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Sample records for matrix protein binding

  1. Binding of triiodothyronine to rat liver nuclear matrix. influence of thyroid hormones on the phosphorylation of nuclear matrix proteins

    International Nuclear Information System (INIS)

    Adylova, A.T.; Atakhanova, B.A.

    1986-01-01

    The interaction of thyroid hormones with rat liver nuclear matrix proteins was investigated. It was shown that the nuclear matrix contains sites that bind triiodothyronine with high affinity (K = 1.07.10 9 M -1 ) and limited capacity (the maximum binding capacity is equal to 28 /SUP a/ .5 fmoles of triiodothyronine per 100 ug protein). Electrophoretic identification of the matrix proteins that bind triiodothyronine was performed. The molecular weight of the main triiodothyronine-binding fraction is 50,000-52,000. It was shown that the administration of triiodothyronine to thyroidectomized rats stimulates the phosphorylation of all the protein fractions of the nuclear matrix

  2. Binding of recombinant apolipoprotein(a) to extracellular matrix proteins

    NARCIS (Netherlands)

    van der Hoek, Y. Y.; Sangrar, W.; Côté, G. P.; Kastelein, J. J.; Koschinsky, M. L.

    1994-01-01

    Elevated levels of lipoprotein(a), which consists of apolipoprotein(a) [apo(a)] covalently linked to a low-density lipoprotein-like moiety, is an independent risk factor for the development of atherosclerosis. We show that a recombinant form of apo(a) [r-apo(a)] binds strongly to fibronectin and

  3. Adhesion properties of Lactobacillus rhamnosus mucus-binding factor to mucin and extracellular matrix proteins.

    Science.gov (United States)

    Nishiyama, Keita; Nakamata, Koichi; Ueno, Shintaro; Terao, Akari; Aryantini, Ni Putu Desy; Sujaya, I Nengah; Fukuda, Kenji; Urashima, Tadasu; Yamamoto, Yuji; Mukai, Takao

    2015-01-01

    We previously described potential probiotic Lactobacillus rhamnosus strains, isolated from fermented mare milk produced in Sumbawa Island, Indonesia, which showed high adhesion to porcine colonic mucin (PCM) and extracellular matrix (ECM) proteins. Recently, mucus-binding factor (MBF) was found in the GG strain of L. rhamnosus as a mucin-binding protein. In this study, we assessed the ability of recombinant MBF protein from the FSMM22 strain, one of the isolates of L. rhamnosus from fermented Sumbawa mare milk, to adhere to PCM and ECM proteins by overlay dot blot and Biacore assays. MBF bound to PCM, laminin, collagen IV, and fibronectin with submicromolar dissociation constants. Adhesion of the FSMM22 mbf mutant strain to PCM and ECM proteins was significantly less than that of the wild-type strain. Collectively, these results suggested that MBF contribute to L. rhamnosus host colonization via mucin and ECM protein binding.

  4. Binding of a cementum attachment protein to extracellular matrix components and to dental surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Pitaru, S; Hekmati, H [Department of Oral Biology, Goldschleger School of Dental Medicine, Tel Aviv University (Israel); Savion, N [Goldschleger Eye Institute, Sackler School of Medicine, Tel Aviv University (Israel); Olsen, S; Narayanan, S A [Department of Pathology, School of Medicine, University of Washington, Seattle, Washington (United States)

    1992-01-01

    Cementum proteins (CP) have been shown to mediate cell attachment. Among these, a 55 kDa protein was isolated. The purpose of the present study was to assess the capacity of CP to bind to non-demineralized and demineralized root surfaces and to support cell attachment to dentin. CP were prepared by sequential extraction of bovine cementum with 25 mM EDTA, 0.5 M acetic acid followed by 4 M guanidine HCl. The latter was subjected to ion exchange chromatography on a DEAE-3SW column and eluted stepwise with a 0-0.5 M NaCl gradient. CP were labelled with [sup 125]I and the capacity of [sup 125]I-CP to bind to mineralized and partially demineralized dentin, synthetic hydroxyapatite, collagen, fibronectin and fibrillar collagen-fibronectin cimplex was assessed. It was found that CP bind specifically to mineralized dentin and synthetic hydroxyapatite but not to demineralized dentin. The specific binding was 60% of the total binding. SDS-PAGE analysis of the proteins bound to dentin indicated that the main bound protein had a molecular weight of 55 kDa. CP exhibited high affinity for fibronectin (k[sub D] = 1.56 x 10[sup -10] M) and fibronectincollagen complex, but their binding to either molecular or fibrillar collagen was negligible. It is suggested that CP may play an important role in the attachment of cells of the periodontium to cementum extracellular matrix during homeostasis and regeneration. (au).

  5. Assessment of algorithms for inferring positional weight matrix motifs of transcription factor binding sites using protein binding microarray data.

    Directory of Open Access Journals (Sweden)

    Yaron Orenstein

    Full Text Available The new technology of protein binding microarrays (PBMs allows simultaneous measurement of the binding intensities of a transcription factor to tens of thousands of synthetic double-stranded DNA probes, covering all possible 10-mers. A key computational challenge is inferring the binding motif from these data. We present a systematic comparison of four methods developed specifically for reconstructing a binding site motif represented as a positional weight matrix from PBM data. The reconstructed motifs were evaluated in terms of three criteria: concordance with reference motifs from the literature and ability to predict in vivo and in vitro bindings. The evaluation encompassed over 200 transcription factors and some 300 assays. The results show a tradeoff between how the methods perform according to the different criteria, and a dichotomy of method types. Algorithms that construct motifs with low information content predict PBM probe ranking more faithfully, while methods that produce highly informative motifs match reference motifs better. Interestingly, in predicting high-affinity binding, all methods give far poorer results for in vivo assays compared to in vitro assays.

  6. The anchorless adhesin Eap (extracellular adherence protein) from Staphylococcus aureus selectively recognizes extracellular matrix aggregates but binds promiscuously to monomeric matrix macromolecules.

    Science.gov (United States)

    Hansen, Uwe; Hussain, Muzaffar; Villone, Daniela; Herrmann, Mathias; Robenek, Horst; Peters, Georg; Sinha, Bhanu; Bruckner, Peter

    2006-05-01

    Besides a number of cell wall-anchored adhesins, the majority of Staphylococcus aureus strains produce anchorless, cell wall-associated proteins, such as Eap (extracellular adherence protein). Eap contains four to six tandem repeat (EAP)-domains. Eap mediates diverse biological functions, including adherence and immunomodulation, thus contributing to S. aureus pathogenesis. Eap binding to host macromolecules is unusually promiscuous and includes matrix or matricellular proteins as well as plasma proteins. The structural basis of this promiscuity is poorly understood. Here, we show that in spite of the preferential location of the binding epitopes within triple helical regions in some collagens there is a striking specificity of Eap binding to different collagen types. Collagen I, but not collagen II, is a binding substrate in monomolecular form. However, collagen I is virtually unrecognized by Eap when incorporated into banded fibrils. By contrast, microfibrils containing collagen VI as well as basement membrane-associated networks containing collagen IV, or aggregates containing fibronectin bound Eap as effectively as the monomeric proteins. Therefore, Eap-binding to extracellular matrix ligands is promiscuous at the molecular level but not indiscriminate with respect to supramolecular structures containing the same macromolecules. In addition, Eap bound to banded fibrils after their partial disintegration by matrix-degrading proteinases, including matrix metalloproteinase 1. Therefore, adherence to matrix suprastructures by S. aureus can be supported by inflammatory reactions.

  7. Structure and self-assembly of the calcium binding matrix protein of human metapneumovirus.

    Science.gov (United States)

    Leyrat, Cedric; Renner, Max; Harlos, Karl; Huiskonen, Juha T; Grimes, Jonathan M

    2014-01-07

    The matrix protein (M) of paramyxoviruses plays a key role in determining virion morphology by directing viral assembly and budding. Here, we report the crystal structure of the human metapneumovirus M at 2.8 Å resolution in its native dimeric state. The structure reveals the presence of a high-affinity Ca²⁺ binding site. Molecular dynamics simulations (MDS) predict a secondary lower-affinity site that correlates well with data from fluorescence-based thermal shift assays. By combining small-angle X-ray scattering with MDS and ensemble analysis, we captured the structure and dynamics of M in solution. Our analysis reveals a large positively charged patch on the protein surface that is involved in membrane interaction. Structural analysis of DOPC-induced polymerization of M into helical filaments using electron microscopy leads to a model of M self-assembly. The conservation of the Ca²⁺ binding sites suggests a role for calcium in the replication and morphogenesis of pneumoviruses. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Staphylococcus aureus manganese transport protein C (MntC is an extracellular matrix- and plasminogen-binding protein.

    Directory of Open Access Journals (Sweden)

    Natália Salazar

    Full Text Available Infections caused by Staphylococcus aureus--particularly nosocomial infections--represent a great concern. Usually, the early stage of pathogenesis consists on asymptomatic nasopharynx colonization, which could result in dissemination to other mucosal niches or invasion of sterile sites, such as blood. This pathogenic route depends on scavenging of nutrients as well as binding to and disrupting extracellular matrix (ECM. Manganese transport protein C (MntC, a conserved manganese-binding protein, takes part in this infectious scenario as an ion-scavenging factor and surprisingly as an ECM and coagulation cascade binding protein, as revealed in this work. This study showed a marked ability of MntC to bind to several ECM and coagulation cascade components, including laminin, collagen type IV, cellular and plasma fibronectin, plasminogen and fibrinogen by ELISA. The MntC binding to plasminogen appears to be related to the presence of surface-exposed lysines, since previous incubation with an analogue of lysine residue, ε-aminocaproic acid, or increasing ionic strength affected the interaction between MntC and plasminogen. MntC-bound plasminogen was converted to active plasmin in the presence of urokinase plasminogen activator (uPA. The newly released plasmin, in turn, acted in the cleavage of the α and β chains of fibrinogen. In conclusion, we describe a novel function for MntC that may help staphylococcal mucosal colonization and establishment of invasive disease, through the interaction with ECM and coagulation cascade host proteins. These data suggest that this potential virulence factor could be an adequate candidate to compose an anti-staphylococcal human vaccine formulation.

  9. Identification of the proteins responsible for SAR DNA binding in nuclear matrix of ''Cucurbita pepo''

    International Nuclear Information System (INIS)

    Rzepecki, R.; Markiewicz, E.; Szopa, J.

    1995-01-01

    The nuclear matrices from White bush (''Cucurbita pepo var. patisonina'') cell nuclei have been isolated using three methods: I, standard procedure involving extraction of cell nuclei with 2 M NaCl and 1% Triton X-100; II, the same with pre-treatment of cell nuclei with 0.5 mM CuSO 4 (stabilisation step); and III, method with extraction by lithium diiodosalicylate (LIS), and compared the polypeptide pattern. The isolated matrices specifically bind SAR DNA derived from human β-interferon gene in the exogenous SAR binding assay and in the gel mobility shift assay. Using IgG against the 32 kDa endonuclease we have found in the DNA-protein blot assay that this protein is one of the proteins binding SAR DNA. We have identified three proteins with molecular mass of 65 kDa, 60 kDa and 32 kDa which are responsible for SAR DNA binding in the gel mobility shift assay experiments. (author). 21 refs, 3 figs

  10. The structure of myristoylated Mason-Pfizer monkey virus matrix protein and the role of phosphatidylinositol-(4,5)-bisphosphate in its membrane binding.

    Science.gov (United States)

    Prchal, Jan; Srb, Pavel; Hunter, Eric; Ruml, Tomáš; Hrabal, Richard

    2012-10-26

    We determined the solution structure of myristoylated Mason-Pfizer monkey virus matrix protein by NMR spectroscopy. The myristoyl group is buried inside the protein and causes a slight reorientation of the helices. This reorientation leads to the creation of a binding site for phosphatidylinositols. The interaction between the matrix protein and phosphatidylinositols carrying C(8) fatty acid chains was monitored by observation of concentration-dependent chemical shift changes of the affected amino acid residues, a saturation transfer difference experiment and changes in (31)P chemical shifts. No differences in the binding mode or affinity were observed with differently phosphorylated phosphatidylinositols. The structure of the matrix protein-phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)] complex was then calculated with HADDOCK software based on the intermolecular nuclear Overhauser enhancement contacts between the ligand and the matrix protein obtained from a (13)C-filtered/(13)C-edited nuclear Overhauser enhancement spectroscopy experiment. PI(4,5)P(2) binding was not strong enough for triggering of the myristoyl-switch. The structural changes of the myristoylated matrix protein were also found to result in a drop in the oligomerization capacity of the protein. Copyright © 2012. Published by Elsevier Ltd.

  11. Microfibril-associated Protein 4 Binds to Surfactant Protein A (SP-A) and Colocalizes with SP-A in the Extracellular Matrix of the Lung

    DEFF Research Database (Denmark)

    Schlosser, Anders; Thomsen, Theresa H.; Shipley, J. Michael

    2006-01-01

    for phagocytes. Here we describe the molecular interaction between the extracellular matrix protein microfibril-associated protein 4 (MFAP4) and SP-A. MFAP4 is a collagen-binding molecule containing a C-terminal fibrinogen-like domain and a N-terminal located integrin-binding motif. We produced recombinant MFAP4......-A composed of the neck region and carbohydrate recognition domain of SP-A indicating that the interaction between MFAP4 and SP-A is mediated via the collagen domain of SP-A. Monoclonal antibodies directed against MFAP4 and SP-A were used for immunohistochemical analysis, which demonstrates that the two...... molecules colocalize both on the elastic fibres in the interalveolar septum and in elastic lamina of pulmonary arteries of chronically inflamed lung tissue. We conclude, that MFAP4 interacts with SP-A via the collagen region in vitro, and that MFAP4 and SP-A colocates in different lung compartments...

