (NFIV), which forms a similar DNA-multimeric protein complex. In agreement with enhanced NFI binding, DBP stimulates initiation of adenovirus DNA replication in vitro especially strongly at subsaturating NFI concentrations. We explain our results by assuming that DBP forms a complex with origin DNA that promotes formation of an alternative DNA structure, thereby facilitating the binding of NFI as well as the initiation of DNA replication via NFI. Images PMID:2293667

  10. A calmodulin binding protein from Arabidopsis is induced by ethylene and contains a DNA-binding motif

    NASA Technical Reports Server (NTRS)

    Reddy, A. S.; Reddy, V. S.; Golovkin, M.

    2000-01-01

    Calmodulin (CaM), a key calcium sensor in all eukaryotes, regulates diverse cellular processes by interacting with other proteins. To isolate CaM binding proteins involved in ethylene signal transduction, we screened an expression library prepared from ethylene-treated Arabidopsis seedlings with 35S-labeled CaM. A cDNA clone, EICBP (Ethylene-Induced CaM Binding Protein), encoding a protein that interacts with activated CaM was isolated in this screening. The CaM binding domain in EICBP was mapped to the C-terminus of the protein. These results indicate that calcium, through CaM, could regulate the activity of EICBP. The EICBP is expressed in different tissues and its expression in seedlings is induced by ethylene. The EICBP contains, in addition to a CaM binding domain, several features that are typical of transcription factors. These include a DNA-binding domain at the N terminus, an acidic region at the C terminus, and nuclear localization signals. In database searches a partial cDNA (CG-1) encoding a DNA-binding motif from parsley and an ethylene up-regulated partial cDNA from tomato (ER66) showed significant similarity to EICBP. In addition, five hypothetical proteins in the Arabidopsis genome also showed a very high sequence similarity with EICBP, indicating that there are several EICBP-related proteins in Arabidopsis. The structural features of EICBP are conserved in all EICBP-related proteins in Arabidopsis, suggesting that they may constitute a new family of DNA binding proteins and are likely to be involved in modulating gene expression in the presence of ethylene.

  11. Isolation and characterizations of oxalate-binding proteins in the kidney

    SciTech Connect

    Roop-ngam, Piyachat; Chaiyarit, Sakdithep; Pongsakul, Nutkridta; Thongboonkerd, Visith

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer The first large-scale characterizations of oxalate-binding kidney proteins. Black-Right-Pointing-Pointer The recently developed oxalate-conjugated EAH Sepharose 4B beads were applied. Black-Right-Pointing-Pointer 38 forms of 26 unique oxalate-binding kidney proteins were identified. Black-Right-Pointing-Pointer 25/26 (96%) of identified proteins had 'L-x(3,5)-R-x(2)-[AGILPV]' domain. -- Abstract: Oxalate-binding proteins are thought to serve as potential modulators of kidney stone formation. However, only few oxalate-binding proteins have been identified from previous studies. Our present study, therefore, aimed for large-scale identification of oxalate-binding proteins in porcine kidney using an oxalate-affinity column containing oxalate-conjugated EAH Sepharose 4B beads for purification followed by two-dimensional gel electrophoresis (2-DE) to resolve the recovered proteins. Comparing with those obtained from the controlled column containing uncoupled EAH-Sepharose 4B (to subtract the background of non-specific bindings), a total of 38 protein spots were defined as oxalate-binding proteins. These protein spots were successfully identified by quadrupole time-of-flight mass spectrometry (MS) and/or tandem MS (MS/MS) as 26 unique proteins, including several nuclear proteins, mitochondrial proteins, oxidative stress regulatory proteins, metabolic enzymes and others. Identification of oxalate-binding domain using the PRATT tool revealed 'L-x(3,5)-R-x(2)-[AGILPV]' as a functional domain responsible for oxalate-binding in 25 of 26 (96%) unique identified proteins. We report herein, for the first time, large-scale identification and characterizations of oxalate-binding proteins in the kidney. The presence of positively charged arginine residue in the middle of this functional domain suggested its significance for binding to the negatively charged oxalate. These data will enhance future stone research, particularly on stone

  12. Bacterial chitin binding proteins show differential substrate binding and synergy with chitinases.

    PubMed

    Manjeet, Kaur; Purushotham, Pallinti; Neeraja, Chilukoti; Podile, Appa Rao

    2013-08-25

    Glycosyl hydrolase (GH) family 18 chitinases (Chi) and family 33 chitin binding proteins (CBPs) from Bacillus thuringiensis serovar kurstaki (BtChi and BtCBP), B. licheniformis DSM13 (BliChi and BliCBP) and Serratia proteamaculans 568 (SpChiB and SpCBP21) were used to study the efficiency and synergistic action of BtChi, BliChi and SpChiB individually with BtCBP, BliCBP or SpCBP21. Chitinase assay revealed that only BtChi and SpChiB showed synergism in hydrolysis of chitin, while there was no increase in products generated by BliChi, in the presence of the three above mentioned CBPs. This suggests that some (specific) CBPs are able to exert a synergistic effect on (specific) chitinases. A mutant of BliChi, designated as BliGH, was constructed by deleting the C-terminal fibronectin III (FnIII) and carbohydrate binding module 5 (CBM5) to assess the contribution of FnIII and CBM5 domains in the synergistic interactions of GH18 chitinases with CBPs. Chitinase assay with BliGH revealed that the accessory domains play a major role in making BliChi an efficient enzyme. We studied binding of BtCBP and BliCBP to α- and β-chitin. The BtCBP, BliCBP or SpCBP21 did not act synergistically with chitinases in hydrolysis of the chitin, interspersed with other polymers, present in fungal cell walls.

  13. PIP3-binding proteins promote age-dependent protein aggregation and limit survival in C. elegans.

    PubMed

    Ayyadevara, Srinivas; Balasubramaniam, Meenakshisundaram; Johnson, Jay; Alla, Ramani; Mackintosh, Samuel G; Shmookler Reis, Robert J

    2016-08-02

    Class-I phosphatidylinositol 3-kinase (PI3KI) converts phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3). PIP3 comprises two fatty-acid chains that embed in lipid-bilayer membranes, joined by glycerol to inositol triphosphate. Proteins with domains that specifically bind that head-group (e.g. pleckstrin-homology [PH] domains) are thus tethered to the inner plasma-membrane surface where they have an enhanced likelihood of interaction with other PIP3-bound proteins, in particular other components of their signaling pathways. Null alleles of the C. elegans age-1 gene, encoding the catalytic subunit of PI3KI, lack any detectable class-I PI3K activity and so cannot form PIP3. These mutant worms survive almost 10-fold longer than the longest-lived normal control, and are highly resistant to a variety of stresses including oxidative and electrophilic challenges. Traits associated with age-1 mutation are widely believed to be mediated through AKT-1, which requires PIP3 for both tethering and activation. Active AKT complex phosphorylates and thereby inactivates the DAF-16/FOXO transcription factor. However, extensive evidence indicates that pleiotropic effects of age-1-null mutations, including extreme longevity, cannot be explained by insulin like-receptor/AKT/FOXO signaling alone, suggesting involvement of other PIP3-binding proteins. We used ligand-affinity capture to identify membrane-bound proteins downstream of PI3KI that preferentially bind PIP3. Computer modeling supports a subset of candidate proteins predicted to directly bind PIP3 in preference to PIP2, and functional testing by RNAi knockdown confirmed candidates that partially mediate the stress-survival, aggregation-reducing and longevity benefits of PI3KI disruption. PIP3-specific candidate sets are highly enriched for proteins previously reported to affect translation, stress responses, lifespan, proteostasis, and lipid transport.

  14. Characterization and properties of steroid binding protein in Bufo arenarum serum.

    PubMed

    Fernández, S N; Mansilla-Whitacre, Z C; Miceli, D C

    1994-08-01

    Serum steroid binding properties of mature Bufo arenarum females were studied. Binding data obtained using charcoal adsorption assay and equilibrium dialysis methods indicates a single protein, named Bufo arenarum sex binding protein (Ba SBP), which binds 5 alpha-dihydrotestosterone (DHT), testosterone (T), and estradiol-17 beta (E2) with high affinity (10(-7) M-1 - 10(8) M-1) and fair capacity (10(-6) M). Scatchard plot analysis demonstrated the coexistence of two binding sites. Ba SBP has a sedimentation coefficient of 5.2 S in sucrose gradient centrifugation in low salt and under steady-state conditions. The specificity of this protein, determined by competitive binding experiments, is comparable to human SBP. DHT and T bind with higher affinity than E2. Estriol and estrone competed poorly, while diethylstilbestrol and C21 steroids did not compete. The binding capacity of this protein is under estrogenic control.

  15. COVALENT BINDING OF TRICHLOROETHYLENE TO PROTEINS IN HUMAN AND RAT HEPATOCYTES. (R826409)

    EPA Science Inventory

    The environmental contaminant and occupational solvent trichloroethylene is metabolized to a reactive intermediate that covalently binds to specific hepatic proteins in exposed mice and rats. In order to compare covalent binding between humans and rodents, primary hepatocyte c...

  16. Intestinal calcium-binding protein 3 months after massive small bowel resection in the piglet.

    PubMed

    Margolis, A; Ricour, C; Harouchi, A; Guyot, M; Laouari, D; Balsan, S

    1977-12-01

    Changes in intestinal calcium-binding protein and calcium binding activity were studied at resection and 3 months after 90% small bowel resection in piglets and one adult pig. A calcium-binding protein (MW congruent to 11.000) with calcium-dependent eletrophoretic mobility was partially purified from mucosal extract of proximal jejunum, mid-gut, and ileum. The concentration of calcium-binding protein and the calcium-binding activity of the intact animals were found highest in the proximal jejunal segment, lowest in the ileal segment. After resection in the four surviving animals out of nine, a significant increase in calcium-binding activity was observed in the proximal jejunum and in the distal ileal segment. The change in calcium-binding activity was much more marked in the ileum than the jejunum. These data demonstrate that pig intestinal mucosa possesses an adaptive capacity to increase the synthesis of calcium-binding protein after massive small bowel resection.

  17. Screening of the Binding of Small Molecules to Proteins by Desorption Electrospray Ionization Mass Spectrometry Combined with Protein Microarray

    NASA Astrophysics Data System (ADS)

    Yao, Chenxi; Wang, Tao; Zhang, Buqing; He, Dacheng; Na, Na; Ouyang, Jin

    2015-11-01

    The interaction between bioactive small molecule ligands and proteins is one of the important research areas in proteomics. Herein, a simple and rapid method is established to screen small ligands that bind to proteins. We designed an agarose slide to immobilize different proteins. The protein microarrays were allowed to interact with different small ligands, and after washing, the microarrays were screened by desorption electrospray ionization mass spectrometry (DESI MS). This method can be applied to screen specific protein binding ligands and was shown for seven proteins and 34 known ligands for these proteins. In addition, a high-throughput screening was achieved, with the analysis requiring approximately 4 s for one sample spot. We then applied this method to determine the binding between the important protein matrix metalloproteinase-9 (MMP-9) and 88 small compounds. The molecular docking results confirmed the MS results, demonstrating that this method is suitable for the rapid and accurate screening of ligands binding to proteins.

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

    PubMed

    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.

  19. Ligand clouds around protein clouds: a scenario of ligand binding with intrinsically disordered proteins.

    PubMed

    Jin, Fan; Yu, Chen; Lai, Luhua; Liu, Zhirong

    2013-01-01

    Intrinsically disordered proteins (IDPs) were found to be widely associated with human diseases and may serve as potential drug design targets. However, drug design targeting IDPs is still in the very early stages. Progress in drug design is usually achieved using experimental screening; however, the structural disorder of IDPs makes it difficult to characterize their interaction with ligands using experiments alone. To better understand the structure of IDPs and their interactions with small molecule ligands, we performed extensive simulations on the c-Myc₃₇₀₋₄₀₉ peptide and its binding to a reported small molecule inhibitor, ligand 10074-A4. We found that the conformational space of the apo c-Myc₃₇₀₋₄₀₉ peptide was rather dispersed and that the conformations of the peptide were stabilized mainly by charge interactions and hydrogen bonds. Under the binding of the ligand, c-Myc₃₇₀₋₄₀₉ remained disordered. The ligand was found to bind to c-Myc₃₇₀₋₄₀₉ at different sites along the chain and behaved like a 'ligand cloud'. In contrast to ligand binding to more rigid target proteins that usually results in a dominant bound structure, ligand binding to IDPs may better be described as ligand clouds around protein clouds. Nevertheless, the binding of the ligand and a non-ligand to the c-Myc₃₇₀₋₄₀₉ target could be clearly distinguished. The present study provides insights that will help improve rational drug design that targets IDPs.

  20. A fusicoccin binding protein belongs to the family of 14-3-3 brain protein homologs.

    PubMed Central

    Korthout, H A; de Boer, A H

    1994-01-01

    The fusicoccin binding protein (FCBP) is a highly conserved plasma membrane protein present in all higher plants tested thus far. It exhibits high- and low-affinity binding for the fungal toxin fusicoccin (FC). We purified the active FCBP from a fraction highly enriched in plasma membrane by selective precipitation and anion exchange chromatography. After SDS-PAGE, the two FCBP subunits of 30 and 31 kD were detected as major bands. Amino acid sequence analysis of the 31-kD polypeptide displayed a high degree of identity with so-called 14-3-3 proteins, a class of mammalian brain proteins initially described as regulators of neurotransmitter synthesis and protein kinase C inhibitors. Thereafter, we affinity purified the 30- and 31-kD FCBP subunits, using biotinylated FC in combination with a monomeric avidin column. Immunodecoration of these 30- and 31-kD FCBP subunits with polyclonal antibodies raised against a 14-3-3 homolog from yeast confirmed the identity of the FCBP as a 14-3-3 homolog. Similar to all 14-3-3 protein homologs, the FCBP seems to exist as a dimer in native form. Thus far, the FCBP is the only 14-3-3 homolog with a receptor-like function. The conserved structure of the 14-3-3 protein family is a further indication that the FCBP plays an important role in the physiology of higher plants. PMID:7827499

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

    SciTech Connect

    Wu, Chen; Yao, Guangyin; Zou, Minji; Chen, Guangyu; Wang, Min; Liu, Jingqian; Wang, Jiaxi; Xu, Donggang . E-mail: xudg@nic.bmi.ac.cn

    2007-06-01

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

  2. Convolutional neural network architectures for predicting DNA–protein binding

    PubMed Central

    Zeng, Haoyang; Edwards, Matthew D.; Liu, Ge; Gifford, David K.