  12. Structure and function of ameloblastin as an extracellular matrix protein: adhesion, calcium binding, and CD63 interaction in human and mouse.

    Science.gov (United States)

    Zhang, Xu; Diekwisch, Thomas G H; Luan, Xianghong

    2011-12-01

    The functional significance of extracellular matrix proteins in the life of vertebrates is underscored by a high level of sequence variability in tandem with a substantial degree of conservation in terms of cell-cell and cell-matrix adhesion interactions. Many extracellular matrix proteins feature multiple adhesion domains for successful attachment to substrates, such as integrin, CD63, and heparin. Here we have used homology and ab initio modeling algorithms to compare mouse ameloblastin (mAMBN) and human ameloblastin (hABMN) isoforms and to analyze their potential for cell adhesion and interaction with other matrix molecules as well as calcium binding. Sequence comparison between mAMBN and hAMBN revealed a 26-amino-acid deletion in mAMBN, corresponding to a helix-loop-helix frameshift. The human AMBN domain (174Q-201G), homologous to the mAMBN 157E-178I helix-loop-helix region, formed a helix-loop motif with an extended loop, suggesting a higher degree of flexibility of hAMBN compared with mAMBN, as confirmed by molecular dynamics simulation. Heparin-binding domains, CD63-interaction domains, and calcium-binding sites in both hAMBN and mAMBN support the concept of AMBN as an extracellular matrix protein. The high level of conservation between AMBN functional domains related to adhesion and differentiation was remarkable when compared with only 61% amino acid sequence homology. © 2011 Eur J Oral Sci.

  13. General transfer matrix formalism to calculate DNA-protein-drug binding in gene regulation: application to OR operator of phage lambda.

    Science.gov (United States)

    Teif, Vladimir B

    2007-01-01

    The transfer matrix methodology is proposed as a systematic tool for the statistical-mechanical description of DNA-protein-drug binding involved in gene regulation. We show that a genetic system of several cis-regulatory modules is calculable using this method, considering explicitly the site-overlapping, competitive, cooperative binding of regulatory proteins, their multilayer assembly and DNA looping. In the methodological section, the matrix models are solved for the basic types of short- and long-range interactions between DNA-bound proteins, drugs and nucleosomes. We apply the matrix method to gene regulation at the O(R) operator of phage lambda. The transfer matrix formalism allowed the description of the lambda-switch at a single-nucleotide resolution, taking into account the effects of a range of inter-protein distances. Our calculations confirm previously established roles of the contact CI-Cro-RNAP interactions. Concerning long-range interactions, we show that while the DNA loop between the O(R) and O(L) operators is important at the lysogenic CI concentrations, the interference between the adjacent promoters P(R) and P(RM) becomes more important at small CI concentrations. A large change in the expression pattern may arise in this regime due to anticooperative interactions between DNA-bound RNA polymerases. The applicability of the matrix method to more complex systems is discussed.

  14. Mac-2 binding protein is a cell-adhesive protein of the extracellular matrix which self-assembles into ring-like structures and binds beta1 integrins, collagens and fibronectin

    DEFF Research Database (Denmark)

    Sasaki, T; Brakebusch, C; Engel, J

    1998-01-01

    Human Mac-2 binding protein (M2BP) was prepared in recombinant form from the culture medium of 293 kidney cells and consisted of a 92 kDa subunit. The protein was obtained in a native state as indicated by CD spectroscopy, demonstrating alpha-helical and beta-type structure, and by protease resis...... in the extracellular matrix of several mouse tissues....... in solid-phase assays to collagens IV, V and VI, fibronectin and nidogen, but not to fibrillar collagens I and III or other basement membrane proteins. The protein also mediated adhesion of cell lines at comparable strength with laminin. Adhesion to M2BP was inhibited by antibodies to integrin beta1...

  15. IGF binding proteins.

    Science.gov (United States)

    Bach, Leon A

    2017-12-18

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

  16. Characterization of novel OmpA-like protein of Leptospira interrogans that binds extracellular matrix molecules and plasminogen.

    Science.gov (United States)

    Oliveira, Rosane; de Morais, Zenaide Maria; Gonçales, Amane Paldes; Romero, Eliete Caló; Vasconcellos, Silvio Arruda; Nascimento, Ana L T O

    2011-01-01

    Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease of human and veterinary concern. The identification of novel proteins that mediate host-pathogen interactions is important for understanding the bacterial pathogenesis as well as to identify protective antigens that would help fight the disease. We describe in this work the cloning, expression, purification and characterization of three predicted leptospiral membrane proteins, LIC10258, LIC12880 (Lp30) and LIC12238. We have employed Escherichia coli BL21 (SI) strain as a host expression system. Recently, we have identified LIC12238 as a plasminogen (PLG)-binding receptor. We show now that Lp30 and rLIC10258 are also PLG-receptors of Leptospira, both exhibiting dose-dependent and saturating binding (K(D), 68.8±25.2 nM and 167.39±60.1 nM, for rLIC10258 and rLIC12880, respectively). In addition, LIC10258, which is a novel OmpA-like protein, binds laminin and plasma fibronectin ECM molecules and hence, it was named Lsa66 (Leptospiral surface adhesin of 66 kDa). Binding of Lsa66 to ECM components was determined to be specific, dose-dependent and saturable, with a K(D) of 55.4±15.9 nM to laminin and of 290.8±11.8 nM to plasma fibronectin. Binding of the recombinant proteins to PLG or ECM components was assessed by using antibodies against each of the recombinant proteins obtained in mice and confirmed by monoclonal anti-polyhistidine antibodies. Lsa66 caused partial inhibition on leptospiral adherence to immobilized ECM and PLG. Moreover, this adhesin and rLIC12238 are recognized by antibodies in serum samples of confirmed leptospirosis cases. Thus, Lsa66 is a novel OmpA-like protein with dual activity that may promote the attachment of Leptospira to host tissues and may contribute to the leptospiral invasion. To our knowledge, this is the first leptospiral protein with ECM and PLG binding properties reported to date.

  17. Characterization of novel OmpA-like protein of Leptospira interrogans that binds extracellular matrix molecules and plasminogen.

    Directory of Open Access Journals (Sweden)

    Rosane Oliveira

    Full Text Available Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease of human and veterinary concern. The identification of novel proteins that mediate host-pathogen interactions is important for understanding the bacterial pathogenesis as well as to identify protective antigens that would help fight the disease. We describe in this work the cloning, expression, purification and characterization of three predicted leptospiral membrane proteins, LIC10258, LIC12880 (Lp30 and LIC12238. We have employed Escherichia coli BL21 (SI strain as a host expression system. Recently, we have identified LIC12238 as a plasminogen (PLG-binding receptor. We show now that Lp30 and rLIC10258 are also PLG-receptors of Leptospira, both exhibiting dose-dependent and saturating binding (K(D, 68.8±25.2 nM and 167.39±60.1 nM, for rLIC10258 and rLIC12880, respectively. In addition, LIC10258, which is a novel OmpA-like protein, binds laminin and plasma fibronectin ECM molecules and hence, it was named Lsa66 (Leptospiral surface adhesin of 66 kDa. Binding of Lsa66 to ECM components was determined to be specific, dose-dependent and saturable, with a K(D of 55.4±15.9 nM to laminin and of 290.8±11.8 nM to plasma fibronectin. Binding of the recombinant proteins to PLG or ECM components was assessed by using antibodies against each of the recombinant proteins obtained in mice and confirmed by monoclonal anti-polyhistidine antibodies. Lsa66 caused partial inhibition on leptospiral adherence to immobilized ECM and PLG. Moreover, this adhesin and rLIC12238 are recognized by antibodies in serum samples of confirmed leptospirosis cases. Thus, Lsa66 is a novel OmpA-like protein with dual activity that may promote the attachment of Leptospira to host tissues and may contribute to the leptospiral invasion. To our knowledge, this is the first leptospiral protein with ECM and PLG binding properties reported to date.

  18. Competitive protein binding assay

    International Nuclear Information System (INIS)

    Kaneko, Toshio; Oka, Hiroshi

    1975-01-01

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

  19. Construction of genetically engineered M13K07 helper phage for simultaneous phage display of gold binding peptide 1 and nuclear matrix protein 22 ScFv antibody.

    Science.gov (United States)

    Fatemi, Farnaz; Amini, Seyed Mohammad; Kharrazi, Sharmin; Rasaee, Mohammad Javad; Mazlomi, Mohammad Ali; Asadi-Ghalehni, Majid; Rajabibazl, Masoumeh; Sadroddiny, Esmaeil

    2017-11-01

    The most common techniques of antibody phage display are based on the use of M13 filamentous bacteriophages. This study introduces a new genetically engineered M13K07 helper phage displaying multiple copies of a known gold binding peptide on p8 coat proteins. The recombinant helper phages were used to rescue a phagemid vector encoding the p3 coat protein fused to the nuclear matrix protein 22 (NMP22) ScFv antibody. Transmission electron microscopy (TEM), UV-vis absorbance spectroscopy, and field emission scanning electron microscopy (FE-SEM) with energy dispersive X-ray spectroscopy (EDX) analysis revealed that the expression of gold binding peptide 1 (GBP1) on major coat protein p8 significantly enhances the gold-binding affinity of M13 phages. The recombinant bacteriophages at concentrations above 5×10 4 pfu/ml red-shifted the UV-vis absorbance spectra of gold nanoparticles (AuNPs); however, the surface plasmon resonance of gold nanoparticles was not changed by the wild type bacteriophages at concentrations up to 10 12 pfu/ml. The phage ELISA assay demonstrated the high affinity binding of bifunctional bacteriophages to NMP22 antigen at concentrations of 10 5 and 10 6 pfu/ml. Thus, the p3 end of the bifunctional bacteriophages would be able to bind to specific target antigen, while the AuNPs were assembled along the coat of virus for signal generation. Our results indicated that the complex of antigen-bacteriophages lead to UV-vis spectral changes of AuNPs and NMP22 antigen in concentration range of 10-80μg/ml can be detected by bifunctional bacteriophages at concentration of 10 4 pfu/ml. The ability of bifunctional bacteriophages to bind to antigen and generate signal at the same time, makes this approach applicable for identifying different antigens in immunoassay techniques. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Fusobacterium nucleatum binding to complement regulatory protein CD46 modulates the expression and secretion of cytokines and matrix metalloproteinases by oral epithelial cells.

    Science.gov (United States)

    Mahtout, Hayette; Chandad, Fatiha; Rojo, Jose M; Grenier, Daniel

    2011-02-01

    Periodontitis is a chronic inflammatory disease that results in the destruction of the supporting tissues of the teeth. Gingival epithelial cells are an important mechanical barrier and participate in the host inflammatory response to periodontopathogens. The aim of the present study is to investigate the capacity of Fusobacterium nucleatum to bind to the complement regulatory protein CD46 expressed by oral epithelial cells and to determine the impact of the binding on the gene expression and protein secretion of interleukin (IL)-6, IL-8, and matrix metalloproteinase (MMP)-9 by oral epithelial cells. Binding of recombinant human CD46 to the surface of F. nucleatum was demonstrated by immunologic assays. After stimulation of oral epithelial cells with F. nucleatum, gene expression was determined by real-time polymerase chain reaction analysis while protein secretion was monitored by enzyme-linked immunosorbent assays. Heat and protease treatments of bacterial cells reduced CD46 binding. F. nucleatum-bound CD46 mediated the cleavage of C3b in the presence of factor I. Stimulating oral epithelial cells with F. nucleatum at a multiplicity of infection of 50 resulted in a significant upregulation of the gene expression and protein secretion of IL-6, IL-8, and MMP-9 by oral epithelial cells. However, pretreating the epithelial cells with an anti-CD46 polyclonal antibody attenuated the production of IL-6, IL-8, and MMP-9 in response to F. nucleatum. Such an inhibitory effect was not observed with non-specific antibodies. The present study demonstrates that F. nucleatum can bind the complement regulatory protein CD46. The interaction of F. nucleatum with epithelial cell surface CD46 may contribute to increasing the levels of proinflammatory mediators and MMPs in periodontal sites and consequently modulate tissue destruction.

  1. LipL53, a temperature regulated protein from Leptospira interrogans that binds to extracellular matrix molecules.

    Science.gov (United States)

    Oliveira, Tatiane R; Longhi, Mariana T; Gonçales, Amane P; de Morais, Zenaide M; Vasconcellos, Silvio A; Nascimento, Ana L T O

    2010-03-01

    The regulation of gene expression by environmental signals, such as temperature and osmolarity, has been correlated with virulence. In this study, we characterize the protein LipL53 from Leptospira interrogans, previously shown to react with serum sample of individual diagnosed with leptospirosis and to be up-regulated by shift to physiological osmolarity. The recombinant protein was expressed in Escherichia coli system, in insoluble form, recovered by urea solubilization and further refolded by decreasing the denaturing agent concentration during the purification procedure. The secondary structure content of the recombinant LipL53, as assessed by circular dichroism, showed a mixture of beta-strands and alpha-helix. The presence of LipL53 transcript at 28 degrees C was only detected within the virulent strains. However, upon shifted of attenuated cultures of pathogenic strains from 28 degrees C to 37 degrees C and to 39 degrees C, this transcript could also be observed. LipL53 binds laminin, collagen IV, cellular and plasma fibronectin in dose-dependent and saturable manner. Animal challenge studies showed that LipL53, although immunogenic, elicited only partial protection in hamsters. LipL53 is probably surface exposed as seen through immunofluorescence confocal microscopy. Our results suggest that LipL53 is a novel temperature regulated adhesin of L. interrogans that may be relevant in the leptospiral pathogenesis. Copyright 2009 Elsevier Masson SAS. All rights reserved.

  2. Amphipathic alpha-helices and putative cholesterol binding domains of the influenza virus matrix M1 protein are crucial for virion structure organisation.

    Science.gov (United States)

    Tsfasman, Tatyana; Kost, Vladimir; Markushin, Stanislav; Lotte, Vera; Koptiaeva, Irina; Bogacheva, Elena; Baratova, Ludmila; Radyukhin, Victor

    2015-12-02

    The influenza virus matrix M1 protein is an amphitropic membrane-associated protein, forming the matrix layer immediately beneath the virus raft membrane, thereby ensuring the proper structure of the influenza virion. The objective of this study was to elucidate M1 fine structural characteristics, which determine amphitropic properties and raft membrane activities of the protein, via 3D in silico modelling with subsequent mutational analysis. Computer simulations suggest the amphipathic nature of the M1 α-helices and the existence of putative cholesterol binding (CRAC) motifs on six amphipathic α-helices. Our finding explains for the first time many features of this protein, particularly the amphitropic properties and raft/cholesterol binding potential. To verify these results, we generated mutants of the A/WSN/33 strain via reverse genetics. The M1 mutations included F32Y in the CRAC of α-helix 2, W45Y and W45F in the CRAC of α-helix 3, Y100S in the CRAC of α-helix 6, M128A and M128S in the CRAC of α-helix 8 and a double L103I/L130I mutation in both a putative cholesterol consensus motif and the nuclear localisation signal. All mutations resulted in viruses with unusual filamentous morphology. Previous experimental data regarding the morphology of M1-gene mutant influenza viruses can now be explained in structural terms and are consistent with the pivotal role of the CRAC-domains and amphipathic α-helices in M1-lipid interactions. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Binding of matrix metalloproteinase inhibitors to extracellular matrix: 3D-QSAR analysis.

    Science.gov (United States)

    Zhang, Yufen; Lukacova, Viera; Bartus, Vladimir; Nie, Xiaoping; Sun, Guorong; Manivannan, Ethirajan; Ghorpade, Sandeep R; Jin, Xiaomin; Manyem, Shankar; Sibi, Mukund P; Cook, Gregory R; Balaz, Stefan

    2008-10-01

    Binding to the extracellular matrix, one of the most abundant human protein complexes, significantly affects drug disposition. Specifically, the interactions with extracellular matrix determine the free concentrations of small molecules acting in tissues, including signaling peptides, inhibitors of tissue remodeling enzymes such as matrix metalloproteinases, and other drug candidates. The nature of extracellular matrix binding was elucidated for 63 matrix metalloproteinase inhibitors, for which the association constants to an extracellular matrix mimic were reported here. The data did not correlate with lipophilicity as a common determinant of structure-nonspecific, orientation-averaged binding. A hypothetical structure of the binding site of the solidified extracellular matrix surrogate was analyzed using the Comparative Molecular Field Analysis, which needed to be applied in our multi-mode variant. This fact indicates that the compounds bind to extracellular matrix in multiple modes, which cannot be considered as completely orientation-averaged and exhibit structural dependence. The novel comparative molecular field analysis models, exhibiting satisfactory descriptive and predictive abilities, are suitable for prediction of the extracellular matrix binding for the untested chemicals, which are within applicability domains. The results contribute to a better prediction of the pharmacokinetic parameters such as the distribution volume and the tissue-blood partition coefficients, in addition to a more imminent benefit for the development of more effective matrix metalloproteinase inhibitors.

  4. Cellulose binding domain proteins

    Science.gov (United States)

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

    1998-01-01

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

  5. N-terminal aliphatic residues dictate the structure, stability, assembly, and small molecule binding of the coiled-coil region of cartilage oligomeric matrix protein.

    Science.gov (United States)

    Gunasekar, Susheel K; Asnani, Mukta; Limbad, Chandani; Haghpanah, Jennifer S; Hom, Wendy; Barra, Hanna; Nanda, Soumya; Lu, Min; Montclare, Jin Kim

    2009-09-15

    The coiled-coil domain of cartilage oligomeric matrix protein (COMPcc) assembles into a homopentamer that naturally recognizes the small molecule 1,25-dihydroxyvitamin D(3) (vit D). To identify the residues critical for the structure, stability, oligomerization, and binding to vit D as well as two other small molecules, all-trans-retinol (ATR) and curcumin (CCM), here we perform an alanine scanning mutagenesis study. Ten residues lining the hydrophobic pocket of COMPcc were mutated into alanine; of the mutated residues, the N-terminal aliphatic residues L37, L44, V47, and L51 are responsible for maintaining the structure and function. Furthermore, two polar residues, T40 and Q54, within the N-terminal region when converted into alanine improve the alpha-helical structure, stability, and self-assembly behavior. Helical stability, oligomerization, and binding appear to be linked in a manner in which mutations that abolish helical structure and assembly bind poorly to vit D, ATR, and CCM. These results provide not only insight into COMPcc and its functional role but also useful guidelines for the design of stable, pentameric coiled-coils capable of selectively storing and delivering various small molecules.

  6. [Expression of erythroblastic leukemia viral oncogene homolog 3 (ErbB-3) binding protein-1, matrix metalloproteinases, eplthelial cadherin in adenoid cystic carcinoma and correlation analysis].

    Science.gov (United States)

    Sun, Jian; Yu, You-cheng; Luo, Yi-xi; Tian, Zhen

    2012-12-01

    To investigate the expression of ErbB-3 binding protein-1 (EBP-1), matrix metalloproteinase 9 (MMP-9) and E-cadherin (E-cad) in adenoid cystic carcinoma and their correlation. Immunohistochemistry(PV6000 method) was used to detect EBP-1, MMP-9 and E-cad expression in 66 cases of adenoid cystic carcinoma tissues and matched para-cancerous normal tissues. In this study all cases were successfully followed up. The positive expression rate of EBP-1 in adenoid cystic carcinoma tissues was 85%. EBP-1 expression was significantly correlated to pathological pattern and clinical stage (P correlation between EBP-1 and E-cad expression, and positive correlation between EBP-1 and MMP-9. EBP-1 and its correlation with MMP-9 and E-cad may be used as useful indicators for clinical assessment of tumor biological behavior and prognosis in patients with adenoid cystic carcinoma.

  7. Presence of Insulin-Like Growth Factor Binding Proteins Correlates With Tumor-Promoting Effects of Matrix Metalloproteinase 9 in Breast Cancer

    Directory of Open Access Journals (Sweden)

    Jae-Hyun Park

    2015-05-01

    Full Text Available The stroma of breast cancer can promote the disease’s progression, but whether its composition and functions are shared among different subtypes is poorly explored. We compared stromal components of a luminal [mouse mammary tumor virus (MMTV–Neu] and a triple-negative/basal-like [C3(1–Simian virus 40 large T antigen (Tag] genetically engineered breast cancer mouse model. The types of cytokines and their expression levels were very different in the two models, as was the extent of innate immune cell infiltration; however, both models showed infiltration of innate immune cells that expressed matrix metalloproteinase 9 (MMP9, an extracellular protease linked to the progression of many types of cancer. By intercrossing with Mmp9 null mice, we found that the absence of MMP9 delayed tumor onset in the C3(1-Tag model but had no effect on tumor onset in the MMTV-Neu model. We discovered that protein levels of insulin-like growth factor binding protein-1 (IGFBP-1, an MMP9 substrate, were increased in C3(1-Tag;Mmp9−/− compared to C3(1-Tag;Mmp9+/+ tumors. In contrast, IGFBP-1 protein expression was low in MMTV-Neu tumors regardless of Mmp9 status. IGFBP-1 binds and antagonizes IGFs, preventing them from activating their receptors to promote cell proliferation and survival. Tumors from C3(1-Tag;Mmp9−/− mice had reduced IGF-1 receptor phosphorylation, consistent with slower tumor onset. Finally, gene expression analysis of human breast tumors showed that high expression of IGFBP mRNA was strongly correlated with good prognosis but not when MMP9 mRNA was also highly expressed. In conclusion, MMP9 has different effects on breast cancer progression depending on whether IGFBPs are expressed.

  8. The anchorless adhesin Eap (extracellular adherence protein) from Staphylococcus aureus selectively recognizes extracellular matrix aggregates but binds promiscuously to monomeric matrix macromolecules

    NARCIS (Netherlands)

    Hansen, Uwe; Hussain, Muzaffar; Villone, Daniela; Herrmann, Mathias; Robenek, Horst; Peters, Georg; Sinha, Bhanu; Bruckner, Peter

    Besides a number of cell wall-anchored adhesins, the majority of Staphylococcus aureus strains produce anchorless, cell wall-associated proteins, such as Eap (extracellular adherence protein). Eap contains four to six tandem repeat (EAP)-domains. Eap mediates diverse biological functions, including

  9. MARs Wars: heterogeneity and clustering of DNA-binding domains in the nuclear matrix

    Directory of Open Access Journals (Sweden)

    Ioudinkova E. S.

    2009-12-01

    Full Text Available Aim. CO326 is a chicken nuclear scaffold/matrix attachment region (MAR associated with the nuclear matrix in several types of chicken cells. It contains a binding site for a sequence-specific DNA-binding protein, F326. We have studied its interaction with the nuclear matrix. Methods. We have used an in vitro MAR assay with isolated matrices from chicken HD3 cells. Results. We have found that an oligonucleotide binding site for the F326 inhibits binding of the CO326 to the nuclear matrix. At the same time, the binding of heterologous MARs is enhanced. Conclusions. Taken together, these data suggest that there exist several classes of MARs and MAR-binding domains and that the MAR-binding proteins may be clustered in the nuclear matrix.

  10. Protein binding of psychotropic agents

    International Nuclear Information System (INIS)

    Hassan, H.A.

    1990-01-01

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

  11. The Complement Binding and Inhibitory Protein CbiA of Borrelia miyamotoi Degrades Extracellular Matrix Components by Interacting with Plasmin(ogen

    Directory of Open Access Journals (Sweden)

    Ngoc T. T. Nguyen

    2018-02-01

    Full Text Available The emerging relapsing fever spirochete Borrelia (B. miyamotoi is transmitted by ixodid ticks and causes the so-called hard tick-borne relapsing fever or B. miyamotoi disease (BMD. More recently, we identified a surface-exposed molecule, CbiA exhibiting complement binding and inhibitory capacity and rendering spirochetes resistant to complement-mediated lysis. To gain deeper insight into the molecular principles of B. miyamotoi-host interaction, we examined CbiA as a plasmin(ogen receptor that enables B. miyamotoi to interact with the serine protease plasmin(ogen. Recombinant CbiA was able to bind plasminogen in a dose-dependent fashion. Moreover, lysine residues appear to play a crucial role in the protein-protein interaction as binding of plasminogen was inhibited by the lysine analog tranexamic acid as well as increasing ionic strength. Of relevance, plasminogen bound to CbiA can be converted by urokinase-type plasminogen activator (uPa to active plasmin which cleaved both, the chromogenic substrate S-2251 and its physiologic substrate fibrinogen. Concerning the involvement of specific amino acids in the interaction with plasminogen, lysine residues located at the C-terminus are frequently involved in the binding as reported for various other plasminogen-interacting proteins of Lyme disease spirochetes. Lysine residues located within the C-terminal domain were substituted with alanine to generate single, double, triple, and quadruple point mutants. However, binding of plasminogen to the mutated CbiA proteins was not affected, suggesting that lysine residues distant from the C-terminus might be involved in the interaction.

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

    DEFF Research Database (Denmark)

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

    1999-01-01

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

  13. The specific binding of the thyroid hormones to matrix isolated from rat liver nuclei

    International Nuclear Information System (INIS)

    Wilson, B.D.; Albrecht, C.F.; Wium, C.A.

    1982-01-01

    Specific binding sites for the thyroid hormones have been demonstrated in the liver nuclear matrix, a structural framework of the nucleus. When labelled 3,5,3'-tri-iodo-L-thyronine ([ 125 l]T 3 ) is injected into rats, 5% of the total nucleus bound T 3 is bound to the matrix after 1 hour. However, when either isolated nuclei or isolated nuclear matrices were incubated with[ 125 l]T 3 in vitro, a 3- to 7- fold greater number of specific T 3 binding sites were revealed in the nuclear matrix. The properties of the matrix-associated thyroid hormone binding sites were investigated in vitro. These binding sites showed limited capacity and high affinity for T 3 ; the equilibrium association constant (K(a)) was 1,3X10 M -1 and the binding capacity was 20,2 fmol T 3 per 100 μg matrix protein

  14. Cellulose binding domain fusion proteins

    Science.gov (United States)

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

    1998-01-01

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

  15. When is protein binding important?

    Science.gov (United States)

    Heuberger, Jules; Schmidt, Stephan; Derendorf, Hartmut

    2013-09-01

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

  16. Probing protein phosphatase substrate binding

    DEFF Research Database (Denmark)

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

    2012-01-01

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

  17. Human plasminogen binding protein tetranectin

    DEFF Research Database (Denmark)

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

    1997-01-01

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

  18. A collagen-binding EGFR antibody fragment targeting tumors with a collagen-rich extracellular matrix

    OpenAIRE

    Hui Liang; Xiaoran Li; Bin Wang; Bing Chen; Yannan Zhao; Jie Sun; Yan Zhuang; Jiajia Shi; He Shen; Zhijun Zhang; Jianwu Dai

    2016-01-01

    Many tumors over-express collagen, which constitutes the physical scaffold of tumor microenvironment. Collagen has been considered to be a target for cancer therapy. The collagen-binding domain (CBD) is a short peptide, which could bind to collagen and achieve the sustained release of CBD-fused proteins in collagen scaffold. Here, a collagen-binding EGFR antibody fragment was designed and expressed for targeting the collagen-rich extracellular matrix in tumors. The antibody fragment (Fab) of ...