    2016-01-01

    Motivation: Convolutional neural networks (CNN) have outperformed conventional methods in modeling the sequence specificity of DNA–protein binding. Yet inappropriate CNN architectures can yield poorer performance than simpler models. Thus an in-depth understanding of how to match CNN architecture to a given task is needed to fully harness the power of CNNs for computational biology applications. Results: We present a systematic exploration of CNN architectures for predicting DNA sequence binding using a large compendium of transcription factor datasets. We identify the best-performing architectures by varying CNN width, depth and pooling designs. We find that adding convolutional kernels to a network is important for motif-based tasks. We show the benefits of CNNs in learning rich higher-order sequence features, such as secondary motifs and local sequence context, by comparing network performance on multiple modeling tasks ranging in difficulty. We also demonstrate how careful construction of sequence benchmark datasets, using approaches that control potentially confounding effects like positional or motif strength bias, is critical in making fair comparisons between competing methods. We explore how to establish the sufficiency of training data for these learning tasks, and we have created a flexible cloud-based framework that permits the rapid exploration of alternative neural network architectures for problems in computational biology. Availability and Implementation: All the models analyzed are available at http://cnn.csail.mit.edu. Contact: gifford@mit.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27307608

  3. Z-DNA binding protein from chicken blood nuclei

    NASA Technical Reports Server (NTRS)

    Herbert, A. G.; Spitzner, J. R.; Lowenhaupt, K.; Rich, A.

    1993-01-01

    A protein (Z alpha) that appears to be highly specific for the left-handed Z-DNA conformer has been identified in chicken blood nuclear extracts. Z alpha activity is measured in a band-shift assay by using a radioactive probe consisting of a (dC-dG)35 oligomer that has 50% of the deoxycytosines replaced with 5-bromodeoxycytosine. In the presence of 10 mM Mg2+, the probe converts to the Z-DNA conformation and is bound by Z alpha. The binding of Z alpha to the radioactive probe is specifically blocked by competition with linear poly(dC-dG) stabilized in the Z-DNA form by chemical bromination but not by B-form poly(dC-dG) or boiled salmon-sperm DNA. In addition, the binding activity of Z alpha is competitively blocked by supercoiled plasmids containing a Z-DNA insert but not by either the linearized plasmid or by an equivalent amount of the parental supercoiled plasmid without the Z-DNA-forming insert. Z alpha can be crosslinked to the 32P-labeled brominated probe with UV light, allowing us to estimate that the minimal molecular mass of Z alpha is 39 kDa.

  4. A WD-Repeat Protein Stabilizes ORC Binding to Chromatin

    PubMed Central

    Shen, Zhen; Sathyan, Kizhakke M.; Geng, Yijie; Zheng, Ruiping; Chakraborty, Arindam; Freeman, Brian; Wang, Fei; Prasanth, Kannanganattu V.; Prasanth, Supriya G.

    2015-01-01

    SUMMARY Origin recognition complex (ORC) plays critical roles in the initiation of DNA replication and cell-cycle progression. In metazoans, ORC associates with origin DNA during G1 and with heterochromatin in postreplicated cells. However, what regulates the binding of ORC to chromatin is not understood. We have identified a highly conserved, leucine-rich repeats and WD40 repeat domain-containing protein 1 (LRWD1) or ORC-associated (ORCA) in human cells that interacts with ORC and modulates chromatin association of ORC. ORCA colocalizes with ORC and shows similar cell-cycle dynamics. We demonstrate that ORCA efficiently recruits ORC to chromatin. Depletion of ORCA in human primary cells and embryonic stem cells results in loss of ORC association to chromatin, concomitant reduction of MCM binding, and a subsequent accumulation in G1 phase. Our results suggest ORCA-mediated association of ORC to chromatin is critical to initiate preRC assembly in G1 and chromatin organization in post-G1 cells. PMID:20932478

  5. Small Molecules Engage Hot Spots through Cooperative Binding To Inhibit a Tight Protein-Protein Interaction.

    PubMed

    Liu, Degang; Xu, David; Liu, Min; Knabe, William Eric; Yuan, Cai; Zhou, Donghui; Huang, Mingdong; Meroueh, Samy O

    2017-03-28

    Protein-protein interactions drive every aspect of cell signaling, yet only a few small-molecule inhibitors of these interactions exist. Despite our ability to identify critical residues known as hot spots, little is known about how to effectively engage them to disrupt protein-protein interactions. Here, we take advantage of the ease of preparation and stability of pyrrolinone 1, a small-molecule inhibitor of the tight interaction between the urokinase receptor (uPAR) and its binding partner, the urokinase-type plasminogen activator uPA, to synthesize more than 40 derivatives and explore their effect on the protein-protein interaction. We report the crystal structure of uPAR bound to previously discovered pyrazole 3 and to pyrrolinone 12. While both 3 and 12 bind to uPAR and compete with a fluorescently labeled peptide probe, only 12 and its derivatives inhibit the full uPAR·uPA interaction. Compounds 3 and 12 mimic and engage different hot-spot residues on uPA and uPAR, respectively. Interestingly, 12 is involved in a π-cation interaction with Arg-53, which is not considered a hot spot. Explicit-solvent molecular dynamics simulations reveal that 3 and 12 exhibit dramatically different correlations of motion with residues on uPAR. Free energy calculations for the wild-type and mutant uPAR bound to uPA or 12 show that Arg-53 interacts with uPA or with 12 in a highly cooperative manner, thereby altering the contributions of hot spots to uPAR binding. The direct engagement of peripheral residues not considered hot spots through π-cation or salt-bridge interactions could provide new opportunities for enhanced small-molecule engagement of hot spots to disrupt challenging protein-protein interactions.

  6. Dynamical DNA accessibility induced by chromatin remodeling and protein binding

    NASA Astrophysics Data System (ADS)

    Montel, F.; Faivre-Moskalenko, C.; Castelnovo, M.

    2014-11-01

    Chromatin remodeling factors are enzymes being able to alter locally chromatin structure at the nucleosomal level and they actively participate in the regulation of gene expression. Using simple rules for individual nucleosome motion induced by a remodeling factor, we designed simulations of the remodeling of oligomeric chromatin, in order to address quantitatively collective effects in DNA accessibility upon nucleosome mobilization. Our results suggest that accessibility profiles are inhomogeneous thanks to borders effects like protein binding. Remarkably, we show that the accessibility lifetime of DNA sequence is roughly doubled in the vicinity of borders as compared to its value in bulk regions far from the borders. These results are quantitatively interpreted as resulting from the confined diffusion of a large nucleosome depleted region.

  7. RNA Binding Proteins in the miRNA Pathway.

    PubMed

    Connerty, Patrick; Ahadi, Alireza; Hutvagner, Gyorgy

    2015-12-26

    microRNAs (miRNAs) are short ~22 nucleotides (nt) ribonucleic acids which post-transcriptionally regulate gene expression. miRNAs are key regulators of all cellular processes, and the correct expression of miRNAs in an organism is crucial for proper development and cellular function. As a result, the miRNA biogenesis pathway is highly regulated. In this review, we outline the basic steps of miRNA biogenesis and miRNA mediated gene regulation focusing on the role of RNA binding proteins (RBPs). We also describe multiple mechanisms that regulate the canonical miRNA pathway, which depends on a wide range of RBPs. Moreover, we hypothesise that the interaction between miRNA regulation and RBPs is potentially more widespread based on the analysis of available high-throughput datasets.

  8. RNA-Binding Proteins: Splicing Factors and Disease

    PubMed Central

    Fredericks, Alger M.; Cygan, Kamil J.; Brown, Brian A.; Fairbrother, William G.

    2015-01-01

    Pre-mRNA splicing is mediated by interactions of the Core Spliceosome and an array of accessory RNA binding proteins with cis-sequence elements. Splicing is a major regulatory component in higher eukaryotes. Disruptions in splicing are a major contributor to human disease. One in three hereditary disease alleles are believed to cause aberrant splicing. Hereditary disease alleles can alter splicing by disrupting a splicing element, creating a toxic RNA, or affecting splicing factors. One of the challenges of medical genetics is identifying causal variants from the thousands of possibilities discovered in a clinical sequencing experiment. Here we review the basic biochemistry of splicing, the mechanisms of splicing mutations, the methods for identifying splicing mutants, and the potential of therapeutic interventions. PMID:25985083

  9. Glycosylation status of vitamin D binding protein in cancer patients

    PubMed Central

    Rehder, Douglas S; Nelson, Randall W; Borges, Chad R

    2009-01-01

    On the basis of the results of activity studies, previous reports have suggested that vitamin D binding protein (DBP) is significantly or even completely deglycosylated in cancer patients, eliminating the molecular precursor of the immunologically important Gc macrophage activating factor (GcMAF), a glycosidase-derived product of DBP. The purpose of this investigation was to directly determine the relative degree of O-linked trisaccharide glycosylation of serum-derived DBP in human breast, colorectal, pancreatic, and prostate cancer patients. Results obtained by electrospray ionization-based mass spectrometric immunoassay showed that there was no significant depletion of DBP trisaccharide glycosylation in the 56 cancer patients examined relative to healthy controls. These results suggest that alternative hypotheses regarding the molecular and/or structural origins of GcMAF must be considered to explain the relative inability of cancer patient serum to activate macrophages. PMID:19642159

  10. Glycosylation status of vitamin D binding protein in cancer patients.

    PubMed

    Rehder, Douglas S; Nelson, Randall W; Borges, Chad R

    2009-10-01

    On the basis of the results of activity studies, previous reports have suggested that vitamin D binding protein (DBP) is significantly or even completely deglycosylated in cancer patients, eliminating the molecular precursor of the immunologically important Gc macrophage activating factor (GcMAF), a glycosidase-derived product of DBP. The purpose of this investigation was to directly determine the relative degree of O-linked trisaccharide glycosylation of serum-derived DBP in human breast, colorectal, pancreatic, and prostate cancer patients. Results obtained by electrospray ionization-based mass spectrometric immunoassay showed that there was no significant depletion of DBP trisaccharide glycosylation in the 56 cancer patients examined relative to healthy controls. These results suggest that alternative hypotheses regarding the molecular and/or structural origins of GcMAF must be considered to explain the relative inability of cancer patient serum to activate macrophages.

  11. Protein binding properties of surface-modified porous polyethylene membranes.

    PubMed

    Greene, George; Radhakrishna, Harish; Tannenbaum, Rina

    2005-10-01

    In this study, we quantified the adsorption of immunoglobulin G (IgG) protein onto several polyelectrolyte-modified sintered porous polyethylene (PPE) membranes. The polymer surfaces had both cationic and anionic charges obtained via the adsorption of polyethylenimine (PEI) and polyacrylic acid (PAA), respectively, onto plasma-activated PPE. The amount of IgG adsorption was determined by measuring the gamma radiation emitted by [125I]-IgG radio labeled protein. By studying the impact of pH and ionic strength on IgG adsorption, we attempted to characterize the role and nature of the electrostatic interactions involved in the adsorption process to better understand how these interactions were influenced by the charge and structure of immobilized polyelectrolyte complexes at modified membrane surfaces. We were able to show that surface modification of PPE membranes with adsorbed PEI monolayers and PEI-PAA bilayers can greatly improve the IgG binding ability of the membrane under optimized conditions. We also showed that the observed improvement in the IgG binding is derived from electrostatic interactions between IgG and the polyelectrolyte surface. In addition, we found that the greatest IgG adsorption occurred when the IgG and the surface possessed predominantly opposite charges, rather than when the surface possessed the greatest electrostatic charge. Finally, we have found that the molecular weight of the terminating polyelectrolyte has a noticeable effect upon the electrostatic interactions between IgG and the PEI-PAA bilayer-modified PPE surfaces.

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

    PubMed

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

    2012-10-15

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

  13. Acanthamoeba castellanii contains a ribosomal RNA enhancer binding protein which stimulates TIF-IB binding and transcription under stringent conditions.

    PubMed Central

    Yang, Q; Radebaugh, C A; Kubaska, W; Geiss, G K; Paule, M R

    1995-01-01

    The intergenic spacer (IGS) of Acanthamoeba castellanii rRNA genes contains repeated elements which are weak enhancers for transcription by RNA polymerase I. A protein, EBF, was identified and partially purified which binds to the enhancers and to several other sequences within the IGS, but not to other DNA fragments, including the rRNA core promoter. No consensus binding sequence could be discerned in these fragments and bound factor is in rapid equilibrium with unbound. EBF has functional characteristics similar to vertebrate upstream binding factors (UBF). Not only does it bind to the enhancer and other IGS elements, but it also stimulates binding of TIF-IB, the fundamental transcription initiation factor, to the core promoter and stimulates transcription from the promoter. Attempts to identify polypeptides with epitopes similar to rat or Xenopus laevis UBF suggest that structurally the protein from A.castellanii is not closely related to vertebrate UBF. Images PMID:7501455

  14. Acanthamoeba castellanii contains a ribosomal RNA enhancer binding protein which stimulates TIF-IB binding and transcription under stringent conditions.

    PubMed

    Yang, Q; Radebaugh, C A; Kubaska, W; Geiss, G K; Paule, M R

    1995-11-11

    The intergenic spacer (IGS) of Acanthamoeba castellanii rRNA genes contains repeated elements which are weak enhancers for transcription by RNA polymerase I. A protein, EBF, was identified and partially purified which binds to the enhancers and to several other sequences within the IGS, but not to other DNA fragments, including the rRNA core promoter. No consensus binding sequence could be discerned in these fragments and bound factor is in rapid equilibrium with unbound. EBF has functional characteristics similar to vertebrate upstream binding factors (UBF). Not only does it bind to the enhancer and other IGS elements, but it also stimulates binding of TIF-IB, the fundamental transcription initiation factor, to the core promoter and stimulates transcription from the promoter. Attempts to identify polypeptides with epitopes similar to rat or Xenopus laevis UBF suggest that structurally the protein from A.castellanii is not closely related to vertebrate UBF.

  15. Structural basis for cooperative DNA binding by two dimers of the multidrug-binding protein QacR

    PubMed Central

    Schumacher, Maria A.; Miller, Marshall C.; Grkovic, Steve; Brown, Melissa H.; Skurray, Ronald A.; Brennan, Richard G.

    2002-01-01

    The Staphylococcus aureus multidrug-binding protein QacR represses transcription of the qacA multidrug transporter gene and is induced by multiple structurally dissimilar drugs. QacR is a member of the TetR/CamR family of transcriptional regulators, which share highly homologous N-terminal DNA-binding domains connected to seemingly non-homologous ligand-binding domains. Unlike other TetR members, which bind ∼15 bp operators, QacR recognizes an unusually long 28 bp operator, IR1, which it appears to bind cooperatively. To elucidate the DNA-binding mechanism of QacR, we determined the 2.90 Å resolution crystal structure of a QacR–IR1 complex. Strikingly, our data reveal that the DNA recognition mode of QacR is distinct from TetR and involves the binding of a pair of QacR dimers. In this unique binding mode, recognition at each IR1 half-site is mediated by a complement of DNA contacts made by two helix–turn–helix motifs. The inferred cooperativity does not arise from cross-dimer protein–protein contacts, but from the global undertwisting and major groove widening elicited by the binding of two QacR dimers. PMID:11867549

  16. A calmodulin-binding/CGCG box DNA-binding protein family involved in multiple signaling pathways in plants

    NASA Technical Reports Server (NTRS)

    Yang, Tianbao; Poovaiah, B. W.