  19. cAMP-response Element-binding Protein (CREB) and NF-κB Transcription Factors Are Activated during Prolonged Hypoxia and Cooperatively Regulate the Induction of Matrix Metalloproteinase MMP1*

    Science.gov (United States)

    Nakayama, Koh

    2013-01-01

    Responses to low levels of oxygen (hypoxia) are essential to maintain homeostasis. During the hypoxic response, gene expression is altered by various transcription factors. The transcription factor, hypoxia-inducible factor (HIF), plays a central role in the hypoxic response. The α subunit of HIF, which is actively degraded during normoxia, becomes stabilized during hypoxia, which leads to HIF activation. A microarray analysis of HeLa cells showed that expression of matrix metalloproteinase 1 (MMP1) was markedly induced during prolonged hypoxia. CREB and NF-κB binding sites were identified in the MMP1 promoter region between 1945 and 1896 nucleotides upstream of the transcription start site. Assays with luciferase reporters demonstrated that HIF activity was induced during the early phase of hypoxia, whereas CREB and NF-κB were activated during the later (prolonged) phase. Depletion of CREB and/or NF-κB reduced MMP1 induction during prolonged hypoxia both at the mRNA and protein levels. A chromatin immunoprecipitation assay demonstrated binding of CREB and NF-κB to the MMP1 promoter. Finally, cell migration and invasion on a collagen matrix and pulmonary metastasis in nude mice were inhibited after depletion of CREB and NF-κB in MDA-MB-231 cells. Taken together, these results suggest that the cooperative action of CREB and NF-κB plays an important role to induce MMP1 expression during prolonged hypoxia and regulates cell migration and invasion in cancer cells. PMID:23775082

  20. Polymeric competitive protein binding adsorbents for radioassay

    International Nuclear Information System (INIS)

    Adams, R.J.

    1976-01-01

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

  1. Lead-Binding Proteins: A Review

    Directory of Open Access Journals (Sweden)

    Harvey C. Gonick

    2011-01-01

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

  2. What Happened to the IGF Binding Proteins?

    Science.gov (United States)

    Bach, Leon A

    2018-02-01

    Insulinlike growth factor (IGF) binding proteins (IGFBPs) 1 to 6 are high-affinity regulators of IGF activity. They generally inhibit IGF actions by preventing binding to the IGF-I receptor but can also enhance their actions under some conditions. Posttranslational modifications such as glycosylation and phosphorylation modulate IGFBP properties, and IGFBP proteolysis results in IGF release. IGFBPs have more recently been shown to have IGF-independent actions. A number of mechanisms are involved, including modulation of other growth factor pathways, nuclear localization and transcriptional regulation, interaction with the sphingolipid pathway, and binding to non-IGF biomolecules in the extracellular space and matrix, on the cell surface and intracellularly. IGFBPs modulate important biological processes, including cell proliferation, survival, migration, senescence, autophagy, and angiogenesis. Their actions have been implicated in growth, metabolism, cancer, stem cell maintenance and differentiation, and immune regulation. Recent studies have shown that epigenetic mechanisms are involved in the regulation of IGFBP abundance. A more complete understanding of IGFBP biology is necessary to further define their cellular roles and determine their therapeutic potential. Copyright © 2018 Endocrine Society.

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

    DEFF Research Database (Denmark)

    Birn, Henrik; Zhai, Xiaoyue; Holm, Jan

    2005-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Yu-Ru Zhang

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

  5. Preferential binding of DNA primase to the nuclear matrix

    International Nuclear Information System (INIS)

    Wood, S.H.; Collins, J.M.

    1986-01-01

    Several lines of research have stimulated interest in the nuclear matrix as the subcellular site of DNA replication. The authors have recently reported a relationship between rates of DNA synthesis and the differential binding of polymerase α to the nuclear matrix. They now report the detection of DNA primase bound to the HeLa nuclear matrix. Matrix-bound primase can be measured either indirectly, by the incorporation of [ 32 P] dAMP into an unprimed single-stranded template, or directly, by the incorporation of [ 3 H] AMP into matrix DNA. Characteristics of this system include a requirement for ATP, inhibition by adenosine-5'-0-(3'-thiotriphosphate), a primase inhibitor, and insensitivity to aphidicolin and α-amanitine, inhibitors of polymerase α and RNA polymerase, respectively. Subcellular quantification of primase and polymerase α activity revealed that while a majority of primase activity is bound to the matrix (72%), only 32% of polymerase α activity is matrix-bound. Treatment of the nuclear matrix with β-D-Octylglucoside allowed the solubilization of 54% of primase activity and 39% of polymerase α activity. This data provides further evidence of a structural and functional role for the nuclear matrix in DNA replication. The ability to solubilize matrix-bound replicative enzymes may prove to be an important tool in the elucidation of the spatial organization of DNA replication

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

    International Nuclear Information System (INIS)

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

    1986-01-01

    Last year the authors reported that upon freezing and thawing mitochondria from rats injected with [ 75 Se]Na 2 SeO 3 ( 75 Se-selenite), a 75 Se-binding protein (SeBP) was released. They have studied further in vitro labelling of SeBP. This matrix protein was labelled in vitro when lysed mitochondria (containing non-matrix material) were incubated with 75 Se-selenite but not when matrix material alone was incubated with 75 Se-selenite. Thus, there are one or more promoters of in vitro SeBP labelling in the non-matrix fraction. SeBP was also labelled in vitro when 75 Se-selenite was added to matrix alone and dialyzed. Dialysis tubing, and not the dialysis process, promoted labelling by affecting SeBP and not by affecting 75 Se-selenite. Labelling did not occur when matrix alone and 75 Se-selenite were incubated (not dialyzed) in a glass test tube but did occur in a polystyrene test tube. They hypothesize that non-covalent interactions occur between SeBP and dialysis tubing or polystyrene that expose Se binding sites on the protein. A similar mechanism involving mitochondrial non-matrix material may function in vivo. Non-denaturing disc gel electrophoresis of partially purified SeBP labelled in vivo or in vitro suggested that the same protein was labelled in both conditions. Using in vitro binding techniques, SeBP was also found in sheep liver mitochondrial matrix. This supports the theory that SeBP is important in Se metabolism

  7. Ubiquitination of specific mitochondrial matrix proteins

    International Nuclear Information System (INIS)

    Lehmann, Gilad; Ziv, Tamar; Braten, Ori; Admon, Arie; Udasin, Ronald G.; Ciechanover, Aaron

    2016-01-01

    Several protein quality control systems in bacteria and/or mitochondrial matrix from lower eukaryotes are absent in higher eukaryotes. These are transfer-messenger RNA (tmRNA), The N-end rule ATP-dependent protease ClpAP, and two more ATP-dependent proteases, HslUV and ClpXP (in yeast). The lost proteases resemble the 26S proteasome and the role of tmRNA and the N-end rule in eukaryotic cytosol is performed by the ubiquitin proteasome system (UPS). Therefore, we hypothesized that the UPS might have substituted these systems – at least partially – in the mitochondrial matrix of higher eukaryotes. Using three independent experimental approaches, we demonstrated the presence of ubiquitinated proteins in the matrix of isolated yeast mitochondria. First, we show that isolated mitochondria contain ubiquitin (Ub) conjugates, which remained intact after trypsin digestion. Second, we demonstrate that the mitochondrial soluble fraction contains Ub-conjugates, several of which were identified by mass spectrometry and are localized to the matrix. Third, using immunoaffinity enrichment by specific antibodies recognizing digested ubiquitinated peptides, we identified a group of Ub-modified matrix proteins. The modification was further substantiated by separation on SDS-PAGE and immunoblots. Last, we attempted to identify the ubiquitin ligase(s) involved, and identified Dma1p as a trypsin-resistant protein in our mitochondrial preparations. Taken together, these data suggest a yet undefined role for the UPS in regulation of the mitochondrial matrix proteins. -- Highlights: •Mitochondrial matrix contains ubiquitinated proteins. •Ubiquitination occurs most probably in the matrix. •Dma1p is a ubiquitin ligase present in mitochondrial preparations.

  8. Ubiquitination of specific mitochondrial matrix proteins

    Energy Technology Data Exchange (ETDEWEB)

    Lehmann, Gilad [The Janet and David Polak Tumor and Vascular Biology Research Center and the Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Haifa, 31096 (Israel); Ziv, Tamar [The Smoler Proteomics Center, Faculty of Biology – Technion-Israel Institute of Technology, Haifa, 32000 (Israel); Braten, Ori [The Janet and David Polak Tumor and Vascular Biology Research Center and the Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Haifa, 31096 (Israel); Admon, Arie [The Smoler Proteomics Center, Faculty of Biology – Technion-Israel Institute of Technology, Haifa, 32000 (Israel); Udasin, Ronald G. [The Janet and David Polak Tumor and Vascular Biology Research Center and the Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Haifa, 31096 (Israel); Ciechanover, Aaron, E-mail: aaroncie@tx.technion.ac.il [The Janet and David Polak Tumor and Vascular Biology Research Center and the Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Haifa, 31096 (Israel)

    2016-06-17

    Several protein quality control systems in bacteria and/or mitochondrial matrix from lower eukaryotes are absent in higher eukaryotes. These are transfer-messenger RNA (tmRNA), The N-end rule ATP-dependent protease ClpAP, and two more ATP-dependent proteases, HslUV and ClpXP (in yeast). The lost proteases resemble the 26S proteasome and the role of tmRNA and the N-end rule in eukaryotic cytosol is performed by the ubiquitin proteasome system (UPS). Therefore, we hypothesized that the UPS might have substituted these systems – at least partially – in the mitochondrial matrix of higher eukaryotes. Using three independent experimental approaches, we demonstrated the presence of ubiquitinated proteins in the matrix of isolated yeast mitochondria. First, we show that isolated mitochondria contain ubiquitin (Ub) conjugates, which remained intact after trypsin digestion. Second, we demonstrate that the mitochondrial soluble fraction contains Ub-conjugates, several of which were identified by mass spectrometry and are localized to the matrix. Third, using immunoaffinity enrichment by specific antibodies recognizing digested ubiquitinated peptides, we identified a group of Ub-modified matrix proteins. The modification was further substantiated by separation on SDS-PAGE and immunoblots. Last, we attempted to identify the ubiquitin ligase(s) involved, and identified Dma1p as a trypsin-resistant protein in our mitochondrial preparations. Taken together, these data suggest a yet undefined role for the UPS in regulation of the mitochondrial matrix proteins. -- Highlights: •Mitochondrial matrix contains ubiquitinated proteins. •Ubiquitination occurs most probably in the matrix. •Dma1p is a ubiquitin ligase present in mitochondrial preparations.

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

    International Nuclear Information System (INIS)

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

    1982-01-01

    A minor cow's whey protein associated with β-lactoglobulin is used as binding protein in the competitive radioassay for serum and erythrocyte folate. Seeking to optimize the assay, we tested the performance of binder solutions of increasing purity. The folate binding protein was isolated from cow's whey by means of CM-Sepharose CL-6B cation-exchange chromatography, and further purified on a methotrexate-AH-Sepharose 4B affinity matrix. In contrast to β-lactoglobulin, the purified protein did not bind folate unless the detergents cetyltrimethylammonium (10 mmol/Ll) or Triton X-100 (1 g/L) were present. Such detergent activation was not needed in the presence of serum. There seems to be a striking analogy between these phenomena and the well-known reactivation of certain purified membrane-derived enzymes by surfactants

  10. Osmotic regulation of expression of two extracellular matrix-binding proteins and a haemolysin of Leptospira interrogans: differential effects on LigA and Sph2 extracellular release.

    Science.gov (United States)

    Matsunaga, James; Medeiros, Marco A; Sanchez, Yolanda; Werneid, Kristian F; Ko, Albert I

    2007-10-01

    The life cycle of the pathogen Leptospira interrogans involves stages outside and inside the host. Entry of L. interrogans from moist environments into the host is likely to be accompanied by the induction of genes encoding virulence determinants and the concomitant repression of genes encoding products required for survival outside of the host. The expression of the adhesin LigA, the haemolysin Sph2 (Lk73.5) and the outer-membrane lipoprotein LipL36 of pathogenic Leptospira species have been reported to be regulated by mammalian host signals. A previous study demonstrated that raising the osmolarity of the leptospiral growth medium to physiological levels encountered in the host by addition of various salts enhanced the levels of cell-associated LigA and LigB and extracellular LigA. In this study, we systematically examined the effects of osmotic upshift with ionic and non-ionic solutes on expression of the known mammalian host-regulated leptospiral genes. The levels of cell-associated LigA, LigB and Sph2 increased at physiological osmolarity, whereas LipL36 levels decreased, corresponding to changes in specific transcript levels. These changes in expression occurred irrespective of whether sodium chloride or sucrose was used as the solute. The increase of cellular LigA, LigB and Sph2 protein levels occurred within hours of adding sodium chloride. Extracellular Sph2 levels increased when either sodium chloride or sucrose was added to achieve physiological osmolarity. In contrast, enhanced levels of extracellular LigA were observed only with an increase in ionic strength. These results indicate that the mechanisms for release of LigA and Sph2 differ during host infection. Thus, osmolarity not only affects leptospiral gene expression by affecting transcript levels of putative virulence determinants but also affects the release of such proteins into the surroundings.