    2002-01-01

    We reported earlier that the tobacco early ethylene-responsive gene NtER1 encodes a calmodulin-binding protein (Yang, T., and Poovaiah, B. W. (2000) J. Biol. Chem. 275, 38467-38473). Here we demonstrate that there is one NtER1 homolog as well as five related genes in Arabidopsis. These six genes are rapidly and differentially induced by environmental signals such as temperature extremes, UVB, salt, and wounding; hormones such as ethylene and abscisic acid; and signal molecules such as methyl jasmonate, H(2)O(2), and salicylic acid. Hence, they were designated as AtSR1-6 (Arabidopsis thaliana signal-responsive genes). Ca(2+)/calmodulin binds to all AtSRs, and their calmodulin-binding regions are located on a conserved basic amphiphilic alpha-helical motif in the C terminus. AtSR1 targets the nucleus and specifically recognizes a novel 6-bp CGCG box (A/C/G)CGCG(G/T/C). The multiple CGCG cis-elements are found in promoters of genes such as those involved in ethylene signaling, abscisic acid signaling, and light signal perception. The DNA-binding domain in AtSR1 is located on the N-terminal 146 bp where all AtSR1-related proteins share high similarity but have no similarity to other known DNA-binding proteins. The calmodulin-binding nuclear proteins isolated from wounded leaves exhibit specific CGCG box DNA binding activities. These results suggest that the AtSR gene family encodes a family of calmodulin-binding/DNA-binding proteins involved in multiple signal transduction pathways in plants.

  17. The ubiquitous octamer-binding protein(s) is sufficient for transcription of immunoglobulin genes.

    PubMed Central

    Johnson, D G; Carayannopoulos, L; Capra, J D; Tucker, P W; Hanke, J H

    1990-01-01

    All immunoglobulin genes contain a conserved octanucleotide promoter element, ATGCAAAT, which has been shown to be required for their normal B-cell-specific transcription. Proteins that bind this octamer have been purified, and cDNAs encoding octamer-binding proteins have been cloned. Some of these proteins (referred to as OTF-2) are lymphoid specific, whereas at least one other, and possibly more (referred to as OTF-1), is found ubiquitously in all cell types. The exact role of these different proteins in directing the tissue-specific expression of immunoglobulin genes is unclear. We have identified two human pre-B-cell lines that contain extremely low levels of OTF-2 yet still express high levels of steady-state immunoglobulin heavy-chain mRNA in vivo and efficiently transcribe an immunoglobulin gene in vitro. Addition of a highly enriched preparation of OTF-1 made from one of these pre-B cells or from HeLa cells specifically stimulated in vitro transcription of an immunoglobulin gene. Furthermore, OFT-1 appeared to have approximately the same transactivation ability as OTF-2 when normalized for binding activity. These results suggest that OTF-1, without OTF-2, is sufficient for transcription of immunoglobulin genes and that OTF-2 alone is not responsible for the B-cell-specific regulation of immunoglobulin gene expression. Images PMID:2304473

  18. Macromolecular crowding effects on protein-protein binding affinity and specificity

    NASA Astrophysics Data System (ADS)

    Kim, Young C.; Best, Robert B.; Mittal, Jeetain

    2010-11-01

    Macromolecular crowding in cells is recognized to have a significant impact on biological function, yet quantitative models for its effects are relatively undeveloped. The influence of crowding on protein-protein interactions is of particular interest, since these mediate many processes in the cell, including the self-assembly of larger complexes, recognition, and signaling. We use a residue-level coarse-grained model to investigate the effects of macromolecular crowding on the assembly of protein-protein complexes. Interactions between the proteins are treated using a fully transferable energy function, and interactions of protein residues with the spherical crowders are repulsive. We show that the binding free energy for two protein complexes, ubiquitin/UIM1 and cytochrome c/cytochrome c peroxidase, decreases modestly as the concentration of crowding agents increases. To obtain a quantitative description of the stabilizing effect, we map the aspherical individual proteins and protein complexes onto spheres whose radii are calculated from the crowder-excluded protein volumes. With this correspondence, we find that the change in the binding free energy due to crowding can be quantitatively described by the scaled particle theory model without any fitting parameters. The effects of a mixture of different-size crowders—as would be found in a real cell—are predicted by the same model with an additivity ansatz. We also obtain the remarkable result that crowding increases the fraction of specific complexes at the expense of nonspecific transient encounter complexes in a crowded environment. This result, due to the greater excluded volume of the nonspecific complexes, demonstrates that macromolecular crowding can have subtle functional effects beyond the relative stability of bound and unbound complexes.

  19. Characterization of a mitochondrial protein binding to single-stranded DNA.

    PubMed Central

    Mignotte, B; Barat, M; Mounolou, J C

    1985-01-01

    A DNA-binding protein from Xenopus laevis oocyte mitochondria which has been found associated with the D-loop also shows a strong preference for single-stranded DNA. The binding to polynucleotides is dependent on the base composition, but no sequence specificity was found. This protein, called mtSSB, binds tightly and cooperatively to single-stranded DNA. By its amino-acid composition and its binding properties it appears to be similar to the single-stranded DNA-binding proteins found in prokaryotes. PMID:4039816

  20. Visualization of coupled protein folding and binding in bacteria and purification of the heterodimeric complex

    PubMed Central

    Wang, Haoyong; Chong, Shaorong

    2003-01-01

    During overexpression of recombinant proteins in Escherichia coli, misfolded proteins often aggregate and form inclusion bodies. If an aggregation-prone recombinant protein is fused upstream (as an N-terminal fusion) to GFP, aggregation of the recombinant protein domain also leads to misfolding of the downstream GFP domain, resulting in a decrease or loss of fluorescence. We investigated whether the GFP domain could fold correctly if aggregation of the upstream protein domain was prevented in vivo by a coupled protein folding and binding interaction. Such interaction has been previously shown to occur between the E. coli integration host factors α and β, and between the domains of the general transcriptional coactivator cAMP response element binding protein (CREB)-binding protein and the activator for thyroid hormone and retinoid receptors. In this study, fusion of integration host factor β or the CREB-binding protein domain upstream to GFP resulted in aggregation of the fusion protein. Coexpression of their respective partners, on the other hand, allowed soluble expression of the fusion protein and a dramatic increase in fluorescence. The study demonstrated that coupled protein folding and binding could be correlated to GFP fluorescence. A modified miniintein containing an affinity tag was inserted between the upstream protein domain and GFP to allow rapid purification and identification of the heterodimeric complex. The GFP coexpression fusion system may be used to identify novel protein–protein interactions that involve coupled folding and binding or protein partners that can solubilize aggregation-prone recombinant proteins. PMID:12515863

  1. Identification of residues in the human guanylate-binding protein 1 critical for nucleotide binding and cooperative GTP hydrolysis.

    PubMed

    Praefcke, Gerrit J K; Kloep, Stephan; Benscheid, Utz; Lilie, Hauke; Prakash, Balaji; Herrmann, Christian

    2004-11-12

    The guanylate-binding proteins (GBPs) form a group of interferon-gamma inducible GTP-binding proteins which belong to the family of dynamin-related proteins. Like other members of this family, human guanylate-binding protein 1 (hGBP1) shows nucleotide-dependent oligomerisation that stimulates the GTPase activity of the protein. A unique feature of the GBPs is their ability to hydrolyse GTP to GDP and GMP. In order to elucidate the relationship between these findings, we designed point mutants in the phosphate-binding loop (P-loop) as well as in the switch I and switch II regions of the protein based on the crystal structure of hGBP1. These mutant proteins were analysed for their interaction with guanine nucleotides labeled with a fluorescence dye and for their ability to hydrolyse GTP in a cooperative manner. We identified mutations of amino acid residues that decrease GTPase activity by orders of magnitude a part of which are conserved in GTP-binding proteins. In addition, mutants in the P-loop were characterized that strongly impair binding of nucleotide. In consequence, together with altered GTPase activity and given cellular nucleotide concentrations this results in hGBP1 mutants prevailingly resting in the nucleotide-free (K51A and S52N) or the GTP bound form (R48A), respectively. Using size-exclusion chromatography and analytical ultracentrifugation we addressed the impact on protein oligomerisation. In summary, mutants of hGBP1 were identified and biochemically characterized providing hGBP1 locked in defined states in order to investigate their functional role in future cell biology studies.

  2. Membrane-Protein Binding Measured with Solution-Phase Plasmonic Nanocube Sensors

    PubMed Central

    Wu, Hung-Jen; Henzie, Joel; Lin, Wan-Chen; Rhodes, Christopher; Li, Zhu; Sartorel, Elodie; Thorner, Jeremy; Yang, Peidong; Groves, Jay. T.

    2013-01-01

    We describe a solution-phase sensor of lipid-protein binding based on localized surface plasmon resonance (LSPR) of silver nanocubes. When silica-coated nanocubes are mixed into a suspension of lipid vesicles, supported membranes spontaneously assemble on their surfaces. Using a standard laboratory spectrophotometer, we calibrate the LSPR peak shift due to protein binding to the membrane surface and then characterize the lipid-binding specificity of a pleckstrin-homology domain protein. PMID:23085614

  3. Regulating Prostate Cancer Sensitivity to Chemotherapy through Translational Control of CCAAT Enhancer Binding Proteins

    DTIC Science & Technology

    2015-08-01

    Binding Proteins PRINCIPAL INVESTIGATOR: David J. Barakat Ph.D. CONTRACTING ORGANIZATION: Johns Hopkins University Baltimore, MD 21205-1832...Enhancer Binding Proteins 5a. CONTRACT NUMBER W81XWH-14-1-0209 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) David J. Barakat, Samuel R... binding protein (C/EBP) family are regulated through translation mechanisms by mTOR activity and have been linked to PCa survival and metastatic gene

  4. QM/MM Molecular Dynamics Studies of Metal Binding Proteins

    PubMed Central

    Vidossich, Pietro; Magistrato, Alessandra

    2014-01-01

    Mixed quantum-classical (quantum mechanical/molecular mechanical (QM/MM)) simulations have strongly contributed to providing insights into the understanding of several structural and mechanistic aspects of biological molecules. They played a particularly important role in metal binding proteins, where the electronic effects of transition metals have to be explicitly taken into account for the correct representation of the underlying biochemical process. In this review, after a brief description of the basic concepts of the QM/MM method, we provide an overview of its capabilities using selected examples taken from our work. Specifically, we will focus on heme peroxidases, metallo-β-lactamases, α-synuclein and ligase ribozymes to show how this approach is capable of describing the catalytic and/or structural role played by transition (Fe, Zn or Cu) and main group (Mg) metals. Applications will reveal how metal ions influence the formation and reduction of high redox intermediates in catalytic cycles and enhance drug metabolism, amyloidogenic aggregate formation and nucleic acid synthesis. In turn, it will become manifest that the protein frame directs and modulates the properties and reactivity of the metal ions. PMID:25006697

  5. Pathophysiological role of guanylate-binding proteins in gastrointestinal diseases.

    PubMed

    Britzen-Laurent, Nathalie; Herrmann, Christian; Naschberger, Elisabeth; Croner, Roland S; Stürzl, Michael

    2016-07-28

    Guanylate-binding proteins (GBPs) are interferon-stimulated factors involved in the defense against cellular pathogens and inflammation. These proteins, particularly GBP-1, the most prominent member of the family, have been established as reliable markers of interferon-γ-activated cells in various diseases, including colorectal carcinoma (CRC) and inflammatory bowel diseases (IBDs). In CRC, GBP-1 expression is associated with a Th1-dominated angiostatic micromilieu and is correlated with a better outcome. Inhibition of tumor growth by GBP-1 is the result of its strong anti-angiogenic activity as well as its direct anti-tumorigenic effect on tumor cells. In IBD, GBP-1 mediates the anti-proliferative effects of interferon-γ on intestinal epithelial cells. In addition, it plays a protective role on the mucosa by preventing cell apoptosis, by inhibiting angiogenesis and by regulating the T-cell receptor signaling. These functions rely to a large extent on the ability of GBP-1 to interact with and remodel the actin cytoskeleton.

  6. Pathophysiological role of guanylate-binding proteins in gastrointestinal diseases

    PubMed Central

    Britzen-Laurent, Nathalie; Herrmann, Christian; Naschberger, Elisabeth; Croner, Roland S; Stürzl, Michael

    2016-01-01

    Guanylate-binding proteins (GBPs) are interferon-stimulated factors involved in the defense against cellular pathogens and inflammation. These proteins, particularly GBP-1, the most prominent member of the family, have been established as reliable markers of interferon-γ-activated cells in various diseases, including colorectal carcinoma (CRC) and inflammatory bowel diseases (IBDs). In CRC, GBP-1 expression is associated with a Th1-dominated angiostatic micromilieu and is correlated with a better outcome. Inhibition of tumor growth by GBP-1 is the result of its strong anti-angiogenic activity as well as its direct anti-tumorigenic effect on tumor cells. In IBD, GBP-1 mediates the anti-proliferative effects of interferon-γ on intestinal epithelial cells. In addition, it plays a protective role on the mucosa by preventing cell apoptosis, by inhibiting angiogenesis and by regulating the T-cell receptor signaling. These functions rely to a large extent on the ability of GBP-1 to interact with and remodel the actin cytoskeleton. PMID:27605879

  7. A statistical model of protein binding in parallel actin bundles

    NASA Astrophysics Data System (ADS)

    Shin, Homin; Grason, Gregory; Purdy Drew, Kirstin; Wong, Gerard

    2010-03-01

    We propose a coarse-grained lattice model of cross-linking proteins in parallel actin bundles. Based on this model that captures the interplay between geometrical frustration of binding and the intrinsic flexibility of filaments and linkers, we predict a unique regular ground-state structure of fully cross-linked bundles. We also discuss the linker-dependent thermodynamic transition of actin filaments from their native state to the overtwisted state and map out the ``twist-state'' phase diagram in terms of linker flexibility as well as the chemical potential. A flexible linker regime exhibits a continuous spectrum of intermediate twist states, while a stiff linker regime only allows for untwisted actin filaments and fully overtwisted bundles. Our predictions compare well with small-angle scattering studies of bundles formed in the presence of two types of reconstituted cross-linking proteins, fascin and espin. Additionally, this study reveals how subtle differences in crosslinking agents themselves may be used by cells to achieve self-organized bundles with dramatically different properties.

  8. The latent transforming growth factor beta binding protein (LTBP) family.

    PubMed Central

    Oklü, R; Hesketh, R

    2000-01-01

    The transforming growth factor beta (TGFbeta) cytokines are a multi-functional family that exert a wide variety of effects on both normal and transformed mammalian cells. The secretion and activation of TGFbetas is regulated by their association with latency-associated proteins and latent TGFbeta binding proteins (LTBPs). Over the past few years, three members of the LTBP family have been identified, in addition to the protoype LTBP1 first sequenced in 1990. Three of the LTBP family are expressed in a variety of isoforms as a consequence of alternative splicing. This review summarizes the differences between the isoforms in terms of the effects on domain structure and hence possible function. The close identity between LTBPs and members of the fibrillin family, mutations in which have been linked directly to Marfan's syndrome, suggests that anomalous expression of LTBPs may be associated with disease. Recent data indicating that differential expression of LTBP1 isoforms occurs during the development of coronary heart disease is considered, together with evidence that modulation of LTBP function, and hence of TGFbeta activity, is associated with a variety of cancers. PMID:11104663

  9. Nuclear actin-binding proteins as modulators of gene transcription.

    PubMed

    Gettemans, Jan; Van Impe, Katrien; Delanote, Veerle; Hubert, Thomas; Vandekerckhove, Joël; De Corte, Veerle

    2005-10-01

    Dynamic transformations in the organization of the cellular microfilament system are the driving force behind fundamental biological processes such as cellular motility, cytokinesis, wound healing and secretion. Eukaryotic cells express a plethora of actin-binding proteins (ABPs) allowing cells to control the organization of the actin cytoskeleton in a flexible manner. These structural proteins were, not surprisingly, originally described as (major) constituents of the cytoplasm. However, in recent years, there has been a steady flow of reports detailing not only translocation of ABPs into and out of the nucleus but also describing their role in the nuclear compartment. This review focuses on recent developments pertaining to nucleocytoplasmic transport of ABPs, including their mode of translocation and nuclear function. In particular, evidence that structurally and functionally unrelated cytoplasmic ABPs regulate transcription activation by various nuclear (steroid hormone) receptors is steadily accruing. Furthermore, the recent finding that actin is a necessary component of the RNA polymerase II-containing preinitiation complex opens up new opportunities for nuclear ABPs in gene transcription regulation.