  11. Interaction between the enamel matrix proteins amelogenin and ameloblastin

    International Nuclear Information System (INIS)

    Ravindranath, Hanumanth H.; Chen, Li-Sha; Zeichner-David, Margaret; Ishima, Rieko; Ravindranath, Rajeswari M.H.

    2004-01-01

    Enamel matrix consists of amelogenin and non-amelogenins. Though amelogenin is not involved in nucleation of minerals, the enamel mineralization is impaired when amelogenin or other matrix protein (ameloblastin/enamelin) genes are mutated. We hypothesize that amelogenin may promote enamel mineralization by interacting with the calcium-binding matrix proteins. Specific binding of amelogenin to N-acetylglucosamine (GlcNAc), GlcNAc-mimicking peptides (GMps), and their carrier proteins and the identification of amelogenin-trityrosyl-motif-peptide (ATMP) as a GlcNAc/GMp-binding domain in amelogenin favor the hypothesis. This study tested the interaction of amelogenin with ameloblastin, a carrier of GMp sequence at intermittent sites. Neither GlcNAc nor sialic acids were identified in the recombinant-ameloblastin. Amelogenin bound to recombinant-ameloblastin in both Western blots and in ELISA. More specifically, [ 3 H]ATMP bound to both recombinant and native ameloblastins. Dosimetry and Scatchard analyses showed the specific interaction between ATMP and ameloblastin, suggesting that amelogenin may interact with ameloblastin to form a heteromolecular assembly

  12. Interaction between the enamel matrix proteins amelogenin and ameloblastin.

    Science.gov (United States)

    Ravindranath, Hanumanth H; Chen, Li-Sha; Zeichner-David, Margaret; Ishima, Rieko; Ravindranath, Rajeswari M H

    2004-10-22

    Enamel matrix consists of amelogenin and non-amelogenins. Though amelogenin is not involved in nucleation of minerals, the enamel mineralization is impaired when amelogenin or other matrix protein (ameloblastin/enamelin) genes are mutated. We hypothesize that amelogenin may promote enamel mineralization by interacting with the calcium-binding matrix proteins. Specific binding of amelogenin to N-acetylglucosamine (GlcNAc), GlcNAc-mimicking peptides (GMps), and their carrier proteins and the identification of amelogenin-trityrosyl-motif-peptide (ATMP) as a GlcNAc/GMp-binding domain in amelogenin favor the hypothesis. This study tested the interaction of amelogenin with ameloblastin, a carrier of GMp sequence at intermittent sites. Neither GlcNAc nor sialic acids were identified in the recombinant-ameloblastin. Amelogenin bound to recombinant-ameloblastin in both Western blots and in ELISA. More specifically, [(3)H]ATMP bound to both recombinant and native ameloblastins. Dosimetry and Scatchard analyses showed the specific interaction between ATMP and ameloblastin, suggesting that amelogenin may interact with ameloblastin to form a heteromolecular assembly.

  13. Determination of insoluble avian eggshell matrix proteins

    Czech Academy of Sciences Publication Activity Database

    Mikšík, Ivan; Sedláková, Pavla; Lacinová, Kateřina; Pataridis, Statis; Eckhardt, Adam

    2010-01-01

    Roč. 397, č. 1 (2010), s. 205-214 ISSN 1618-2642 R&D Projects: GA MŠk(CZ) 1M0510; GA ČR(CZ) GA203/09/0675; GA ČR(CZ) GA203/08/1428 Institutional research plan: CEZ:AV0Z50110509 Keywords : eggshell proteins * insoluble proteins * matrix proteins Subject RIV: CE - Biochemistry Impact factor: 3.841, year: 2010

  14. Radiation damage to DNA-binding proteins

    International Nuclear Information System (INIS)

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

    2003-01-01

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

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

    International Nuclear Information System (INIS)

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

    1989-01-01

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

  16. Fatty Acid Binding Proteins in Prostate Cancer

    National Research Council Canada - National Science Library

    Jett, Marti

    2000-01-01

    We have shown that there is a distinct pattern of fatty acid binding protein (FAEP) expression in prostate cancer vs normal cells and that finding has be confirmed in patient samples of biopsy specimens...

  17. Bacterial binding to extracellular proteins - in vitro adhesion

    DEFF Research Database (Denmark)

    Schou, C.; Fiehn, N.-E.

    1999-01-01

    Viridans streptococci, bacterial adherence, extracellular matrix proteins, surface receptors, endocarditis......Viridans streptococci, bacterial adherence, extracellular matrix proteins, surface receptors, endocarditis...

  18. Extracellular and intracellular steroid binding proteins

    International Nuclear Information System (INIS)

    Wagner, R.K.

    1978-01-01

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

  19. Interaction of bovine gallbladder mucin and calcium-binding protein: effects on calcium phosphate precipitation

    NARCIS (Netherlands)

    Afdhal, N. H.; Ostrow, J. D.; Koehler, R.; Niu, N.; Groen, A. K.; Veis, A.; Nunes, D. P.; Offner, G. D.

    1995-01-01

    Gallstones consist of calcium salts and cholesterol crystals, arrayed on a matrix of gallbladder mucin (GBM), and regulatory proteins like calcium-binding protein (CBP). To determine if interactions between CBP and GBM follow a biomineralization scheme, their mutual binding and effects on CaHPO4

  20. Domain organizations of modular extracellular matrix proteins and their evolution.

    Science.gov (United States)

    Engel, J

    1996-11-01

    Multidomain proteins which are composed of modular units are a rather recent invention of evolution. Domains are defined as autonomously folding regions of a protein, and many of them are similar in sequence and structure, indicating common ancestry. Their modular nature is emphasized by frequent repetitions in identical or in different proteins and by a large number of different combinations with other domains. The extracellular matrix is perhaps the largest biological system composed of modular mosaic proteins, and its astonishing complexity and diversity are based on them. A cluster of minireviews on modular proteins is being published in Matrix Biology. These deal with the evolution of modular proteins, the three-dimensional structure of domains and the ways in which these interact in a multidomain protein. They discuss structure-function relationships in calcium binding domains, collagen helices, alpha-helical coiled-coil domains and C-lectins. The present minireview is focused on some general aspects and serves as an introduction to the cluster.

  1. Molecular events in matrix protein metabolism in the aging kidney

    Science.gov (United States)

    Sataranatarajan, Kavithalakshmi; Feliers, Denis; Mariappan, Meenalakshmi M.; Lee, Hak Joo; Lee, Myung Ja; Day, Robert T.; Yalamanchili, Hima Bindu; Choudhury, Goutam G.; Barnes, Jeffrey L.; Van Remmen, Holly; Richardson, Arlan; Kasinath, Balakuntalam S.

    2018-01-01

    Summary We explored molecular events associated with aging-induced matrix changes in the kidney. C57BL6 mice were studied in youth, middle age, and old age. Albuminuria and serum cystatin C level (an index of glomerular filtration) increased with aging. Renal hypertrophy was evident in middle-aged and old mice and was associated with glomerulomegaly and increase in mesangial fraction occupied by extracellular matrix. Content of collagen types I and III and fibronectin was increased with aging; increment in their mRNA varied with the phase of aging. The content of ZEB1 and ZEB2, collagen type I transcription inhibitors, and their binding to the collagen type Iα2 promoter by ChIP assay also showed age-phase-specific changes. Lack of increase in mRNA and data from polysome assay suggested decreased degradation as a potential mechanism for kidney collagen type I accumulation in the middle-aged mice. These changes occurred with increment in TGFβ mRNA and protein and activation of its SMAD3 pathway; SMAD3 binding to the collagen type Iα2 promoter was also increased. TGFβ-regulated microRNAs (miRs) exhibited selective regulation. The renal cortical content of miR-21 and miR-200c, but not miR-192, miR-200a, or miR-200b, was increased with aging. Increased miR-21 and miR-200c contents were associated with reduced expression of their targets, Sprouty-1 and ZEB2, respectively. These data show that aging is associated with complex molecular events in the kidney that are already evident in the middle age and progress to old age. Agephase-specific regulation of matrix protein synthesis occurs and involves matrix protein-specific transcriptional and post-transcriptional mechanisms. PMID:23020145

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

    Science.gov (United States)

    Piłat, Zofia; Antosiewicz, Jan M

    2008-11-27

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

  3. Protein Binding Capacity of Different Forages Tannin

    Science.gov (United States)

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

    2018-02-01

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

  4. ALG-2, a multifunctional calcium binding protein?

    DEFF Research Database (Denmark)

    Tarabykina, Svetlana; Mollerup, Jens; Winding Gojkovic, P.

    2004-01-01

    ALG-2 was originally discovered as a pro-apoptotic protein in a genetic screen. Due to its ability to bind calcium with high affinity it was postulated to provide a link between the known effect of calcium in programmed cell death and the molecular death execution machinery. This review article...

  5. Plant ice-binding (antifreeze) proteins

    Science.gov (United States)

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

  6. Monomeric Yeast Frataxin is an Iron-Binding Protein

    International Nuclear Information System (INIS)

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

    2006-01-01

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

  7. Structure-Guided Design of an Engineered Streptavidin with Reusability to Purify Streptavidin-Binding Peptide Tagged Proteins or Biotinylated Proteins

    OpenAIRE

    Wu, Sau-Ching; Wong, Sui-Lam

    2013-01-01

    Development of a high-affinity streptavidin-binding peptide (SBP) tag allows the tagged recombinant proteins to be affinity purified using the streptavidin matrix without the need of biotinylation. The major limitation of this powerful technology is the requirement to use biotin to elute the SBP-tagged proteins from the streptavidin matrix. Tight biotin binding by streptavidin essentially allows the matrix to be used only once. To address this problem, differences in interactions of biotin an...

  8. Quantifying drug-protein binding in vivo

    International Nuclear Information System (INIS)

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

    2004-01-01

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

  9. A structural classification of substrate-binding proteins

    NARCIS (Netherlands)

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

    2010-01-01

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

  10. Human pentraxin 3 binds to the complement regulator c4b-binding protein.

    Directory of Open Access Journals (Sweden)

    Anne Braunschweig

    Full Text Available The long pentraxin 3 (PTX3 is a soluble recognition molecule with multiple functions including innate immune defense against certain microbes and the clearance of apoptotic cells. PTX3 interacts with recognition molecules of the classical and lectin complement pathways and thus initiates complement activation. In addition, binding of PTX3 to the alternative complement pathway regulator factor H was shown. Here, we show that PTX3 binds to the classical and lectin pathway regulator C4b-binding protein (C4BP. A PTX3-binding site was identified within short consensus repeats 1-3 of the C4BP α-chain. PTX3 did not interfere with the cofactor activity of C4BP in the fluid phase and C4BP maintained its complement regulatory activity when bound to PTX3 on surfaces. While C4BP and factor H did not compete for PTX3 binding, the interaction of C4BP with PTX3 was inhibited by C1q and by L-ficolin. PTX3 bound to human fibroblast- and endothelial cell-derived extracellular matrices and recruited functionally active C4BP to these surfaces. Whereas PTX3 enhanced the activation of the classical/lectin pathway and caused enhanced C3 deposition on extracellular matrix, deposition of terminal pathway components and the generation of the inflammatory mediator C5a were not increased. Furthermore, PTX3 enhanced the binding of C4BP to late apoptotic cells, which resulted in an increased rate of inactivation of cell surface bound C4b and a reduction in the deposition of C5b-9. Thus, in addition to complement activators, PTX3 interacts with complement inhibitors including C4BP. This balanced interaction on extracellular matrix and on apoptotic cells may prevent excessive local complement activation that would otherwise lead to inflammation and host tissue damage.

  11. RNA-Binding Proteins in Plant Immunity

    Directory of Open Access Journals (Sweden)

    Virginia Woloshen

    2011-01-01

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

  12. Binding proteins of somatomedins and their functions

    International Nuclear Information System (INIS)

    Kostelecka, Z.; Blahovec, J.

    1998-01-01

    In this paper the functions of binding proteins are discussed. One variable that provides insulin-like growth factors (IGFs) control at the extracellular level is the presence of high-affinity, soluble insulin-like growth factor proteins (IGFBPs). IGFBP-1 inhibits IGF effect on human osteosarcoma cells. Increased concentration of IGFBP-3 inhibits the proliferation of breast cancer cell line MCF 7 either directly or by competition for IGF receptors. Maybe IGFBPs work as anti-mitogens and IGFs are potential promotors of cancer growth

  13. Graphene oxide as a protein matrix: influence on protein biophysical properties.

    Science.gov (United States)

    Hernández-Cancel, Griselle; Suazo-Dávila, Dámaris; Ojeda-Cruzado, Axel J; García-Torres, Desiree; Cabrera, Carlos R; Griebenow, Kai

    2015-10-19

    This study provides fundamental information on the influence of graphene oxide (GO) nanosheets and glycans on protein catalytic activity, dynamics, and thermal stability. We provide evidence of protein stabilization by glycans and how this strategy could be implemented when GO nanosheets is used as protein immobilization matrix. A series of bioconjugates was constructed using two different strategies: adsorbing or covalently attaching native and glycosylated bilirubin oxidase (BOD) to GO. Bioconjugate formation was followed by FT-IR, zeta-potential, and X-ray photoelectron spectroscopy measurements. Enzyme kinetic parameters (k(m) and k(cat)) revealed that the substrate binding affinity was not affected by glycosylation and immobilization on GO, but the rate of enzyme catalysis was reduced. Structural analysis by circular dichroism showed that glycosylation did not affect the tertiary or the secondary structure of BOD. However, GO produced slight changes in the secondary structure. To shed light into the biophysical consequence of protein glycosylation and protein immobilization on GO nanosheets, we studied structural protein dynamical changes by FT-IR H/D exchange and thermal inactivation. It was found that glycosylation caused a reduction in structural dynamics that resulted in an increase in thermostability and a decrease in the catalytic activity for both, glycoconjugate and immobilized enzyme. These results establish the usefulness of chemical glycosylation to modulate protein structural dynamics and stability to develop a more stable GO-protein matrix.

  14. Structure and assembly of a paramyxovirus matrix protein.

    Science.gov (United States)

    Battisti, Anthony J; Meng, Geng; Winkler, Dennis C; McGinnes, Lori W; Plevka, Pavel; Steven, Alasdair C; Morrison, Trudy G; Rossmann, Michael G

    2012-08-28

    Many pleomorphic, lipid-enveloped viruses encode matrix proteins that direct their assembly and budding, but the mechanism of this process is unclear. We have combined X-ray crystallography and cryoelectron tomography to show that the matrix protein of Newcastle disease virus, a paramyxovirus and relative of measles virus, forms dimers that assemble into pseudotetrameric arrays that generate the membrane curvature necessary for virus budding. We show that the glycoproteins are anchored in the gaps between the matrix proteins and that the helical nucleocapsids are associated in register with the matrix arrays. About 90% of virions lack matrix arrays, suggesting that, in agreement with previous biological observations, the matrix protein needs to dissociate from the viral membrane during maturation, as is required for fusion and release of the nucleocapsid into the host's cytoplasm. Structure and sequence conservation imply that other paramyxovirus matrix proteins function similarly.

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

    Science.gov (United States)

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

    2012-11-01

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

  16. A collagen-binding EGFR antibody fragment targeting tumors with a collagen-rich extracellular matrix.

    Science.gov (United States)

    Liang, Hui; Li, Xiaoran; Wang, Bin; Chen, Bing; Zhao, Yannan; Sun, Jie; Zhuang, Yan; Shi, Jiajia; Shen, He; Zhang, Zhijun; Dai, Jianwu

    2016-02-17

    Many tumors over-express collagen, which constitutes the physical scaffold of tumor microenvironment. Collagen has been considered to be a target for cancer therapy. The collagen-binding domain (CBD) is a short peptide, which could bind to collagen and achieve the sustained release of CBD-fused proteins in collagen scaffold. Here, a collagen-binding EGFR antibody fragment was designed and expressed for targeting the collagen-rich extracellular matrix in tumors. The antibody fragment (Fab) of cetuximab was fused with CBD (CBD-Fab) and expressed in Pichia pastoris. CBD-Fab maintained antigen binding and anti-tumor activity of cetuximab and obtained a collagen-binding ability in vitro. The results also showed CBD-Fab was mainly enriched in tumors and had longer retention time in tumors in A431 s.c. xenografts. Furthermore, CBD-Fab showed a similar therapeutic efficacy as cetuximab in A431 xenografts. Although CBD-Fab hasn't showed better therapeutic effects than cetuximab, its smaller molecular and special target may be applicable as antibody-drug conjugates (ADC) or immunotoxins.

  17. Protein-anchoring therapy to target extracellular matrix proteins to their physiological destinations.

    Science.gov (United States)

    Ito, Mikako; Ohno, Kinji

    2018-02-20

    Endplate acetylcholinesterase (AChE) deficiency is a form of congenital myasthenic syndrome (CMS) caused by mutations in COLQ, which encodes collagen Q (ColQ). ColQ is an extracellular matrix (ECM) protein that anchors AChE to the synaptic basal lamina. Biglycan, encoded by BGN, is another ECM protein that binds to the dystrophin-associated protein complex (DAPC) on skeletal muscle, which links the actin cytoskeleton and ECM proteins to stabilize the sarcolemma during repeated muscle contractions. Upregulation of biglycan stabilizes the DPAC. Gene therapy can potentially ameliorate any disease that can be recapitulated in cultured cells. However, the difficulty of tissue-specific and developmental stage-specific regulated expression of transgenes, as well as the difficulty of introducing a transgene into all cells in a specific tissue, prevents us from successfully applying gene therapy to many human diseases. In contrast to intracellular proteins, an ECM protein is anchored to the target tissue via its specific binding affinity for protein(s) expressed on the cell surface within the target tissue. Exploiting this unique feature of ECM proteins, we developed protein-anchoring therapy in which a transgene product expressed even in remote tissues can be delivered and anchored to a target tissue using specific binding signals. We demonstrate the application of protein-anchoring therapy to two disease models. First, intravenous administration of adeno-associated virus (AAV) serotype 8-COLQ to Colq-deficient mice, resulting in specific anchoring of ectopically expressed ColQ-AChE at the NMJ, markedly improved motor functions, synaptic transmission, and the ultrastructure of the neuromuscular junction (NMJ). In the second example, Mdx mice, a model for Duchenne muscular dystrophy, were intravenously injected with AAV8-BGN. The treatment ameliorated motor deficits, mitigated muscle histopathologies, decreased plasma creatine kinase activities, and upregulated expression

  18. Cobalamin and its binding protein in rat milk

    DEFF Research Database (Denmark)

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

    1989-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Yu Zhu

    2015-11-01

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

  20. Photoaffinity labeling of the oxysterol binding protein

    International Nuclear Information System (INIS)

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

    1986-01-01

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

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

    Science.gov (United States)

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

    2012-03-01

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

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

    Science.gov (United States)

    Oshiro, Satoshi; Honda, Shinya

    2014-04-18

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

  3. Characterization of membrane association of Rinderpest virus matrix protein

    International Nuclear Information System (INIS)

    Subhashri, R.; Shaila, M.S.

    2007-01-01

    Paramyxovirus matrix protein is believed to play a crucial role in the assembly and maturation of the virus particle by bringing the major viral components together at the budding site in the host cell. The membrane association capability of many enveloped virus matrix proteins has been characterized to be their intrinsic property. In this work, we have characterized the membrane association of Rinderpest virus matrix (M) protein. The M protein of Rinderpest virus when expressed in the absence of other viral proteins is present both in the cytoplasm and plasma membrane. When expressed as GFP fusion protein, the M protein gets localized into plasma membrane protrusions. High salt and alkaline conditions resulted in partial dissociation of M protein from cell membrane. Thus, M protein behaves like an integral membrane protein although its primary structure suggests it to be a peripheral membrane protein

  4. Apolipoprotein B is a calcium binding protein

    International Nuclear Information System (INIS)

    Dashti, N.; Lee, D.M.; Mok, T.

    1986-01-01

    Human hepatocarcinoma Hep G2 cells were grown in culture medium containing [ 45 Ca 2+ ]. The secreted lipoproteins of d 45 Ca] from the gels showed that the peak of radioactivity corresponded to the apolipoprotein B band. The molar ratio of the incorporated [ 45 Ca 2+ ] and apolipoprotein B was close to unity. No radioactivity was found associated with any other secreted apolipoproteins. To confirm these findings, apolipoprotein B-containing lipoproteins were precipitated with anti-apolipoprotein B and high density lipoproteins were precipitated with anti-apolipoprotein A-I. Only the former precipitate was radioactive. These results suggest that apolipoprotein B is a calcium binding protein

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

    Science.gov (United States)

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

    2010-01-01

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

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

    Science.gov (United States)

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

    2000-08-01

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

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

    Science.gov (United States)

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

    2014-01-01

    Since the discovery of group-specific component and its polymorphism by Hirschfeld in 1959, research has put spotlight on this multifunctional transport protein (vitamin D binding protein, DBP). Besides the transport of vitamin D metabolites, DBP is a plasma glycoprotein with many important functions, including sequestration of actin, modulation of immune and inflammatory responses, binding of fatty acids, and control of bone development. A considerable DBP polymorphism has been described with a specific allele distribution in different geographic area. Multiple studies have shed light on the interesting relationship between polymorphisms of the DBP gene and the susceptibility to diseases. In this review, we give an overview of the multifunctional character of DBP and describe the clinical importance of DBP and its polymorphisms. Finally, we discuss the possibilities to use DBP as a novel therapeutic agent.

  8. Calculation of protein-ligand binding affinities.

    Science.gov (United States)

    Gilson, Michael K; Zhou, Huan-Xiang

    2007-01-01

    Accurate methods of computing the affinity of a small molecule with a protein are needed to speed the discovery of new medications and biological probes. This paper reviews physics-based models of binding, beginning with a summary of the changes in potential energy, solvation energy, and configurational entropy that influence affinity, and a theoretical overview to frame the discussion of specific computational approaches. Important advances are reported in modeling protein-ligand energetics, such as the incorporation of electronic polarization and the use of quantum mechanical methods. Recent calculations suggest that changes in configurational entropy strongly oppose binding and must be included if accurate affinities are to be obtained. The linear interaction energy (LIE) and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) methods are analyzed, as are free energy pathway methods, which show promise and may be ready for more extensive testing. Ultimately, major improvements in modeling accuracy will likely require advances on multiple fronts, as well as continued validation against experiment.

  9. LRPPRC is a mitochondrial matrix protein that is conserved in metazoans

    International Nuclear Information System (INIS)

    Sterky, Fredrik H.; Ruzzenente, Benedetta; Gustafsson, Claes M.; Samuelsson, Tore; Larsson, Nils-Goeran

    2010-01-01

    Research highlights: → LRPPRC orthologs are restricted to metazoans. → LRPPRC is imported to the mitochondrial matrix. → No evidence of nuclear isoform. -- Abstract: LRPPRC (also called LRP130) is an RNA-binding pentatricopeptide repeat protein. LRPPRC has been recognized as a mitochondrial protein, but has also been shown to regulate nuclear gene transcription and to bind specific RNA molecules in both the nucleus and the cytoplasm. We here present a bioinformatic analysis of the LRPPRC primary sequence, which reveals that orthologs to the LRPPRC gene are restricted to metazoan cells and that all of the corresponding proteins contain mitochondrial targeting signals. To address the subcellular localization further, we have carefully analyzed LRPPRC in mammalian cells and identified a single isoform that is exclusively localized to mitochondria. The LRPPRC protein is imported to the mitochondrial matrix and its mitochondrial targeting sequence is cleaved upon entry.

  10. DB-PABP: a database of polyanion-binding proteins.

    Science.gov (United States)

    Fang, Jianwen; Dong, Yinghua; Salamat-Miller, Nazila; Middaugh, C Russell

    2008-01-01

    The interactions between polyanions (PAs) and polyanion-binding proteins (PABPs) have been found to play significant roles in many essential biological processes including intracellular organization, transport and protein folding. Furthermore, many neurodegenerative disease-related proteins are PABPs. Thus, a better understanding of PA/PABP interactions may not only enhance our understandings of biological systems but also provide new clues to these deadly diseases. The literature in this field is widely scattered, suggesting the need for a comprehensive and searchable database of PABPs. The DB-PABP is a comprehensive, manually curated and searchable database of experimentally characterized PABPs. It is freely available and can be accessed online at http://pabp.bcf.ku.edu/DB_PABP/. The DB-PABP was implemented as a MySQL relational database. An interactive web interface was created using Java Server Pages (JSP). The search page of the database is organized into a main search form and a section for utilities. The main search form enables custom searches via four menus: protein names, polyanion names, the source species of the proteins and the methods used to discover the interactions. Available utilities include a commonality matrix, a function of listing PABPs by the number of interacting polyanions and a string search for author surnames. The DB-PABP is maintained at the University of Kansas. We encourage users to provide feedback and submit new data and references.

  11. Penicillin-binding proteins in Actinobacteria.

    Science.gov (United States)

    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.

  12. The RNA-binding protein repertoire of Arabidopsis thaliana

    KAUST Repository

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

    2016-01-01

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

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

    NARCIS (Netherlands)

    BEINTEMA, JJ

    1994-01-01

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

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

    Science.gov (United States)

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

    2015-12-29

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

  15. Rhabdovirus matrix protein structures reveal a novel mode of self-association.

    Directory of Open Access Journals (Sweden)

    Stephen C Graham

    2008-12-01

    Full Text Available The matrix (M proteins of rhabdoviruses are multifunctional proteins essential for virus maturation and budding that also regulate the expression of viral and host proteins. We have solved the structures of M from the vesicular stomatitis virus serotype New Jersey (genus: Vesiculovirus and from Lagos bat virus (genus: Lyssavirus, revealing that both share a common fold despite sharing no identifiable sequence homology. Strikingly, in both structures a stretch of residues from the otherwise-disordered N terminus of a crystallographically adjacent molecule is observed binding to a hydrophobic cavity on the surface of the protein, thereby forming non-covalent linear polymers of M in the crystals. While the overall topology of the interaction is conserved between the two structures, the molecular details of the interactions are completely different. The observed interactions provide a compelling model for the flexible self-assembly of the matrix protein during virion morphogenesis and may also modulate interactions with host proteins.