  10. RNA binding proteins in the regulation of heart development.

    PubMed

    Blech-Hermoni, Yotam; Ladd, Andrea N

    2013-11-01

    In vivo, RNA molecules are constantly accompanied by RNA binding proteins (RBPs), which are intimately involved in every step of RNA biology, including transcription, editing, splicing, transport and localization, stability, and translation. RBPs therefore have opportunities to shape gene expression at multiple levels. This capacity is particularly important during development, when dynamic chemical and physical changes give rise to complex organs and tissues. This review discusses RBPs in the context of heart development. Since the targets and functions of most RBPs--in the heart and at large--are not fully understood, this review focuses on the expression and roles of RBPs that have been implicated in specific stages of heart development or developmental pathology. RBPs are involved in nearly every stage of cardiogenesis, including the formation, morphogenesis, and maturation of the heart. A fuller understanding of the roles and substrates of these proteins could ultimately provide attractive targets for the design of therapies for congenital heart defects, cardiovascular disease, or cardiac tissue repair.

  11. The RIPper case: identification of RNA-binding protein targets by RNA immunoprecipitation.

    PubMed

    Köster, Tino; Haas, Meike; Staiger, Dorothee

    2014-01-01

    Control at the posttranscriptional level emerges as an important layer of regulation in the circadian timing system. RNA-binding proteins that specifically interact with cis-regulatory motifs within pre-mRNAs are key elements of this regulation. While the ability to interact with RNA in vitro has been demonstrated for numerous Arabidopsis RNA-binding proteins, a full understanding of posttranscriptional networks controlled by an RNA-binding protein requires the identification of its immediate in vivo targets. Here we describe differential RNA immunoprecipitation in transgenic Arabidopsis thaliana plants expressing RNA-binding protein variants epitope-tagged with green fluorescent protein. To control for RNAs that nonspecifically co-purify with the RNA-binding protein, transgenic plants are generated with a mutated version of the RNA-binding protein that is not capable of binding to its target RNAs. The RNA-binding protein variants are expressed under the control of their authentic promoter and cis-regulatory motifs. Incubation of the plants with formaldehyde in vivo cross-links the proteins to their RNA targets. A whole-cell extract is then prepared and subjected to immunoprecipitation with an antibody against the GFP tag and to mock precipitation with an antibody against the unrelated red fluorescent protein. The RNAs coprecipitating with the proteins are eluted from the immunoprecipitate and identified via reverse transcription-PCR.

  12. The PDZ Domain of the LIM Protein Enigma Binds to β-Tropomyosin

    PubMed Central

    Guy, Pamela M.; Kenny, Daryn A.; Gill, Gordon N.

    1999-01-01

    PDZ and LIM domains are modular protein interaction motifs present in proteins with diverse functions. Enigma is representative of a family of proteins composed of a series of conserved PDZ and LIM domains. The LIM domains of Enigma and its most related family member, Enigma homology protein, bind to protein kinases, whereas the PDZ domains of Enigma and family member actin-associated LIM protein bind to actin filaments. Enigma localizes to actin filaments in fibroblasts via its PDZ domain, and actin-associated LIM protein binds to and colocalizes with the actin-binding protein α-actinin-2 at Z lines in skeletal muscle. We show that Enigma is present at the Z line in skeletal muscle and that the PDZ domain of Enigma binds to a skeletal muscle target, the actin-binding protein tropomyosin (skeletal β-TM). The interaction between Enigma and skeletal β-TM was specific for the PDZ domain of Enigma, was abolished by mutations in the PDZ domain, and required the PDZ-binding consensus sequence (Thr-Ser-Leu) at the extreme carboxyl terminus of skeletal β-TM. Enigma interacted with isoforms of tropomyosin expressed in C2C12 myotubes and formed an immunoprecipitable complex with skeletal β-TM in transfected cells. The association of Enigma with skeletal β-TM suggests a role for Enigma as an adapter protein that directs LIM-binding proteins to actin filaments of muscle cells. PMID:10359609

  13. Purification of recombinant nacre-associated mineralization protein AP7 fused with maltose-binding protein.

    PubMed

    Huang, Yu-Chieh; Chang, Hsun-Hui; Mou, Yun; Chi, Peter; Chan, Jerry Chun Chung; Luo, Shih-Chi

    2014-08-01

    Formation of biominerals often involves specific proteins that modulate the process of matrix assembly, nucleation, and crystal growth. AP7 is an aragonite-associated protein of 7 kDa and is intrinsically disordered. The structural disorder of AP7 makes it very difficult to express in Escherchiacoli. In this work, we report the first successful expression and purification of recombinant AP7 using the maltose-binding protein (MBP) fusion approach. We obtain a high-yield production of recombinant MBP-AP7 protein inE. coli (∼60 mg/L). We also establish an efficient protocol to remove the MBP fusion protein by Factor Xa, followed by purification using size-exclusion chromatography. Characterization of the recombinant AP7 protein has been carried out using MALDI-TOF, peptide mass fingerprinting, and circular dichroism (CD). The mass data confirm that the purified recombinant protein is AP7. The CD data suggest that the recombinant AP7 protein exists as partially disordered structure at neutral pH. The calcium carbonate precipitation assay shows that both MBP-AP7 and AP7 exhibit morphological modification on calcite crystallites. The co-precipitation of MBP-tagged AP7 derivatives and calcium carbonate generate different types of AP7 composite calcite and vaterite crystals. This system should be helpful to establish a model for understanding the structure/function relationship between the protein and inorganic mineral interaction.

  14. Insights into molecular plasticity of choline binding proteins (pneumococcal surface proteins) by SAXS.

    PubMed

    Buey, Rubén M; Monterroso, Begoña; Menéndez, Margarita; Diakun, Greg; Chacón, Pablo; Hermoso, Juan Antonio; Díaz, J Fernando

    2007-01-12

    Phosphocholine moieties decorating the pneumococcal surface are used as a docking station for a family of modular proteins, the so-called choline binding proteins or CBPs. Choline recognition is essential for CBPs function and may also be a determinant for their quaternary structure. There is little knowledge about modular arrangement or oligomeric structures in this family. Therefore, we have used the small angle X-ray scattering (SAXS) technique combined with analytical ultracentrifugation in order to model the three-dimensional envelope of two highly different CBPs: the phage encoded Cpl-1 lysozyme and the pneumococcal phosphorylcholine esterase Pce. Both enzymes have an N-terminal catalytic module and a C-terminal choline-binding module (CBM) that attaches them to the bacterial surface and comprises six and ten sequence repeats in Cpl-1 and Pce, respectively. SAXS experiments have shown an inherent conformational plasticity in Cpl-1 that accounts for the different relative position of these regions in the solution and crystal structures. Dimerization of Cpl-1 upon choline binding has been also visualised for the first time, and monomer-monomer interactions take place through the first CBR where a non-canonical choline binding site has now been identified. This mode of association seems to be independent of the absence or presence of the Cpl-1 catalytic module and reveals that the arrangement of the monomers differs from that previously found in the isolated CBM dimer of pneumococcal LytA amidase. In contrast, Pce displays the same modular disposition in the solution and crystal structures, and remains almost invariant upon choline binding. The present results suggest that protein dimerization and duplication of CBRs may be alternative but not equivalent ways of improving cell wall recognition by CBPs, since they provide different interaction geometries for choline residues present in (lipo)teichoic acids.

  15. Microseparation techniques for the study of the enantioselectivity of drug-plasma protein binding.

    PubMed

    Escuder-Gilabert, Laura; Martínez-Gómez, María Amparo; Villanueva-Camañas, Rosa María; Sagrado, Salvador; Medina-Hernández, María José

    2009-03-01

    Stereoselectivity in protein binding can have a significant effect on the pharmacokinetic and pharmacodynamic properties of chiral drugs. The investigation of enantioselectivity of drugs in their binding with human plasma proteins and the identification of the molecular mechanisms involved in the stereodiscrimination by the proteins represent a great challenge for clinical pharmacology. In this review, the separation techniques used for enantioselective protein binding experiments are described and compared. An overview of studies on enantiomer-protein interactions, enantiomer-enantiomer interactions as well as chiral drug-drug interactions, including allosteric effects, is presented. The contribution of individual plasma proteins to the overall enantioselective binding and the animal species variability in drug-plasma protein binding stereoselectivity are reviewed.

  16. Adsorption of DNA binding proteins to functionalized carbon nanotube surfaces with and without DNA wrapping.

    PubMed

    Ishibashi, Yu; Oura, Shusuke; Umemura, Kazuo

    2017-02-15

    We examined the adsorption of DNA binding proteins on functionalized, single-walled carbon nanotubes (SWNTs). When SWNTs were functionalized with polyethylene glycol (PEG-SWNT), moderate adsorption of protein molecules was observed. In contrast, nanotubes functionalized with CONH2 groups (CONH2-SWNT) exhibited very strong interactions between the CONH2-SWNT and DNA binding proteins. Instead, when these SWNT surfaces were wrapped with DNA molecules (thymine 30-mers), protein binding was a little decreased. Our results revealed that DNA wrapped PEG-SWNT was one of the most promising candidates to realize DNA nanodevices involving protein reactions on DNA-SWNT surfaces. In addition, the DNA binding protein RecA was more adhesive than single-stranded DNA binding proteins to the functionalized SWNT surfaces.

  17. Multicopper manganese oxidase accessory proteins bind Cu and heme.

    PubMed

    Butterfield, Cristina N; Tao, Lizhi; Chacón, Kelly N; Spiro, Thomas G; Blackburn, Ninian J; Casey, William H; Britt, R David; Tebo, Bradley M

    2015-12-01

    Multicopper oxidases (MCOs) catalyze the oxidation of a diverse group of metal ions and organic substrates by successive single-electron transfers to O2 via four bound Cu ions. MnxG, which catalyzes MnO2 mineralization by oxidizing both Mn(II) and Mn(III), is unique among multicopper oxidases in that it carries out two energetically distinct electron transfers and is tightly bound to accessory proteins. There are two of these, MnxE and MnxF, both approximately 12kDa. Although their sequences are similar to those found in the genomes of several Mn-oxidizing Bacillus species, they are dissimilar to those of proteins with known function. Here, MnxE and MnxF are co-expressed independent of MnxG and are found to oligomerize into a higher order stoichiometry, likely a hexamer. They bind copper and heme, which have been characterized by electron paramagnetic resonance (EPR), X-ray absorption spectroscopy (XAS), and UV-visible (UV-vis) spectrophotometry. Cu is found in two distinct type 2 (T2) copper centers, one of which appears to be novel; heme is bound as a low-spin species, implying coordination by two axial ligands. MnxE and MnxF do not oxidize Mn in the absence of MnxG and are the first accessory proteins to be required by an MCO. This may indicate that Cu and heme play roles in electron transfer and/or Cu trafficking.

  18. NikA binds heme: a new role for an Escherichia coli periplasmic nickel-binding protein.

    PubMed

    Shepherd, Mark; Heath, Mathew D; Poole, Robert K

    2007-05-01

    NikA is a periplasmic binding protein involved in nickel uptake in Escherichia coli. NikA was identified as a heme-binding protein in the periplasm of anaerobically grown cells overexpressing CydDC, an ABC transporter that exports reductant to the periplasm. CydDC-overexpressing cells accumulate a heme biosynthesis-derived pigment, P-574. For further biochemical and spectroscopic analysis, unliganded NikA was overexpressed and purified. NikA was found to comigrate with both hemin and protoporphyrin IX during gel filtration. Furthermore, tryptophan fluorescence quenching titrations demonstrated that both hemin and protoporphyrin IX bind to NikA with similar affinity. The binding affinity of NikA for these pigments (Kd approximately 0.5 microM) was unaltered in the presence and absence of saturating concentrations of nickel, suggesting that these tetrapyrroles bind to NikA in a manner independent of nickel. To test the hypothesis that NikA is required for periplasmic heme protein assembly, the effects of a nikA mutation (nikA::Tn5, Km(R) insertion) on accumulation of P-574 by CydDC-overexpressing cells was assessed. This mutation significantly lowered P-574 levels, implying that NikA may be involved in P-574 production. Thus, in the reducing environment of the periplasm, NikA may serve as a heme chaperone as well as a periplasmic nickel-binding protein. The docking of heme onto NikA was modeled using the published crystal structure; many of the predicted complexes exhibit a heme-binding cleft remote from the nickel-binding site, which is consistent with the independent binding of nickel and heme. This work has implications for the incorporation of heme into b- and c-type cytochromes.

  19. Enhanced protective antibody to a mutant meningococcal factor H-binding protein with low-factor H binding

    PubMed Central

    Granoff, Dan M.; Giuntini, Serena; Gowans, Flor A.; Lujan, Eduardo; Sharkey, Kelsey; Beernink, Peter T.

    2016-01-01

    Meningococcal factor H-binding protein (FHbp) is an antigen in 2 serogroup B meningococcal vaccines. FHbp specifically binds human and some nonhuman primate complement FH. To investigate the effect of binding of FH to FHbp on protective antibody responses, we immunized infant rhesus macaques with either a control recombinant FHbp antigen that bound macaque FH or a mutant antigen with 2 amino acid substitutions and >250-fold lower affinity for FH. The mutant antigen elicited 3-fold higher serum IgG anti-FHbp titers and up to 15-fold higher serum bactericidal titers than the control FHbp vaccine. When comparing sera with similar IgG anti-FHbp titers, the antibodies elicited by the mutant antigen gave greater deposition of complement component C4b on live meningococci (classical complement pathway) and inhibited binding of FH, while the anti-FHbp antibodies elicited by the control vaccine enhanced FH binding. Thus, the mutant FHbp vaccine elicited an anti-FHbp antibody repertoire directed at FHbp epitopes within the FH binding site, which resulted in greater protective activity than the antibodies elicited by the control vaccine, which targeted FHbp epitopes outside of the FH combining site. Binding of a host protein to a vaccine antigen impairs protective antibody responses, which can be overcome with low-binding mutant antigens. PMID:27668287

  20. Identification of a heparin-binding protein using monoclonal antibodies that block heparin binding to porcine aortic endothelial cells.