  16. Distinct profile of vascular progenitor attachment to extracellular matrix proteins in cancer patients.

    Science.gov (United States)

    Labonté, Laura; Li, Yuhua; Addison, Christina L; Brand, Marjorie; Javidnia, Hedyeh; Corsten, Martin; Burns, Kevin; Allan, David S

    2012-04-01

    Vascular progenitor cells (VPCs) facilitate angiogenesis and initiate vascular repair by homing in on sites of damage and adhering to extracellular matrix (ECM) proteins. VPCs also contribute to tumor angiogenesis and induce angiogenic switching in sites of metastatic cancer. In this study, the binding of attaching cells in VPC clusters that form in vitro on specific ECM proteins was investigated. VPC cluster assays were performed in vitro on ECM proteins enriched in cancer cells and in remodelling tissue. Profiles of VPC clusters from patients with cancer were compared to healthy controls. The role of VEGF and integrin-specific binding of angiogenic attaching cells was addressed. VPC clusters from cancer patients were markedly increased on fibronectin relative to other ECM proteins tested, in contrast to VPC clusters from control subjects, which formed preferentially on laminin. Specific integrin-mediated binding of attaching cells in VPC clusters was matrix protein-dependent. Furthermore, cancer patients had elevated plasma VEGF levels compared to healthy controls and VEGF facilitated preferential VPC cluster formation on fibronectin. Incubating cells from healthy controls with VEGF induced a switch from the 'healthy' VPC binding profile to the profile observed in cancer patients with a marked increase in VPC cluster formation on fibronectin. The ECM proteins laminin and fibronectin support VPC cluster formation via specific integrins on attaching cells and can facilitate patterns of VPC cluster formation that are distinct in cancer patients. Larger studies, however, are needed to gain insight on how tumor angiogenesis may differ from normal repair processes.

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

    International Nuclear Information System (INIS)

    Matsumoto, Hideaki; Takahashi, Eiichi

    1976-01-01

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

  18. Evaluation of a Solid Phase DNA Binding Matrix for Downstream PCR Analysis

    National Research Council Canada - National Science Library

    Bader, Douglas E; Fisher, Glen R; Stratilo, Chad W

    2005-01-01

    A commercially available solid-phase DNA binding matrix (FTA cards) was evaluated for its ability to capture and release DNA for downstream gene amplification and detection assays using polymerase chain reaction (PCR...

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

    Directory of Open Access Journals (Sweden)

    Elisa E. Figueroa-Angulo

    2015-11-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

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

    International Nuclear Information System (INIS)

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

    1979-01-01

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

  2. Development of radioimmunoassay for prolactin binding protein

    Energy Technology Data Exchange (ETDEWEB)

    Raikar, R.S.; Sheth, A.R. (Institute for Research in Reproduction, Bombay (India))

    1982-01-01

    Using a homogenous prolactin binding protein (PBP) preparations from rat seminal vesicle secretion, a sensitive and specific radioimmunoassay (RIA) for PBP has been developed. The assay was highly specific and showed no cross-reaction with other protein hormones from various species. The antiserum had an affinity constant (Ka) of 2.66 x 10/sup 10/ M/sup -1/. The assay sensitivity was in the range of 0.5-1.0 ng of pure PBP per assay tube and the intra- and inter-assay coefficients of variations were 6-8% and 12-14.5% respectively. The overall recovery of PBP to the rat seminal vesicle secretion was 96.8%. Using this RIA, PBP levels in various biological fluids and reproductive tissues were measured. Azoospermic human semen contained significantly higher levels of PBP than normospermic semen. The seminal vesicle of rat exhibited the highest concentration of PBP. Administration of antiserum to PBP to mature male rats resulted in a significant reduction in the weight of ventral prostrate and serum prolactin levels were significantly elevated in these animals suggesting that the antibody raised against the PBP was capable of blocking prolactin receptors.

  3. Deletion of the calmodulin-binding domain of Grb7 impairs cell attachment to the extracellular matrix and migration

    Energy Technology Data Exchange (ETDEWEB)

    García-Palmero, Irene; Villalobo, Antonio, E-mail: antonio.villalobo@iib.uam.es

    2013-06-28

    Highlights: •Grb7 is a calmodulin (CaM)-binding protein. •Deleting the CaM-binding site impairs cell attachment and migration. •CaM antagonists inhibit Grb7-mediated cell migration. •We conclude that CaM controls Grb7-mediated cell migration. -- Abstract: The adaptor Grb7 is a calmodulin (CaM)-binding protein that participates in signaling pathways involved in cell migration, proliferation and the control of angiogenesis, and plays a significant role in tumor growth, its metastatic spread and tumor-associated neo-vasculature formation. In this report we show that deletion of the CaM-binding site of Grb7, located in the proximal region of its pleckstrin homology (PH) domain, impairs cell migration, cell attachment to the extracellular matrix, and the reorganization of the actin cytoskeleton occurring during this process. Moreover, we show that the cell-permeable CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide (W-13) both retard the migration of cells expressing wild type Grb7, but not the migration of cells expressing the mutant protein lacking the CaM-binding site (Grb7Δ), underscoring the proactive role of CaM binding to Grb7 during this process.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-07-01

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

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

    International Nuclear Information System (INIS)

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

    2014-01-01

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

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

    International Nuclear Information System (INIS)

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

    1989-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Sung Un eHuh

    2013-12-01

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

  8. Partial characterization of GTP-binding proteins in Neurospora

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  9. Von Willebrand protein binds to extracellular matrices independently of collagen.

    OpenAIRE

    Wagner, D D; Urban-Pickering, M; Marder, V J

    1984-01-01

    Von Willebrand protein is present in the extracellular matrix of endothelial cells where it codistributes with fibronectin and types IV and V collagen. Bacterial collagenase digestion of endothelial cells removed fibrillar collagen, but the pattern of fibronectin and of von Willebrand protein remained undisturbed. Exogenous von Willebrand protein bound to matrices of different cells, whether rich or poor in collagen. von Willebrand protein also decorated the matrix of cells grown in the prese...

  10. Cartilage oligomeric matrix protein in patients with juvenile idiopathic arthritis

    DEFF Research Database (Denmark)

    Bjørnhart, Birgitte; Juul, Anders; Nielsen, Susan

    2009-01-01

    Cartilage oligomeric matrix protein (COMP) has been identified as a prognostic marker of progressive joint destruction in rheumatoid arthritis. In this population based study we evaluated associations between plasma concentrations of COMP, disease activity, and growth velocity in patients...

  11. In vivo extracellular matrix protein expression by human periodontal ...

    African Journals Online (AJOL)

    ONOS

    2010-08-23

    Aug 23, 2010 ... Extracellular matrix proteins (ECM) are described as molecular regulators of these events. ..... zation and adhesive interaction of cells (Yamada, 1983). .... periodontal ligament fibroblasts after simulation of orthodontic force.

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

    DEFF Research Database (Denmark)

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

    1996-01-01

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

  13. Surface-associated plasminogen binding of Cryptococcus neoformans promotes extracellular matrix invasion.

    Directory of Open Access Journals (Sweden)

    Jamal Stie

    2009-06-01

    Full Text Available The fungal pathogen Cryptococcus neoformans is a leading cause of illness and death in persons with predisposing factors, including: malignancies, solid organ transplants, and corticosteroid use. C. neoformans is ubiquitous in the environment and enters into the lungs via inhalation, where it can disseminate through the bloodstream and penetrate the central nervous system (CNS, resulting in a difficult to treat and often-fatal infection of the brain, called meningoencephalitis. Plasminogen is a highly abundant protein found in the plasma component of blood and is necessary for the degradation of fibrin, collagen, and other structural components of tissues. This fibrinolytic system is utilized by cancer cells during metastasis and several pathogenic species of bacteria have been found to manipulate the host plasminogen system to facilitate invasion of tissues during infection by modifying the activation of this process through the binding of plasminogen at their surface.The invasion of the brain and the central nervous system by penetration of the protective blood-brain barrier is a prerequisite to the establishment of meningoencephalitis by the opportunistic fungal pathogen C. neoformans. In this study, we examined the ability of C. neoformans to subvert the host plasminogen system to facilitate tissue barrier invasion. Through a combination of biochemical, cell biology, and proteomic approaches, we have shown that C. neoformans utilizes the host plasminogen system to cross tissue barriers, providing support for the hypothesis that plasminogen-binding may contribute to the invasion of the blood-brain barrier by penetration of the brain endothelial cells and underlying matrix. In addition, we have identified the cell wall-associated proteins that serve as plasminogen receptors and characterized both the plasminogen-binding and plasmin-activation potential for this significant human pathogen.The results of this study provide evidence for the

  14. Polyether sulfone/hydroxyapatite mixed matrix membranes for protein purification

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Junfen, E-mail: junfensun@dhu.edu.cn [State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of Material Science and Engineering, Donghua University, North People Road 2999, Shanghai 201620 (China); Wu, Lishun [Department of Chemistry and Chemical Engineering, Heze University, Daxue Road 2269, Heze, Shandong Province 274015 (China)

    2014-07-01

    This work proposes a novel approach for protein purification from solution using mixed matrix membranes (MMMs) comprising of hydroxyapatite (HAP) inside polyether sulfone (PES) matrix. The influence of HAP particle loading on membrane morphology is studied. The MMMs are further characterized concerning permeability and adsorption capacity. The MMMs show purification of protein via both diffusion as well as adsorption, and show the potential of using MMMs for improvements in protein purification techniques. The bovine serum albumin (BSA) was used as a model protein. The properties and structures of MMMs prepared by immersion phase separation process were characterized by pure water flux, BSA adsorption and scanning electron microscopy (SEM).

  15. Matrix proteins of Nipah and Hendra viruses interact with beta subunits of AP-3 complexes.

    Science.gov (United States)

    Sun, Weina; McCrory, Thomas S; Khaw, Wei Young; Petzing, Stephanie; Myers, Terrell; Schmitt, Anthony P

    2014-11-01

    Paramyxoviruses and other negative-strand RNA viruses encode matrix proteins that coordinate the virus assembly process. The matrix proteins link the viral glycoproteins and the viral ribonucleoproteins at virus assembly sites and often recruit host machinery that facilitates the budding process. Using a co-affinity purification strategy, we have identified the beta subunit of the AP-3 adapter protein complex, AP3B1, as a binding partner for the M proteins of the zoonotic paramyxoviruses Nipah virus and Hendra virus. Binding function was localized to the serine-rich and acidic Hinge domain of AP3B1, and a 29-amino-acid Hinge-derived polypeptide was sufficient for M protein binding in coimmunoprecipitation assays. Virus-like particle (VLP) production assays were used to assess the relationship between AP3B1 binding and M protein function. We found that for both Nipah virus and Hendra virus, M protein expression in the absence of any other viral proteins led to the efficient production of VLPs in transfected cells, and this VLP production was potently inhibited upon overexpression of short M-binding polypeptides derived from the Hinge region of AP3B1. Both human and bat (Pteropus alecto) AP3B1-derived polypeptides were highly effective at inhibiting the production of VLPs. VLP production was also impaired through small interfering RNA (siRNA)-mediated depletion of AP3B1 from cells. These findings suggest that AP-3-directed trafficking processes are important for henipavirus particle production and identify a new host protein-virus protein binding interface that could become a useful target in future efforts to develop small molecule inhibitors to combat paramyxoviral infections. Henipaviruses cause deadly infections in humans, with a mortality rate of about 40%. Hendra virus outbreaks in Australia, all involving horses and some involving transmission to humans, have been a continuing problem. Nipah virus caused a large outbreak in Malaysia in 1998, killing 109 people

  16. Interleukin-18 and interleukin-18 Binding Protein

    Directory of Open Access Journals (Sweden)

    Charles eDinarello

    2013-10-01

    Full Text Available Interleukin-18 (IL 18 is a member of the IL 1 family of cytokines. Increasing reports have expanded the role of IL 18 in mediating inflammation in animal models of disease using IL 18 deficient mice, neutralization of IL 18 or deficiency in the IL 18 receptor alpha chain. Similar to IL 1β, IL 18 is synthesized as an inactive precursor requiering processing by caspase 1 into an active cytokine but unlike IL 1β, the IL 18 precursor is constitutively present in nearly all cells in healthy humans and animals. The activity of IL 18 is balanced by the presence of a high-affinity naturally occuring IL 18 binding protein (IL 18BP. In humans, disease increased disease severity can be associated with an imbalance of IL 18 to IL 18BP such that the levels of free IL 18 are elevated in the circulation. A role for IL 18 has been implicated in several autoimmune diseases, myocardial function, emphysema, metabolic syndromes, psoriasis, inflammatory bowel disease, hemophagocytic syndromes, macrophage activation syndrome, sepsis and acute kidney injury, although in some diseases, IL 18 is protective. IL 18 plays a major role in the production of interferon-g from natural killer cells. The IL 18BP has been used safely in humans and clinical trials of IL 18BP as well as neutralizing anti-IL 18 antibodies are in clinical trials. This review updates the biology of IL 18 as well as its role in human disease

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

    Science.gov (United States)

    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

  18. Collagen XII and XIV, New Partners of Cartilage Oligomeric Matrix Protein in the Skin Extracellular Matrix Suprastructure*

    Science.gov (United States)

    Agarwal, Pallavi; Zwolanek, Daniela; Keene, Douglas R.; Schulz, Jan-Niklas; Blumbach, Katrin; Heinegård, Dick; Zaucke, Frank; Paulsson, Mats; Krieg, Thomas; Koch, Manuel; Eckes, Beate

    2012-01-01

    The tensile and scaffolding properties of skin rely on the complex extracellular matrix (ECM) that surrounds cells, vasculature, nerves, and adnexus structures and supports the epidermis. In the skin, collagen I fibrils are the major structural component of the dermal ECM, decorated by proteoglycans and by fibril-associated collagens with interrupted triple helices such as collagens XII and XIV. Here we show that the cartilage oligomeric matrix protein (COMP), an abundant component of cartilage ECM, is expressed in healthy human skin. COMP expression is detected in the dermal compartment of skin and in cultured fibroblasts, whereas epidermis and HaCaT cells are negative. In addition to binding collagen I, COMP binds to collagens XII and XIV via their C-terminal collagenous domains. All three proteins codistribute in a characteristic narrow zone in the superficial papillary dermis of healthy human skin. Ultrastructural analysis by immunogold labeling confirmed colocalization and further revealed the presence of COMP along with collagens XII and XIV in anchoring plaques. On the basis of these observations, we postulate that COMP functions as an adapter protein in human skin, similar to its function in cartilage ECM, by organizing collagen I fibrils into a suprastructure, mainly in the vicinity of anchoring plaques that stabilize the cohesion between the upper dermis and the basement membrane zone. PMID:22573329

  19. Collagen XII and XIV, new partners of cartilage oligomeric matrix protein in the skin extracellular matrix suprastructure.