    PubMed Central

    Patton, W A; Granzow, C A; Getts, L A; Thomas, S C; Zotter, L M; Gunzel, K A; Lowe-Krentz, L J

    1995-01-01

    The binding of heparin or heparan sulphate to a variety of cell types results in specific changes in cell function. Endothelial cells treated with heparin alter their synthesis of heparan sulphate proteoglycans and extracellular matrix proteins. In order to identify a putative endothelial cell heparin receptor that could be involved in heparin signalling, anti-(endothelial cell) monoclonal antibodies that significantly inhibit heparin binding to endothelial cells were prepared. Four of these antibodies were employed in affinity-chromatographic isolation of a heparin-binding protein from detergent-solubilized endothelial cells. The heparin-binding protein isolated from porcine aortic endothelial cells using four different monoclonal antibodies has an M(r) of 45,000 assessed by SDS/PAGE. The 45,000-M(r) heparin-binding polypeptide is isolated as a multimer. The antibody-isolated protein binds to heparin-affinity columns as does the pure 45,000-M(r) polypeptide, consistent with its identification as a putative endothelial heparin receptor. Images Figure 2 Figure 3 PMID:7487882

  1. Using protein-binding microarrays to study transcription factor specificity: homologs, isoforms and complexes

    PubMed Central

    Andrilenas, Kellen K.; Penvose, Ashley

    2015-01-01

    Protein–DNA binding is central to specificity in gene regulation, and methods for characterizing transcription factor (TF)–DNA binding remain crucial to studies of regulatory specificity. High-throughput (HT) technologies have revolutionized our ability to characterize protein–DNA binding by significantly increasing the number of binding measurements that can be performed. Protein-binding microarrays (PBMs) are a robust and powerful HT platform for studying DNA-binding specificity of TFs. Analysis of PBM-determined DNA-binding profiles has provided new insight into the scope and mechanisms of TF binding diversity. In this review, we focus specifically on the PBM technique and discuss its application to the study of TF specificity, in particular, the binding diversity of TF homologs and multi-protein complexes. PMID:25431149

  2. Nonconsensus Protein Binding to Repetitive DNA Sequence Elements Significantly Affects Eukaryotic Genomes

    PubMed Central

    Barber-Zucker, Shiran; Gordân, Raluca; Lukatsky, David B.

    2015-01-01

    Recent genome-wide experiments in different eukaryotic genomes provide an unprecedented view of transcription factor (TF) binding locations and of nucleosome occupancy. These experiments revealed that a large fraction of TF binding events occur in regions where only a small number of specific TF binding sites (TFBSs) have been detected. Furthermore, in vitro protein-DNA binding measurements performed for hundreds of TFs indicate that TFs are bound with wide range of affinities to different DNA sequences that lack known consensus motifs. These observations have thus challenged the classical picture of specific protein-DNA binding and strongly suggest the existence of additional recognition mechanisms that affect protein-DNA binding preferences. We have previously demonstrated that repetitive DNA sequence elements characterized by certain symmetries statistically affect protein-DNA binding preferences. We call this binding mechanism nonconsensus protein-DNA binding in order to emphasize the point that specific consensus TFBSs do not contribute to this effect. In this paper, using the simple statistical mechanics model developed previously, we calculate the nonconsensus protein-DNA binding free energy for the entire C. elegans and D. melanogaster genomes. Using the available chromatin immunoprecipitation followed by sequencing (ChIP-seq) results on TF-DNA binding preferences for ~100 TFs, we show that DNA sequences characterized by low predicted free energy of nonconsensus binding have statistically higher experimental TF occupancy and lower nucleosome occupancy than sequences characterized by high free energy of nonconsensus binding. This is in agreement with our previous analysis performed for the yeast genome. We suggest therefore that nonconsensus protein-DNA binding assists the formation of nucleosome-free regions, as TFs outcompete nucleosomes at genomic locations with enhanced nonconsensus binding. In addition, here we perform a new, large-scale analysis using

  3. Ankyrin-independent membrane protein-binding sites for brain and erythrocyte spectrin.

    PubMed

    Steiner, J P; Bennett, V

    1988-10-05

    Brain spectrin reassociates in in vitro binding assays with protein(s) in highly extracted brain membranes quantitatively depleted of ankyrin and spectrin. These newly described membrane sites for spectrin are biologically significant and involve a protein since (a) binding occurs optimally at physiological pH (6.7-6.9) and salt concentrations (50 mM), (b) binding is abolished by digestion of membranes with alpha-chymotrypsin, (c) Scatchard analysis is consistent with a binding capacity of at least 50 pmol/mg total membrane protein, and highest affinity of 3 nM. The major ankyrin-independent binding activity of brain spectrin is localized to the beta subunit of spectrin. Brain membranes also contain high affinity binding sites for erythrocyte spectrin, but a 3-4 fold lower capacity than for brain spectrin. Some spectrin-binding sites associate preferentially with brain spectrin, some with erythrocyte spectrin, and some associate with both types of spectrin. Erythrocyte spectrin contains distinct binding domains for ankyrin and brain membrane protein sites, since the Mr = 72,000 spectrin-binding fragment of ankyrin does not compete for binding of spectrin to brain membranes. Spectrin binds to a small number of ankyrin-independent sites in erythrocyte membranes present in about 10,000-15,000 copies/cell or 10% of the number of sites for ankyrin. Brain spectrin binds to these sites better than erythrocyte spectrin suggesting that erythrocytes have residual binding sites for nonerythroid spectrin. Ankyrin-independent-binding proteins that selectively bind to certain isoforms of spectrin provide a potentially important flexibility in cellular localization and time of synthesis of proteins involved in spectrin-membrane interactions. This flexibility has implications for assembly of the membrane skeleton and targeting of spectrin isoforms to specialized regions of cells.

  4. Dual labeling of a binding protein allows for specific fluorescence detection of native protein.

    PubMed

    Karlström, A; Nygren, P A

    2001-08-01

    Fluorescence resonance energy transfer has been investigated in the context of specific detection of unlabeled proteins. A model system based on the staphylococcal protein A (SPA)-IgG interaction was designed, in which a single domain was engineered to facilitate site-specific incorporation of fluorophores. An Asn23Cys mutant of the B domain from SPA was expressed in Escherichia coli and subsequently labeled at the introduced unique thiol and at an amino group, using N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS) and succinimidyl 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate (NBD-X, SE), respectively. Biosensor analysis of purified doubly labeled protein showed that high-affinity binding to the Fc region of IgG was retained. The fluorescence emission spectrum of the doubly labeled protein showed a shift in the relative emission of the two fluorophores in the presence of Fc3(1) fragments, which bind specifically to the B domain. In addition, the fluorescence emission ratio 480/525 nm was shown to increase with increasing concentration of Fc3(1), whereas the presence of a control protein did not affect the emission ratio over the same concentration range.

  5. Alignment-free ultra-high-throughput comparison of druggable protein-ligand binding sites.

    PubMed

    Weill, Nathanaël; Rognan, Didier

    2010-01-01

    Inferring the biological function of a protein from its three-dimensional structure as well as explaining why a drug may bind to various targets is of crucial importance to modern drug discovery. Here we present a generic 4833-integer vector describing druggable protein-ligand binding sites that can be applied to any protein and any binding cavity. The fingerprint registers counts of pharmacophoric triplets from the Calpha atomic coordinates of binding-site-lining residues. Starting from a customized data set of diverse protein-ligand binding site pairs, the most appropriate metric and a similarity threshold could be defined for similar binding sites. The method (FuzCav) has been used in various scenarios: (i) screening a collection of 6000 binding sites for similarity to different queries; (ii) classifying protein families (serine endopeptidases, protein kinases) by binding site diversity; (iii) discriminating adenine-binding cavities from decoys. The fingerprint generation and comparison supports ultra-high throughput (ca. 1000 measures/s), does not require prior alignment of protein binding sites, and is able to detect local similarity among subpockets. It is thus particularly well suited to the functional annotation of novel genomic structures with low sequence identity to known X-ray templates.

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

    PubMed

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

    2015-04-20

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

  7. GEFs: structural basis for their activation of small GTP-binding proteins.

    PubMed

    Cherfils, J; Chardin, P

    1999-08-01

    Small GTP-binding proteins of the Ras superfamily function as molecular switches in fundamental events such as signal transduction, cytoskeleton dynamics and intracellular trafficking. Guanine-nucleotide-exchange factors (GEFs) positively regulate these GTP-binding proteins in response to a variety of signals. GEFs catalyze the dissociation of GDP from the inactive GTP-binding proteins. GTP can then bind and induce structural changes that allow interaction with effectors. Representative structures of four main classes of exchange factors have been described recently and, in two cases, structures of the GTP-binding protein-GEF complex have been solved. These structures, together with biochemical studies, have allowed a deeper understanding of the mechanisms of activation of Ras-like GTP-binding proteins and suggested how they might represent targets for therapeutic intervention.

  8. Changes in GDP binding to brown adipose tissue mitochondria and the uncoupling protein

    SciTech Connect

    Swick, A.G.; Swick, R.W. )

    1988-12-01

    Incubation in vitro of brown adipose tissue (BAT) mitochondria with divalent cations, spermine, or alkaline phosphatase led to a marked increase in the binding of ({sup 3}H)GDP. The effect of Mg{sup 2+} appeared to be the most specific and led to the largest increase in GDP binding. A simplified method was developed for measuring GDP binding to purified uncoupling protein from rat BAT mitochondria. Application of this method indicates that uncoupling protein from cold-acclimated rats binds twice as much GDP as uncoupling protein from cold-acclimated rats that were briefly returned to thermoneutrality, paralleling changes in GDP binding to the mitochondria. Incubation of BAT mitochondria with Mg{sup 2+} led to a smaller increase in GDP binding to the subsequently purified uncoupling protein, suggesting that divalent cations may somehow participate in the regulation of the activity of the uncoupling protein.

  9. Influence of binding pH and protein solubility on the dynamic binding capacity in hydrophobic interaction chromatography.

    PubMed

    Baumann, Pascal; Baumgartner, Kai; Hubbuch, Jürgen

    2015-05-29

    Hydrophobic interaction chromatography (HIC) is one of the most frequently used purification methods in biopharmaceutical industry. A major drawback of HIC, however, is the rather low dynamic binding capacity (DBC) obtained when compared to e.g. ion exchange chromatography (IEX). The typical purification procedure for HIC includes binding at neutral pH, independently of the proteins nature and isoelectric point. Most approaches to process intensification are based on resin and salt screenings. In this paper a combination of protein solubility data and varying binding pH leads to a clear enhancement of dynamic binding capacity. This is shown for three proteins of acidic, neutral, and alkaline isoelectric points. High-throughput solubility screenings as well as miniaturized and parallelized breakthrough curves on Media Scout RoboColumns (Atoll, Germany) were conducted at pH 3-10 on a fully automated robotic workstation. The screening results show a correlation between the DBC and the operational pH, the protein's isoelectric point and the overall solubility. Also, an inverse relationship of DBC in HIC and the binding kinetics was observed. By changing the operational pH, the DBC could be increased up to 30% compared to the standard purification procedure performed at neutral pH. As structural changes of the protein are reported during HIC processes, the applied samples and the elution fractions were proven not to be irreversibly unfolded.

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

    SciTech Connect

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

    2009-08-14

    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 {approx}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.

  11. Fatty acid-binding site environments of serum vitamin D-binding protein and albumin are different

    PubMed Central

    Swamy, Narasimha; Ray, Rahul

    2008-01-01

    Vitamin D-binding protein (DBP) and albumin (ALB) are abundant serum proteins and both possess high-affinity binding for saturated and unsaturated fatty acids. However, certain differences exist. We surmised that in cases where serum albumin level is low, DBP presumably can act as a transporter of fatty acids. To explore this possibility we synthesized several alkylating derivatives of 14C-palmitic acid to probe the fatty acid binding pockets of DBP and ALB. We observed that N-ethyl-5-phenylisooxazolium-3′-sulfonate-ester (WRK ester) of 14C-palmitic acid specifically labeled DBP; but p-nitrophenyl- and N-hydroxysuccinimidyl-esters failed to do so. However, p-nitrophenyl ester of 14C-palmitic acid specifically labeled bovine ALB, indicating that the micro-environment of the fatty acid-binding domains of DBP and ALB may be different; and DBP may not replace ALB as a transporter of fatty acids. PMID:18374965

  12. An arsenic fluorescent compound as a novel probe to study arsenic-binding proteins.

    PubMed

    Femia, A Lis; Temprana, C Facundo; Santos, Javier; Carbajal, María Laura; Amor, María Silvia; Grasselli, Mariano; Alonso, Silvia Del V

    2012-12-01

    Arsenic-binding proteins are under continuous research. Their identification and the elucidation of arsenic/protein interaction mechanisms are important because the biological effects of these complexes may be related not only to arsenic but also to the arsenic/protein structure. Although many proteins bearing a CXXC motif have been found to bind arsenic in vivo, new tools are necessary to identify new arsenic targets and allow research on protein/arsenic complexes. In this work, we analyzed the performance of the fluorescent compound APAO-FITC (synthesized from p-aminophenylarsenoxide, APAO, and fluorescein isothiocyanate, FITC) in arsenic/protein binding assays using thioredoxin 1 (Trx) as an arsenic-binding protein model. The Trx-APAO-FITC complex was studied through different spectroscopic techniques involving UV-Vis, fluorescence, atomic absorption, infrared and circular dichroism. Our results show that APAO-FITC binds efficiently and specifically to the Trx binding site, labeling the protein fluorescently, without altering its structure and activity. In summary, we were able to study a protein/arsenic complex model, using APAO-FITC as a labeling probe. The use of APAO-FITC in the identification of different protein and cell targets, as well as in in vivo biodistribution studies, conformational studies of arsenic-binding proteins, and studies for the design of drug delivery systems for arsenic anti-cancer therapies, is highly promising.

  13. Development of a protein microarray using sequence-specific DNA binding domain on DNA chip surface

    SciTech Connect

    Choi, Yoo Seong; Pack, Seung Pil; Yoo, Young Je . E-mail: yjyoo@snu.ac.kr

    2005-04-22

    A protein microarray based on DNA microarray platform was developed to identify protein-protein interactions in vitro. The conventional DNA chip surface by 156-bp PCR product was prepared for a substrate of protein microarray. High-affinity sequence-specific DNA binding domain, GAL4 DNA binding domain, was introduced to the protein microarray as fusion partner of a target model protein, enhanced green fluorescent protein. The target protein was oriented immobilized directly on the DNA chip surface. Finally, monoclonal antibody of the target protein was used to identify the immobilized protein on the surface. This study shows that the conventional DNA chip can be used to make a protein microarray directly, and this novel protein microarray can be applicable as a tool for identifying protein-protein interactions.

  14. RsiteDB: a database of protein binding pockets that interact with RNA nucleotide bases.

    PubMed

    Shulman-Peleg, Alexandra; Nussinov, Ruth; Wolfson, Haim J

    2009-01-01

    We present a new database and an on-line search engine, which store and query the protein binding pockets that interact with single-stranded RNA nucleotide bases. The database consists of a classification of binding sites derived from protein-RNA complexes. Each binding site is assigned to a cluster of similar binding sites in other protein-RNA complexes. Cluster members share similar spatial arrangements of physico-chemical properties, thus can reveal novel similarity between proteins and RNAs with different sequences and folds. The clusters provide 3D consensus binding patterns important for protein-nucleotide recognition. The database search engine allows two types of useful queries: first, given a PDB code of a protein-RNA complex, RsiteDB can detail and classify the properties of the protein binding pockets accommodating extruded RNA nucleotides not involved in local RNA base pairing. Second, given an unbound protein structure, RsiteDB can perform an on-line structural search against the constructed database of 3D consensus binding patterns. Regions similar to known patterns are predicted to serve as binding sites. Alignment of the query to these patterns with their corresponding RNA nucleotides allows making unique predictions of the protein-RNA interactions at the atomic level of detail. This database is accessible at http://bioinfo3d.cs.tau.ac.il/RsiteDB.

  15. Arabidopsis Acyl-CoA-binding protein ACBP2 interacts with an ethylene-responsive element-binding protein, AtEBP, via its ankyrin repeats.

    PubMed

    Li, Hong-Ye; Chye, Mee-Len

    2004-01-01

    Cytosolic acyl-CoA-binding proteins (ACBP) bind long-chain acyl-CoAs and act as intracellular acyl-CoA transporters and maintain acyl-CoA pools. Arabidopsis thaliana ACBP2 shows conservation at the acyl-CoA-binding domain to cytosolic ACBPs but is distinct by the presence of an N-terminal transmembrane domain and C-terminal ankyrin repeats. The function of the acyl-CoA-binding domain in ACBP2 has been confirmed by site-directed mutagenesis and four conserved residues crucial for palmitoyl-CoA binding have been identified. Results from ACBP2:GFP fusions transiently expressed in onion epidermal cells have demonstrated that the transmembrane domain functions in plasma membrane targeting, suggesting that ACBP2 transfers acyl-CoA esters to this membrane. In this study, we investigated the significance of its ankyrin repeats in mediating protein-protein interactions by yeast two-hybrid analysis and in vitro protein-binding assays; we showed that ACBP2 interacts with the A. thaliana ethylene-responsive element-binding protein AtEBP via its ankyrin repeats. This interaction was lacking in yeast two-hybrid analysis upon removal of the ankyrin repeats. When the subcellular localizations of ACBP2 and AtEBP were further investigated using autofluorescent protein fusions in transient expression by agroinfiltration of tobacco leaves, the DsRed:ACBP2 fusion protein was localized to the plasma membrane while the GFP:AtEBP fusion protein was targeted to the nucleus and plasma membrane. Co-expression of DsRed:ACBP2 and GFP:AtEBP showed a common localization of both proteins at the plasma membrane, suggesting that ACBP2 likely interacts with AtEBP at the plasma membrane.

  16. Comparison of rabbit androgen binding protein with testosterone estradiol binding globulin--I. Physical and chemical properties.

    PubMed

    Cheng, S L; Kotite, N; Musto, N A

    1984-12-01

    Rabbit epididymal androgen binding protein (rbABP) and serum testosterone estradiol binding globulin (rbTeBG) were purified and their physicochemical properties compared. Both proteins bound dihydrotestosterone (DHT) with high affinity. Both contained two components, Heavy (H) and Light (L), and their molecular weights and pI values were comparable. rbABP and rbTeBG were different with regard to their ConA-Sepharose binding property. rbABP was not bound by ConA-Sepharose while rbTeBG was found and retained by this lectin; thus, rbABP and rbTeBG differed in their carbohydrate structure. Peptide mapping on SDS-PAGE indicated that the H components of rbABP and rbTeBG were distinct even though they showed a high degree of homology. By contrast, the L components of these two proteins appeared to be identical. The structure of the steroid binding sites of these two proteins was analyzed by peptide mapping of [1,2(3)H]17 beta hydroxy-androsta-4,6-dien-3-one photoaffinity labeled protein. The size distribution of radioactive peptide fragments generated appeared to be identical for these two proteins. However, the distribution of labeled peptides was slightly different when examined by high pressure liquid chromatography (HPLC). The observations suggest that the differences between rbABP and rbTeBG might reside not only in carbohydrate moieties but also in their amino acid sequences.

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

    PubMed

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

    2015-04-30

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

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

    PubMed Central

    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

  19. The Binding of Plasmodium falciparum Adhesins and Erythrocyte Invasion Proteins to Aldolase Is Enhanced by Phosphorylation

    PubMed Central

    Diaz, Suraya A.; Martin, Stephen R.; Howell, Steven A.; Grainger, Munira; Moon, Robert W.; Green, Judith L.

    2016-01-01

    Aldolase has been implicated as a protein coupling the actomyosin motor and cell surface adhesins involved in motility and host cell invasion in the human malaria parasite Plasmodium falciparum. It binds to the cytoplasmic domain (CTD) of type 1 membrane proteins of the thrombospondin-related anonymous protein (TRAP) family. Other type 1 membrane proteins located in the apical organelles of merozoites, the form of the parasite that invades red blood cells, including apical membrane antigen 1 (AMA1) and members of the erythrocyte binding ligand (EBL) and reticulocyte binding homologue (RH) protein families have been implicated in host cell binding and invasion. Using a direct binding method we confirm that TRAP and merozoite TRAP (MTRAP) bind aldolase and show that the interaction is mediated by more than just the C-terminal six amino acid residues identified previously. Single amino acid substitutions in the MTRAP CTD abolished binding to aldolase. The CTDs of AMA1 and members of the EBL and RH protein families also bound to aldolase. MTRAP competed with AMA1 and RH4 for binding to aldolase, indicating overlapping binding sites. MTRAP CTD was phosphorylated in vitro by both calcium dependent kinase 1 (CDPK1) and protein kinase A, and this modification increased the affinity of binding to aldolase by ten-fold. Phosphorylation of the CTD of members of the EBL and RH protein families also increased their affinity for aldolase in some cases. To examine whether or not MTRAP expressed in asexual blood stage parasites is phosphorylated, it was tagged with GFP, purified and analysed, however no phosphorylation was detected. We propose that CTD binding to aldolase may be dynamically modulated by phosphorylation, and there may be competition for aldolase binding between different CTDs. The use and efficiency of alternate invasion pathways may be determined by the affinity of adhesins and cell invasion proteins for aldolase, in addition to their host ligand specificity. PMID

  20. The Binding of Plasmodium falciparum Adhesins and Erythrocyte Invasion Proteins to Aldolase Is Enhanced by Phosphorylation.

    PubMed

    Diaz, Suraya A; Martin, Stephen R; Howell, Steven A; Grainger, Munira; Moon, Robert W; Green, Judith L; Holder, Anthony A

    2016-01-01

    Aldolase has been implicated as a protein coupling the actomyosin motor and cell surface adhesins involved in motility and host cell invasion in the human malaria parasite Plasmodium falciparum. It binds to the cytoplasmic domain (CTD) of type 1 membrane proteins of the thrombospondin-related anonymous protein (TRAP) family. Other type 1 membrane proteins located in the apical organelles of merozoites, the form of the parasite that invades red blood cells, including apical membrane antigen 1 (AMA1) and members of the erythrocyte binding ligand (EBL) and reticulocyte binding homologue (RH) protein families have been implicated in host cell binding and invasion. Using a direct binding method we confirm that TRAP and merozoite TRAP (MTRAP) bind aldolase and show that the interaction is mediated by more than just the C-terminal six amino acid residues identified previously. Single amino acid substitutions in the MTRAP CTD abolished binding to aldolase. The CTDs of AMA1 and members of the EBL and RH protein families also bound to aldolase. MTRAP competed with AMA1 and RH4 for binding to aldolase, indicating overlapping binding sites. MTRAP CTD was phosphorylated in vitro by both calcium dependent kinase 1 (CDPK1) and protein kinase A, and this modification increased the affinity of binding to aldolase by ten-fold. Phosphorylation of the CTD of members of the EBL and RH protein families also increased their affinity for aldolase in some cases. To examine whether or not MTRAP expressed in asexual blood stage parasites is phosphorylated, it was tagged with GFP, purified and analysed, however no phosphorylation was detected. We propose that CTD binding to aldolase may be dynamically modulated by phosphorylation, and there may be competition for aldolase binding between different CTDs. The use and efficiency of alternate invasion pathways may be determined by the affinity of adhesins and cell invasion proteins for aldolase, in addition to their host ligand specificity.

  1. The RNA-binding protein SERBP1 interacts selectively with the signaling protein RACK1.

    PubMed

    Bolger, Graeme B

    2017-03-04

    The RACK1 protein interacts with numerous proteins involved in signal transduction, the cytoskeleton, and mRNA splicing and translation. We used the 2-hybrid system to identify additional proteins interacting with RACK1 and isolated the RNA-binding protein SERBP1. SERPB1 shares amino acid sequence homology with HABP4 (also known as Ki-1/57), a component of the RNA spicing machinery that has been shown previously to interact with RACK1. Several different isoforms of SERBP1, generated by alternative mRNA splicing, interacted with RACK1 with indistinguishable interaction strength, as determined by a 2-hybrid beta-galactosidase assay. Analysis of deletion constructs of SERBP1 showed that the C-terminal third of the SERBP1 protein, which contains one of its two substrate sites for protein arginine N-methyltransferase 1 (PRMT1), is necessary and sufficient for it to interact with RACK1. Analysis of single amino acid substitutions in RACK1, identified in a reverse 2-hybrid screen, showed very substantial overlap with those implicated in the interaction of RACK1 with the cAMP-selective phosphodiesterase PDE4D5. These data are consistent with SERBP1 interacting selectively with RACK1, mediated by an extensive interaction surface on both proteins.

  2. Ligand identification of carbohydrate-binding proteins employing a biotinylated glycan binding assay and tandem mass spectrometry.

    PubMed

    Wuhrer, Manfred; van Remoortere, Alexandra; Balog, Crina I A; Deelder, André M; Hokke, Cornelis H

    2010-11-15

    Characterization of protein-carbohydrate interactions at the molecular level is important for understanding many glycan-mediated processes. Here we present a method for the identification of glycan ligands of carbohydrate-binding proteins. The glycans released from natural sources are labeled with biotinamidocaproyl hydrazide (BACH) and subsequently fractionated by high-performance liquid chromatography. Glycan fractions are screened for binding to carbohydrate-binding proteins (CBPs) using a microtitration plate binding assay; CBPs are immobilized, BACH-glycan fractions are added, and bound BACH-glycans are detected using alkaline phosphatase-conjugated streptavidin. The glycan structures in binding fractions are studied by (tandem) mass spectrometry, exoglycosidase treatment, and rechromatography, thereby revealing the glycan motifs recognized by the CBPs. Subsequent surface plasmon resonance experiments using a reverse setup with immobilization of the BACH-glycan ligands on streptavidin-coated surfaces provide more information on glycan-CBP interactions via association and dissociation curves. The presented method is easy and fast, and the required instrumentation is available in many laboratories. The assay is very sensitive given that both the mass spectrometric analysis and the microtitration plate binding assay can be performed on femtomole amounts of BACH-glycans. This approach should be generally applicable to study and structurally identify carbohydrate ligands of anti-glycan antibodies and lectins.

  3. Drug design for protein kinases and phosphatases: flexible-receptor docking, binding affinity and specificity, and drug-binding kinetics.

    PubMed

    Wong, Chung F; Bairy, Sneha

    2013-01-01

    This article reviews some of our experiences on applying computational techniques to aid the design of drugs targeting protein kinases and phosphatases. It is not a comprehensive review. Rather, it focuses on several less explored approaches or ideas that we have experiences on. It reviews some recent improvements on the Poisson-Boltzmann/Surface Area model for calculating binding affinity and discusses ways to perform calculations that are more tolerant to statistical and systematic errors. Several new ways to incorporate protein flexibility in molecular docking and estimating binding affinity are also discussed. Its discussions also go beyond binding affinity to considering drug-binding kinetics, not only on investigating protein-ligand interactions in isolation, but also on accounting for upstream and downstream influences that can occur in cells, through kinetic modeling of cell signaling. This review also describes a quick molecular simulation method for understanding drug-binding kinetics at the molecular level, with the hope of generating guiding principles for designing drugs with the desired kinetic properties. Sources of drug-binding selectivity that appear obvious but often overlooked are also discussed.

  4. recA protein-catalyzed strand assimilation: stimulation by Escherichia coli single-stranded DNA-binding protein.

    PubMed Central

    McEntee, K; Weinstock, G M; Lehman, I R

    1980-01-01

    The single-stranded DNA-binding protein of Escherichia coli significantly alters the strand assimilation reaction catalyzed by recA protein [McEntee, K., Weinstock, G. M. & Lehman, I. R. (1979) Proc. Natl. Acad. Sci. USA 76, 2615--2619]. The binding protein (i) increases the rate and extent of strand assimilation into homologous duplex DNA, (ii) enhances the formation of a complex between recA protein and duplex DNA in the presence of homologous or heterologous single-stranded DNA, (iii) reduces the rate and extent of ATP hydrolysis catalyzed by recA protein in the presence of single-stranded DNA, (iv) reduces the high concentration of recA protein required for strand assimilation, and (v) permits detection of strand assimilation in the presence of the ATP analog, adenosine 5'-O-(O-thiotriphosphate). Single-stranded DNA-binding protein purified from a binding protein mutant (lexC) is considerably less effective than wild-type binding protein in stimulating strand assimilation, a result which suggests that single-stranded DNA-binding protein participates in general recombination in vivo. PMID:6244589

  5. Mitogen-stimulated TIS21 protein interacts with a protein-kinase-Calpha-binding protein rPICK1.

    PubMed Central

    Lin, W J; Chang, Y F; Wang, W L; Huang, C Y

    2001-01-01

    TIS21 is induced transiently by PMA and a number of extracellular stimuli. Yeast two-hybrid screening has identified three TIS21 interacting clones from a rat cDNA library [Lin, Gary, Yang, Clarke and Herschman (1996) J. Biol. Chem 271, 15034-15044]. The amino acid sequence deduced from clone 5A shows 96.9% identity with the murine PICK1, a protein kinase Calpha (PKCalpha)-binding protein postulated to act as an intracellular receptor for PKC. A fusion protein of glutathione S-transferase and rPICK1 associates with the TIS21 translated in vitro, suggesting a direct physical interaction between these two proteins. TIS21 and rPICK1 are co-immunoprecipitated from NIH 3T3 cells overexpressing these two proteins. This indicates that the interaction also occurs in mammalian cells. Deletion of the PDZ domain at the N-terminus of rPICK1 abolishes its interaction with TIS21. A putative carboxylate-binding loop required for PICK1 to bind PKCalpha [Staudinger, Lu and Olson (1997) J. Biol. Chem 272, 32019-32024] is within this deleted region. Our results suggest a potential competition between TIS21 and PKC for binding to PICK1. We show that recombinant TIS21 is phosphorylated by PKC in vitro. The catalytic activity of PKC towards TIS21 is significantly decreased in the presence of rPICK1, whereas phosphorylation of histone by PKC is not affected. rPICK1 seems to modulate the phosphorylation of TIS21 through specific interactions between these two proteins. TIS21 might have a role in PKC-mediated extracellular signal transduction through its interaction with rPICK1. PMID:11237868

  6. Organization and function of Drosophila odorant binding proteins

    PubMed Central

    Larter, Nikki K; Sun, Jennifer S; Carlson, John R

    2016-01-01

    Odorant binding proteins (Obps) are remarkable in their number, diversity, and abundance, yet their role in olfactory coding remains unclear. They are widely believed to be required for transporting hydrophobic odorants through an aqueous lymph to odorant receptors. We construct a map of the Drosophila antenna, in which the abundant Obps are mapped to olfactory sensilla with defined functions. The results lay a foundation for an incisive analysis of Obp function. The map identifies a sensillum type that contains a single abundant Obp, Obp28a. Surprisingly, deletion of the sole abundant Obp in these sensilla does not reduce the magnitude of their olfactory responses. The results suggest that this Obp is not required for odorant transport and that this sensillum does not require an abundant Obp. The results further suggest a novel role for this Obp in buffering changes in the odor environment, perhaps providing a molecular form of gain control. DOI: http://dx.doi.org/10.7554/eLife.20242.001 PMID:27845621

  7. Host humoral immune response to Leishmania lipid-binding protein.

    PubMed

    Maache, M; Azzouz, S; Diaz de la Guardia, R; Alvarez, P; Gil, R; de Pablos, L M; Osuna, A

    2005-06-01

    SUMMARY We report on the use of Leishmania donovani lipid-binding proteins (LBPs) as antigens capable of being recognized by serum from immunocompetent patients from southern Spain suffering from visceral leishmaniasis and from Peruvian patients with localized cutaneous leishmaniasis caused by Leishmania braziliensis. The absorbance found by immunoenzymatic techniques gave significantly different results for the serum samples from patients with and without leishmaniasis. Specificity by ELISA testing was 93.2% and sensibility 100%. Dot blots from human patient serum samples or naturally infected dogs from Spain gave similarly significant results. All the human serum samples from individuals with visceral leishmaniasis and the Leishmania-positive canine samples recognized two bands, with molecular weights of 8 and 57 kDa. The serum from individuals with cutaneous leishmaniasis caused by L. braziliensis recognized an additional band of 16 kDa. We discuss the role of Leishmania FABP and compare the immunological reactions found with serum samples from other protozoan infections such as toxoplasma and Chagas as well as bacterial infections such as tuberculosis and syphilis.

  8. Functional dissection of Odorant binding protein genes in Drosophila melanogaster

    PubMed Central

    Swarup, S; Williams, T I; Anholt, R R H

    2011-01-01

    Most organisms rely on olfaction for survival and reproduction. The olfactory system of Drosophila melanogaster is one of the best characterized chemosensory systems and serves as a prototype for understanding insect olfaction. Olfaction in Drosophila is mediated by multigene families of odorant receptors and odorant binding proteins (OBPs). Although molecular response profiles of odorant receptors have been well documented, the contributions of OBPs to olfactory behavior remain largely unknown. Here, we used RNAi-mediated suppression of Obp gene expression and measurements of behavioral responses to 16 ecologically relevant odorants to systematically dissect the functions of 17 OBPs. We quantified the effectiveness of RNAi-mediated suppression by quantitative real-time polymerase chain reaction and used a proteomic liquid chromatography and tandem mass spectrometry procedure to show target-specific suppression of OBPs expressed in the antennae. Flies in which expression of a specific OBP is suppressed often show altered behavioral responses to more than one, but not all, odorants, in a sex-dependent manner. Similarly, responses to a specific odorant are frequently affected by suppression of expression of multiple, but not all, OBPs. These results show that OBPs are essential for mediating olfactory behavioral responses and suggest that OBP-dependent odorant recognition is combinatorial. PMID:21605338

  9. Symbiont-induced odorant binding proteins mediate insect host hematopoiesis

    PubMed Central

    Benoit, Joshua B; Vigneron, Aurélien; Broderick, Nichole A; Wu, Yineng; Sun, Jennifer S; Carlson, John R; Aksoy, Serap; Weiss, Brian L

    2017-01-01

    Symbiotic bacteria assist in maintaining homeostasis of the animal immune system. However, the molecular mechanisms that underlie symbiont-mediated host immunity are largely unknown. Tsetse flies (Glossina spp.) house maternally transmitted symbionts that regulate the development and function of their host’s immune system. Herein we demonstrate that the obligate mutualist, Wigglesworthia, up-regulates expression of odorant binding protein six in the gut of intrauterine tsetse larvae. This process is necessary and sufficient to induce systemic expression of the hematopoietic RUNX transcription factor lozenge and the subsequent production of crystal cells, which actuate the melanotic immune response in adult tsetse. Larval Drosophila’s indigenous microbiota, which is acquired from the environment, regulates an orthologous hematopoietic pathway in their host. These findings provide insight into the molecular mechanisms that underlie enteric symbiont-stimulated systemic immune system development, and indicate that these processes are evolutionarily conserved despite the divergent nature of host-symbiont interactions in these model systems. DOI: http://dx.doi.org/10.7554/eLife.19535.001 PMID:28079523

  10. RNA binding proteins, neural development and the addictions

    PubMed Central

    Bryant, Camron D.; Yazdani, Neema

    2016-01-01

    Transcriptional and post-transcriptional regulation of gene expression defines the neurobiological mechanisms that bridge genetic and environmental risk factors with neurobehavioral dysfunction underlying the addictions. More than 1000 genes in the eukaryotic genome code for multifunctional RNA binding proteins (RBPs) that can regulate all levels of RNA biogenesis. More than 50% of these RBPs are expressed in the brain where they regulate alternative splicing, transport, localization, stability, and translation of RNAs during development and adulthood. RBP dysfunction can exert global effects on their targetomes that underlie neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease as well as neurodevelopmental disorders, including autism and schizophrenia. Here, we consider the evidence that RBPs influence key molecular targets, neurodevelopment, synaptic plasticity, and neurobehavioral dysfunction underlying the addictions. Increasingly well-powered genome-wide association studies in humans and mammalian model organisms combined with ever more precise transcriptomic and proteomic approaches will continue to uncover novel and possibly selective roles for RBPs in the addictions. Key challenges include identifying the biological functions of the dynamic RBP targetomes from specific cell types throughout subcellular space (e.g., the nuclear spliceome versus the synaptic translatome) and time and manipulating RBP programs through post-transcriptional modifications to prevent or reverse aberrant neurodevelopment and plasticity underlying the addictions. PMID:26643147

  11. Affinity of cefoperazone for penicillin-binding proteins.

    PubMed Central

    Matsubara, N; Minami, S; Matsuhashi, M; Takaoka, M; Mitsuhashi, S

    1980-01-01

    Cefoperazone (T-1551, CFP) a new semisynthetic cephalosporin, has a broad spectrum of antibacterial activity. We investigated the affinity of CFP to penicillin-binding proteins (PBPs) and the inhibition of peptidoglycan synthesis by CFP. CFP had high affinities for Escherichia coli PBP-3, -1Bs, -2, and -1A, in descending order, and low affinities for PBP-4, -5, and -6. Similarly, CFP showed high affinity for Pseudomonas aeruginosa PBP-3, -1A, -1B, -2, and -4, in descending order. It is known that E. coli PBP-3 and P. aeruginosa PBP-3 participate in cell division. These results are in good agreement with the formation of filamentous cells of E. coli and P. aeruginosa treated with CFP. CFP had lower inhibitory activities on D-alanine carboxypeptidase IA and IB of E. coli than that of penicillin G, but its inhibitory activities on the cross-link formation in peptidoglycan synthesis were the same as those of penicillin G and higher than those of ampicillin. Images PMID:6448021

  12. Control of domain swapping in bovine odorant-binding protein.

    PubMed Central

    Ramoni, Roberto; Vincent, Florence; Ashcroft, Alison E; Accornero, Paolo; Grolli, Stefano; Valencia, Christel; Tegoni, Mariella; Cambillau, Christian

    2002-01-01

    As revealed by the X-ray structure, bovine odorant-binding protein (OBPb) is a domain swapped dimer [Tegoni, Ramoni, Bignetti, Spinelli and Cambillau (1996) Nat. Struct. Biol. 3, 863-867; Bianchet, Bains, Petosi, Pevsner, Snyder, Monaco and Amzel (1996) Nat. Struct. Biol. 3, 934-939]. This contrasts with all known mammalian OBPs, which are monomers, and in particular with porcine OBP (OBPp), sharing 42.3% identity with OBPb. By the mechanism of domain swapping, monomers are proposed to evolve into dimers and oligomers, as observed in human prion. Comparison of bovine and porcine OBP sequences pointed at OBPp glycine 121, in the hinge linking the beta-barrel to the alpha-helix. The absence of this residue in OBPb might explain why the normal lipocalin beta-turn is not formed. In order to decipher the domain swapping determinants we have produced a mutant of OBPb in which a glycine residue was inserted after position 121, and a mutant of OBPp in which glycine 121 was deleted. The latter mutation did not result in dimerization, while OBPb-121Gly+ became monomeric, suggesting that domain swapping was reversed. Careful structural analysis revealed that besides the presence of a glycine in the hinge, the dimer interface formed by the C-termini and by the presence of the lipocalins conserved disulphide bridge may also control domain swapping. PMID:11931632

  13. Interphotoreceptor Retinoid-Binding Protein Protects Retinoids from Photodegradation

    PubMed Central

    Gonzalez-Fernandez, Federico; Betts-Obregon, Brandi; Yust, Brian; Mimun, Joshua; Sung, Dongjin; Sardar, Dhiraj; Tsin, Andrew T.

    2015-01-01

    Retinol degrades rapidly in light into a variety of photoproducts. It is remarkable that visual cycle retinoids can evade photodegradation as they are exchanged between the photoreceptors, retinal pigment epithelium and Müller glia. Within the interphotoreceptor matrix, all-trans retinol, 11-cis retinol and retinal are bound by interphotoreceptor retinoid-binding protein (IRBP). Apart from its role in retinoid trafficking and targeting, could IRBP have a photoprotective function? HPLC was used to evaluate the ability of IRBP to protect all-trans and 11-cis retinols from photodegradation when exposed to incandescent light (0 to 8,842 μW/cm2); time periods of 0 – 60 min, and bIRBP: retinol molar ratios of 1:1 to 1:5. bIRBP afforded a significant prevention of both all-trans and 11-cis retinol to rapid photodegradation. The effect was significant over the entire light intensity range tested, and extended to the bIRBP: retinol ratio 1:5. In view of the continual exposure of the retina to light, and the high oxidative stress in the outer retina, our results suggest IRBP may have an important protective role in the visual cycle by reducing photodegradation of all-trans and 11-cis retinols. This role of IRBP is particularly relevant in the high flux conditions of the cone visual cycle. PMID:25565073

  14. Rabies virus binding to the nicotinic acetylcholine receptor alpha subunit demonstrated by virus overlay protein binding assay.

    PubMed

    Gastka, M; Horvath, J; Lentz, T L

    1996-10-01

    A virus overlay protein binding assay was used to study binding of 125I-labelled rabies virus to the acetylcholine receptor (AChR) from Torpedo californica electric organ membranes. After gel electrophoresis of electric organ membranes and transfer of proteins to nitrocellulose, 125I-labelled alpha-bungarotoxin, a curaremimetic neurotoxin, bound to a 40 kDa band and 125I-labelled rabies virus bound to 51 kDa and 40 kDa bands. Binding of rabies virus to the 40 kDa band was inhibited by unlabelled alpha-bungarotoxin. In blots of affinity-purified AChR, labelled virus bound to the 40 kDa alpha subunit and was competed by alpha-bungarotoxin. Based on binding of rabies virus to the alpha subunit and the ability of alpha-bungarotoxin to compete for binding, rabies virus appears to bind to the neurotoxin-binding site of the nicotinic AChR alpha subunit.

  15. Crystal Structures and Binding Dynamics of Odorant-Binding Protein 3 from two aphid species Megoura viciae and Nasonovia ribisnigri

    PubMed Central

    Northey, Tom; Venthur, Herbert; De Biasio, Filomena; Chauviac, Francois-Xavier; Cole, Ambrose; Ribeiro, Karlos Antonio Lisboa; Grossi, Gerarda; Falabella, Patrizia; Field, Linda M.; Keep, Nicholas H.; Zhou, Jing-Jiang

    2016-01-01

    Aphids use chemical cues to locate hosts and find mates. The vetch aphid Megoura viciae feeds exclusively on the Fabaceae, whereas the currant-lettuce aphid Nasonovia ribisnigri alternates hosts between the Grossulariaceae and Asteraceae. Both species use alarm pheromones to warn of dangers. For N. ribisnigri this pheromone is a single component (E)-β-farnesene but M. viciae uses a mixture of (E)-β-farnesene, (−)-α-pinene, β-pinene, and limonene. Odorant-binding proteins (OBP) are believed to capture and transport such semiochemicals to their receptors. Here, we report the first aphid OBP crystal structures and examine their molecular interactions with the alarm pheromone components. Our study reveals some unique structural features: 1) the lack of an internal ligand binding site; 2) a striking groove in the surface of the proteins as a putative binding site; 3) the N-terminus rather than the C-terminus occupies the site closing off the conventional OBP pocket. The results from fluorescent binding assays, molecular docking and dynamics demonstrate that OBP3 from M. viciae can bind to all four alarm pheromone components and the differential ligand binding between these very similar OBP3s from the two aphid species is determined mainly by the direct π-π interactions between ligands and the aromatic residues of OBP3s in the binding pocket. PMID:27102935

  16. CCAAT box binding protein NF-Y facilitates in vivo recruitment of upstream DNA binding transcription factors.

    PubMed Central

    Wright, K L; Vilen, B J; Itoh-Lindstrom, Y; Moore, T L; Li, G; Criscitiello, M; Cogswell, P; Clarke, J B; Ting, J P

    1994-01-01

    NF-Y binds a CCAAT motif found in many eukaryotic polymerase II-dependent promoters. In the HLA-DRA promoter it has been demonstrated that stereo-specific alignment between this motif and the upstream elements X1 and X2 is required for activation. To study the underlying mechanism for this requirement, a panel of transfected cell lines that maintained integrated, wild-type and mutant promoters were analyzed by in vivo genomic footprinting. Cell lines harboring a mutated CCAAT element exhibited a loss of interactions at the CCAAT site, as expected, and no transcriptional activity. Most importantly, mutation of the CCAAT sequence nearly abolished in vivo binding at the X1 and X2 sites, while mutations of X1 and X2 had little effect on CCAAT box binding. However, X1 and X2 binding was interdependent. In vitro, X1 binding activities are known to be stabilized by NF-Y binding. Interaction between NF-Y and X box binding proteins was demonstrated by reciprocal co-immunoprecipitation in the absence of DNA and co-affinity purification in the presence of DNA. Collectively, these studies indicate that occupancy of the CCAAT element represents an early event affecting other protein-DNA interactions and suggest that NF-Y stabilizes and interacts with X box factors to mediate this function. These findings may represent a common theme among promoters containing a CCAAT element. Images PMID:8076600

  17. Ligand concentration regulates the pathways of coupled protein folding and binding.

    PubMed

    Daniels, Kyle G; Tonthat, Nam K; McClure, David R; Chang, Yu-Chu; Liu, Xin; Schumacher, Maria A; Fierke, Carol A; Schmidler, Scott C; Oas, Terrence G

    2014-01-22

    Coupled ligand binding and conformational change plays a central role in biological regulation. Ligands often regulate protein function by modulating conformational dynamics, yet the order in which binding and conformational change occurs are often hotly debated. Here we show that the "conformational selection versus induced fit" distinction on which this debate is based is a false dichotomy because the mechanism depends on ligand concentration. Using the binding of pyrophosphate (PPi) to Bacillus subtilis RNase P protein as a model, we show that coupled reactions are best understood as a change in flux between competing pathways with distinct orders of binding and conformational change. The degree of partitioning through each pathway depends strongly on PPi concentration, with ligand binding redistributing the conformational ensemble toward the folded state by both increasing folding rates and decreasing unfolding rates. These results indicate that ligand binding induces marked and varied changes in protein conformational dynamics, and that the order of binding and conformational change is ligand concentration dependent.

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

    PubMed Central

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

    2016-01-01

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

  19. Placental Vitamin D-Binding Protein Expression in Human Idiopathic Fetal Growth Restriction

    PubMed Central

    Wookey, Alice F.; Chollangi, Tejasvy; Yong, Hannah E. J.

    2017-01-01

    Vitamin D-binding protein is a multifunctional serum protein with multiple actions related to normal health. Vitamin D-binding protein transports vitamin D and influences the metabolism of this key hormone but it also has additional immunomodulatory and actin-clearing properties. We investigated whether vitamin D-binding protein expression is altered in fetal growth restriction-associated placental dysfunction. Protein was extracted from 35 placentae derived from 17 healthy control subjects and 18 gestation-matched subjects with fetal growth restriction (FGR). FGR subjects were further subdivided as idiopathic (n = 9) and nonidiopathic (n = 9). Vitamin D-binding protein and 25(OH) vitamin D were measured by ELISA and normalized to protein concentration. The results showed significantly reduced levels of placental vitamin D-binding protein (control versus FGR, p < 0.05, Student's t-test) that were strongly associated with idiopathic fetal growth restriction (p < 0.01, Kruskal-Wallis), whereas levels of vitamin D-binding protein were not associated with placental 25(OH) vitamin D stores (p = 0.295, Pearson's correlation). As such, vitamin D-binding protein may be a factor in unexplained placental dysfunction associated with idiopathic fetal growth restriction and may potentially serve as a biomarker of this disease. PMID:28293436

  20. The PSII-S protein of higher plants: a new type of pigment-binding protein.

    PubMed

    Funk, C; Schröder, W P; Napiwotzki, A; Tjus, S E; Renger, G; Andersson, B

    1995-09-05

    An intrinsic 22 kDa protein of photosystem II has been shown to possess high sequence homology with the CAB gene products, but differs from these proteins by an additional putative fourth transmembrane helix. This protein, designated PSII-S in accordance with the assignment of the name psbS to its gene, has been isolated by nonionic detergents and preparative isoelectric focusing in this study. The isolated PSII-S protein was shown to bind 5 chlorophyll molecules (a and b) per protein unit and also several different kinds of carotenoids. The room temperature absorption spectrum of the Qy transition of the chlorophylls bound to the isolated protein is characterized by a broad band with a maximum at 671 nm. The 77 K fluorescence spectrum exhibits a peak at 672 nm. A single photon counting technique was applied to resolve the room temperature decay kinetics of the first excited singlet states in the chlorophyll ensemble of the PSII-S protein. The data can be satisfactorily described by triexponential kinetics with lifetimes of tau 1 = 1.8 ns, tau 2 = 4.4 ns, and tau 3 = 6.1 ns and normalized amplitudes of 0.09, 0.60, and 0.31, respectively. Circular dichroism spectra suggest that, in contrast to LHCII, virtually no pigment coupling exists in the PSII-S protein. Two copies of the PSII-S protein were found per PSII in spinach thylakoids. It displays an unusually extreme lateral heterogeneity, since the PSII beta centers located in the stroma exposed thylakoid regions contained only residual amounts of the PSII-S protein.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. Ice-binding structure and mechanism of an antifreeze protein from winter flounder.

    PubMed

    Sicheri, F; Yang, D S

    1995-06-01

    Antifreeze proteins provide fish with protection against the freezing effect of polar environments by binding to ice surfaces and inhibiting growth of ice crystals. We present the X-ray crystal structure at 1.5 A resolution of a lone alpha-helical antifreeze protein from winter flounder, which provides a detailed look at its ice-binding features. These consist of four repeated ice-binding motifs, the side chains of which are inherently rigid or restrained by pair-wise side-chain interactions to form a flat binding surface. Elaborate amino- and carboxy-terminal cap structures are also present, which explain the protein's rich alpha-helical content in solution. We propose an ice-binding model that accounts for the binding specificity of the antifreeze protein along the <0112> axes of the (2021) ice planes.

  2. Properties of proteins binding plasma progesterone in pregnant Cape porcupines (Hystrix africaeaustralis).

    PubMed

    Louw, A I; van Wyk, V; van Aarde, R J

    1992-09-01

    The properties of progesterone-binding proteins in plasma of pregnant Cape porcupines were investigated using radiolabelled progesterone and either progesterone or cortisol as competing ligands as well as native plasma and heated (60 degrees C for 30 min) plasma. The results demonstrated that plasma from pregnant porcupines contains corticosteroid-binding globulin, but that it constitutes a significant portion of plasma progesterone-binding proteins only during the early stages of pregnancy. Corticosteroid-binding globulin of porcupines appears to be as heat labile as that of guinea-pigs. Concentrations of progesterone-binding proteins in plasma increased during pregnancy to reach concentrations at the eleventh week that were 25 times higher than those of progesterone; concentrations increased significantly (r2 = 0.88) with the increase in progesterone concentration. The results indicate that plasma progesterone-binding proteins in Cape porcupines (Old World hystricomorph) are similar in composition to those in guinea-pigs (New World hystricomorph).

  3. Characterization of single stranded telomeric DNA-binding proteins in cultured soybean (Glycine max) cells.

    PubMed

    Kwon, Chian; Kwon, Kisang; Chung, In Kwon; Kim, Soon Young; Cho, Myeon Haeng; Kang, Bin Goo

    2004-06-30

    We have identified and characterized a protein factor in soybean (Glycine max) nuclear extracts that binds to plant single stranded telomeric DNA repeats. A single DNA-protein complex was detected in gel retardation assays using synthetic telomeres and nuclear extracts. The protein forming this complex was designated soy-bean (Glycine max) single stranded telomeric DNA-binding protein (Gm-STBP). Gm-STBP binds to single stranded telomeric DNA containing more than two repeats. It does not bind to Tetrahymena, human or mutated plant telomere sequences, and its binding activity is not affected by RNase treatment. Gm-STBP activity gradually decreased after suspension cultures entered stationary phase. A slower migrating band was formed with extracts of earlier and later phases of soybean suspension cultures. Our findings suggest that binding of Gm-STBP to plant single stranded telomeric DNA may play a role in the proper functioning of telomeres during development.

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

    PubMed Central

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

    1996-01-01

    Binding studies were conducted to identify the anatomical location of brain target sites for OB protein, the ob gene product. 125I-labeled recombinant mouse OB protein or alkaline phosphatase-OB fusion proteins were used for in vitro and in vivo binding studies. Coronal brain sections or fresh tissue from lean, obese ob/ob, and obese db/db mice as well as lean and obese Zucker rats were probed to identify potential central OB protein-binding sites. We report here that recombinant OB protein binds specifically to the choroid plexus. The binding of OB protein (either radiolabeled or the alkaline phosphatase-OB fusion protein) and its displacement by unlabeled OB protein was similar in lean, obese ob/ob, and obese db/db mice as well as lean and obese Zucker rats. These findings suggest that OB protein binds with high affinity to a specific receptor in the choroid plexus. After binding to the choroid plexus receptor, OB protein may then be transported across the blood-brain barrier into the cerebrospinal fluid. Alternatively, binding of OB protein to a specific receptor in the choroid plexus may activate afferent neural inputs to the neural network that regulates feeding behavior and energy balance or may result in the clearance or degradation of OB protein. The identification of the choroid plexus as a brain binding site for OB protein will provide the basis for the construction of expression libraries and facilitate the rapid cloning of the choroid plexus OB receptor. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8643634

  5. Steady-State Fluorescence Anisotropy to Investigate Flavonoids Binding to Proteins

    ERIC Educational Resources Information Center

    Ingersoll, Christine M.; Strollo, Christen M.

    2007-01-01

    The steady-state fluorescence anisotropy is employed to study the binding of protein of a model protein, human serum albumin, to a commonly used flavonoid, quercetin. The experiment describes the thermodynamics, as well as the biochemical interactions of such binding effectively.

  6. Distribution of PASTA domains in penicillin-binding proteins and serine/threonine kinases of Actinobacteria.

    PubMed

    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.

  7. Direct Determination of Vibrational Density of States Change on Ligand Binding to a Protein

    NASA Astrophysics Data System (ADS)

    Balog, Erika; Becker, Torsten; Oettl, Martin; Lechner, Ruep; Daniel, Roy; Finney, John; Smith, Jeremy C.

    2004-07-01

    The change in the vibrational density of states of a protein (dihydrofolate reductase) on binding a ligand (methotrexate) is determined using inelastic neutron scattering. The vibrations of the complex soften significantly relative to the unbound protein. The resulting free-energy change, which is directly determined by the density of states change, is found to contribute significantly to the binding equilibrium.

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

    PubMed Central

    2014-01-01

    Several animal viruses encode proteins that bind double-stranded RNA (dsRNA) to counteract host dsRNA-dependent antiviral responses. This article discusses the structure and function of the dsRNA-binding proteins of influenza A virus and Ebola viruses (EBOVs). PMID:24905203

  9. An Experimentally Based Computer Search Identifies Unstructured Membrane-binding Sites in Proteins

    PubMed Central

    Brzeska, Hanna; Guag, Jake; Remmert, Kirsten; Chacko, Susan; Korn, Edward D.

    2010-01-01

    Programs exist for searching protein sequences for potential membrane-penetrating segments (hydrophobic regions) and for lipid-binding sites with highly defined tertiary structures, such as PH, FERM, C2, ENTH, and other domains. However, a rapidly growing number of membrane-associated proteins (including cytoskeletal proteins, kinases, GTP-binding proteins, and their effectors) bind lipids through less structured regions. Here, we describe the development and testing of a simple computer search program that identifies unstructured potential membrane-binding sites. Initially, we found that both basic and hydrophobic amino acids, irrespective of sequence, contribute to the binding to acidic phospholipid vesicles of synthetic peptides that correspond to the putative membrane-binding domains of Acanthamoeba class I myosins. Based on these results, we modified a hydrophobicity scale giving Arg- and Lys-positive, rather than negative, values. Using this basic and hydrophobic scale with a standard search algorithm, we successfully identified previously determined unstructured membrane-binding sites in all 16 proteins tested. Importantly, basic and hydrophobic searches identified previously unknown potential membrane-binding sites in class I myosins, PAKs and CARMIL (capping protein, Arp2/3, myosin I linker; a membrane-associated cytoskeletal scaffold protein), and synthetic peptides and protein domains containing these newly identified sites bound to acidic phospholipids in vitro. PMID:20018884

  10. Direct determination of vibrational density of states change on ligand binding to a protein.

    PubMed

    Balog, Erika; Becker, Torsten; Oettl, Martin; Lechner, Ruep; Daniel, Roy; Finney, John; Smith, Jeremy C

    2004-07-09

    The change in the vibrational density of states of a protein (dihydrofolate reductase) on binding a ligand (methotrexate) is determined using inelastic neutron scattering. The vibrations of the complex soften significantly relative to the unbound protein. The resulting free-energy change, which is directly determined by the density of states change, is found to contribute significantly to the binding equilibrium.

  11. Identification of procollagen promoter DNA-binding proteins: effects of dexamethasone

    SciTech Connect

    Sweeney, C.; Cutroneo, K.R.

    1987-05-01

    Glucocorticoids selectively decrease procollagen synthesis by decreasing procollagen mRNA transcription. Dexamethasone coordinately decreased total cellular type I and type III procollagen mRNAs in mouse embryonic skin fibroblasts. Since sequence specific DNA-binding proteins are known to modulate eukaryotic gene expression the authors identified in mouse fibroblasts nuclear proteins which bind to types I and III procollagen promoter DNAs. Nuclear proteins were electrophoresed, blotted onto nitrocellulose and probed with /sup 32/P-end-labeled type I and type III procollagen promoter DNAs in the presence of equimolar amounts of /sup 32/P-end-labeled vector DNA. Differences in total DNA binding were noted by the densitometric scans of the nuclear proteins. Dexamethasone treatment enhanced total DNA binding. Increasing the NaCl concentration decreased the number of promoter DNA-binding proteins without altering the relative specificity for the promoter DNAs. Promoter DNA binding to nuclear proteins was also inhibited by increasing concentrations of E. coli DNA. The number of DNA-binding proteins was greater for type III procollagen promoter DNA. The effect of dexamethasone treatment on promoter DNA binding to nuclear proteins was determined.

  12. Computational Exploration of a Protein Receptor Binding Space with Student Proposed Peptide Ligands

    ERIC Educational Resources Information Center

    King, Matthew D.; Phillips, Paul; Turner, Matthew W.; Katz, Michael; Lew, Sarah; Bradburn, Sarah; Andersen, Tim; McDougal, Owen M.

    2016-01-01

    Computational molecular docking is a fast and effective "in silico" method for the analysis of binding between a protein receptor model and a ligand. The visualization and manipulation of protein to ligand binding in three-dimensional space represents a powerful tool in the biochemistry curriculum to enhance student learning. The…

  13. Structure, Function, and Evolution of Biogenic Amine-binding Proteins in Soft Ticks

    SciTech Connect

    Mans, Ben J.; Ribeiro, Jose M.C.; Andersen, John F.

    2008-08-19

    Two highly abundant lipocalins, monomine and monotonin, have been isolated from the salivary gland of the soft tick Argas monolakensis and shown to bind histamine and 5-hydroxytryptamine (5-HT), respectively. The crystal structures of monomine and a paralog of monotonin were determined in the presence of ligands to compare the determinants of ligand binding. Both the structures and binding measurements indicate that the proteins have a single binding site rather than the two sites previously described for the female-specific histamine-binding protein (FS-HBP), the histamine-binding lipocalin of the tick Rhipicephalus appendiculatus. The binding sites of monomine and monotonin are similar to the lower, low affinity site of FS-HBP. The interaction of the protein with the aliphatic amine group of the ligand is very similar for the all of the proteins, whereas specificity is determined by interactions with the aromatic portion of the ligand. Interestingly, protein interaction with the imidazole ring of histamine differs significantly between the low affinity binding site of FS-HBP and monomine, suggesting that histamine binding has evolved independently in the two lineages. From the conserved features of these proteins, a tick lipocalin biogenic amine-binding motif could be derived that was used to predict biogenic amine-binding function in other tick lipocalins. Heterologous expression of genes from salivary gland libraries led to the discovery of biogenic amine-binding proteins in soft (Ornithodoros) and hard (Ixodes) tick genera. The data generated were used to reconstruct the most probable evolutionary pathway for the evolution of biogenic amine-binding in tick lipocalins.

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

    SciTech Connect

    A Bagaria; D Kumaran; S Burley; S Swaminathan

    2011-12-31

    The APT-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 nontransport 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 pro