    Science.gov (United States)

    Agarwal, Pallavi; Zwolanek, Daniela; Keene, Douglas R; Schulz, Jan-Niklas; Blumbach, Katrin; Heinegård, Dick; Zaucke, Frank; Paulsson, Mats; Krieg, Thomas; Koch, Manuel; Eckes, Beate

    2012-06-29

    The tensile and scaffolding properties of skin rely on the complex extracellular matrix (ECM) that surrounds cells, vasculature, nerves, and adnexus structures and supports the epidermis. In the skin, collagen I fibrils are the major structural component of the dermal ECM, decorated by proteoglycans and by fibril-associated collagens with interrupted triple helices such as collagens XII and XIV. Here we show that the cartilage oligomeric matrix protein (COMP), an abundant component of cartilage ECM, is expressed in healthy human skin. COMP expression is detected in the dermal compartment of skin and in cultured fibroblasts, whereas epidermis and HaCaT cells are negative. In addition to binding collagen I, COMP binds to collagens XII and XIV via their C-terminal collagenous domains. All three proteins codistribute in a characteristic narrow zone in the superficial papillary dermis of healthy human skin. Ultrastructural analysis by immunogold labeling confirmed colocalization and further revealed the presence of COMP along with collagens XII and XIV in anchoring plaques. On the basis of these observations, we postulate that COMP functions as an adapter protein in human skin, similar to its function in cartilage ECM, by organizing collagen I fibrils into a suprastructure, mainly in the vicinity of anchoring plaques that stabilize the cohesion between the upper dermis and the basement membrane zone.

  20. Ligand Binding Domain Protein in Tetracycline-Inducible Expression

    African Journals Online (AJOL)

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

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

    Directory of Open Access Journals (Sweden)

    Anders Gerd

    2008-01-01

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

  2. Clinical relevance of drug binding to plasma proteins

    Science.gov (United States)

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

    2014-12-01

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

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

    International Nuclear Information System (INIS)

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

    1984-01-01

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

  4. GTP-binding proteins in rat liver nuclear envelopes

    International Nuclear Information System (INIS)

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

    1990-01-01

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

  5. CC1, a novel crenarchaeal DNA binding protein.

    Science.gov (United States)

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

    2007-01-01

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

  6. The requirement of matrix ATP for the import of precursor proteins into the mitochondrial matrix and intermembrane space

    NARCIS (Netherlands)

    Stuart, Rosemary A.; Gruhler, Albrecht; Klei, Ida van der; Guiard, Bernard; Koll, Hans; Neupert, Walter

    1994-01-01

    The role of ATP in the matrix for the import of precursor proteins into the various mitochondrial subcompartments was investigated by studying protein translocation at experimentally defined ATP levels. Proteins targeted to the matrix were neither imported or processed when matrix ATP was depleted.

  7. In vivo extracellular matrix protein expression by human periodontal ...

    African Journals Online (AJOL)

    It is well known that the orthodontic force applied to teeth generates a series of events that remodel the periodontal ligament (PDL). Extracellular matrix proteins (ECM) are described as molecular regulators of these events. However, the exact contribution of these proteins in human PDL modeling by orthodontic force ...

  8. Calcium-binding proteins from human platelets

    International Nuclear Information System (INIS)

    Gogstad, G.O.; Krutnes, M.B.; Solum, N.O.

    1983-01-01

    Calcium-binding platelet proteins were examined by crossed immunoelectrophoresis of solubilized platelets against antibodies to whole platelets followed by incubation of the immunoplates with 45 Ca 2 + and autoradiography. When the immunoplates had been pretreated with EDTA at pH 9.0 in order to remove divalent cations, three immunoprecipitates were markedly labelled with 45 Ca 2 + . These corresponded to the glycoprotein IIb-IIIa complex, glycoprotein Ia and a presently unidentified antigen termed G18. These antigens were membrane-bound and surface-oriented. When an excess of EDTA was introduced in the incubation media the results revealed that the glycoprotein IIb-IIIa complex and antigen G18, but not glycoprotein Ia, contained sites with a stronger affinity for calcium than has EDTA at pH 7.4 Immunoprecipitates of the separate glycoproteins IIb and IIIa both bound calcium in the same manner as the glycoprotein IIb-IIIa complex. As another approach, platelet-rich plasma was incubated with 45 Ca 2 + prior to crossed immunoelectrophoresis of the solubilized platelets. A single immunoprecipitate was wekly labelled. This did not correspond to any of the immunoprecipitates which were visible after staining with Coomassie blue. The labelling of this antigen was markedly increased when the platelt-rich plasma had been preincubated with EDTA and in this case a weak labelling of the glycoprotein IIB-IIIa precipitate also became apparent. No increased incorporation of calcium occured in any of these immunoprecipitates when the platelets were aggregated with ADP in the presence of 45 Ca 2 + . (orig.)

  9. Protein structure estimation from NMR data by matrix completion.

    Science.gov (United States)

    Li, Zhicheng; Li, Yang; Lei, Qiang; Zhao, Qing

    2017-09-01

    Knowledge of protein structures is very important to understand their corresponding physical and chemical properties. Nuclear Magnetic Resonance (NMR) spectroscopy is one of the main methods to measure protein structure. In this paper, we propose a two-stage approach to calculate the structure of a protein from a highly incomplete distance matrix, where most data are obtained from NMR. We first randomly "guess" a small part of unobservable distances by utilizing the triangle inequality, which is crucial for the second stage. Then we use matrix completion to calculate the protein structure from the obtained incomplete distance matrix. We apply the accelerated proximal gradient algorithm to solve the corresponding optimization problem. Furthermore, the recovery error of our method is analyzed, and its efficiency is demonstrated by several practical examples.

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

    Science.gov (United States)

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

    2017-09-01

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

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

    Science.gov (United States)

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

    2003-01-01

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

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

    Directory of Open Access Journals (Sweden)

    M.A.M. Marques

    2001-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Waqasuddin Khan

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

  14. Guardian of Genetic Messenger-RNA-Binding Proteins

    Directory of Open Access Journals (Sweden)

    Antje Anji

    2016-01-01

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

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

    Science.gov (United States)

    Lam, Pui-Man; Zhen, Yi

    2011-04-01

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

  16. Characterization of a cocaine binding protein in human placenta

    International Nuclear Information System (INIS)

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

    1990-01-01

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

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

    Science.gov (United States)

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

    2012-02-01

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

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

    DEFF Research Database (Denmark)

    Mandrup, S; Hummel, R; Ravn, S

    1992-01-01

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

  19. Cartilage Acidic Protein 2 a hyperthermostable, high affinity calcium-binding protein.

    Science.gov (United States)

    Anjos, Liliana; Gomes, Ana S; Melo, Eduardo P; Canário, Adelino V; Power, Deborah M

    2013-03-01

    Cartilage Acidic Protein 2 (CRTAC2) is a novel protein present from prokaryotes to vertebrates with abundant expression in the teleost fish pituitary gland and an isoform of CRTAC1, a chondrocyte marker in humans. The two proteins are non-integrins containing N-terminal integrin-like Ca(2+)-binding motifs and their structure and function remain to be assigned. Structural studies of recombinant sea bream (sb)CRTAC2 revealed it is composed of 8.8% α-helix, 33.4% β-sheet and 57.8% unordered protein. sbCRTAC2 bound Ca(2+) with high affinity (K(d)=1.46nM) and favourable Gibbs free energy (∆G=-12.4kcal/mol). The stoichiometry for Ca(2+) bound to sbCRTAC2 at saturation indicated six Ca(2+) ligand-binding sites exist per protein molecule. No conformational change in sbCRTAC2 occurred in the presence of Ca(2+). Fluorescence emission revealed that the tertiary structure of the protein is hyperthermostable between 25°C and 95°C and the fully unfolded state is only induced by chemical denaturing (4M GndCl). sbCRTAC has a widespread tissue distribution and is present as high molecular weight aggregates, although strong reducing conditions promote formation of the monomer. sbCRTAC2 promotes epithelial cell outgrowth in vitro suggesting it may share functional homology with mammalian CRTAC1, recently implicated in cell-cell and cell-matrix interactions. Copyright © 2012 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2015-06-01

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

  1. Analysis of the ligand binding properties of recombinant bovine liver-type fatty acid binding protein

    DEFF Research Database (Denmark)

    Rolf, B; Oudenampsen-Krüger, E; Börchers, T

    1995-01-01

    The coding part of the cDNA for bovine liver-type fatty acid binding protein (L-FABP) has been amplified by RT-PCR, cloned and used for the construction of an Escherichia coli (E. coli) expression system. The recombinant protein made up to 25% of the soluble E. coli proteins and could be isolated...

  2. Cartilage oligomeric matrix protein enhances the vascularization of acellular nerves

    Directory of Open Access Journals (Sweden)

    Wei-ling Cui

    2016-01-01

    Full Text Available Vascularization of acellular nerves has been shown to contribute to nerve bridging. In this study, we used a 10-mm sciatic nerve defect model in rats to determine whether cartilage oligomeric matrix protein enhances the vascularization of injured acellular nerves. The rat nerve defects were treated with acellular nerve grafting (control group alone or acellular nerve grafting combined with intraperitoneal injection of cartilage oligomeric matrix protein (experimental group. As shown through two-dimensional imaging, the vessels began to invade into the acellular nerve graft from both anastomotic ends at day 7 post-operation, and gradually covered the entire graft at day 21. The vascular density, vascular area, and the velocity of revascularization in the experimental group were all higher than those in the control group. These results indicate that cartilage oligomeric matrix protein enhances the vascularization of acellular nerves.

  3. Tissue specificity of the hormonal response in sex accessory tissues is associated with nuclear matrix protein patterns.

    Science.gov (United States)

    Getzenberg, R H; Coffey, D S

    1990-09-01

    The DNA of interphase nuclei have very specific three-dimensional organizations that are different in different cell types, and it is possible that this varying DNA organization is responsible for the tissue specificity of gene expression. The nuclear matrix organizes the three-dimensional structure of the DNA and is believed to be involved in the control of gene expression. This study compares the nuclear structural proteins between two sex accessory tissues in the same animal responding to the same androgen stimulation by the differential expression of major tissue-specific secretory proteins. We demonstrate here that the nuclear matrix is tissue specific in the rat ventral prostate and seminal vesicle, and undergoes characteristic alterations in its protein composition upon androgen withdrawal. Three types of nuclear matrix proteins were observed: 1) nuclear matrix proteins that are different and tissue specific in the rat ventral prostate and seminal vesicle, 2) a set of nuclear matrix proteins that either appear or disappear upon androgen withdrawal, and 3) a set of proteins that are common to both the ventral prostate and seminal vesicle and do not change with the hormonal state of the animal. Since the nuclear matrix is known to bind androgen receptors in a tissue- and steroid-specific manner, we propose that the tissue specificity of the nuclear matrix arranges the DNA in a unique conformation, which may be involved in the specific interaction of transcription factors with DNA sequences, resulting in tissue-specific patterns of secretory protein expression.

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

    International Nuclear Information System (INIS)

    Graham-Lorence, S.E.

    1987-01-01

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

  5. Patchwork structure-function analysis of the Sendai virus matrix protein.

    Science.gov (United States)

    Mottet-Osman, Geneviève; Miazza, Vincent; Vidalain, Pierre-Olivier; Roux, Laurent

    2014-09-01

    Paramyxoviruses contain a bi-lipidic envelope decorated by two transmembrane glycoproteins and carpeted on the inner surface with a layer of matrix proteins (M), thought to bridge the glycoproteins with the viral nucleocapsids. To characterize M structure-function features, a set of M domains were mutated or deleted. The genes encoding these modified M were incorporated into recombinant Sendai viruses and expressed as supplemental proteins. Using a method of integrated suppression complementation system (ISCS), the functions of these M mutants were analyzed in the context of the infection. Cellular membrane association, localization at the cell periphery, nucleocapsid binding, cellular protein interactions and promotion of viral particle formation were characterized in relation with the mutations. At the end, lack of nucleocapsid binding go together with lack of cell surface localization and both features definitely correlate with loss of M global function estimated by viral particle production. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Ligand Binding Domain Protein in Tetracycline-Inducible Expression ...

    African Journals Online (AJOL)

    binding domain proteins in E. coli using a tetracycline inducible system. To allow for ... development of molecular ligands with improved therapeutic windows. Keywords: Nuclear receptor ..... functional recombinant cannabinoid receptor CB2 in ...

  7. Direct detection of ligand binding to Sepharose-immobilised protein using saturation transfer double difference (STDD) NMR spectroscopy

    International Nuclear Information System (INIS)

    Haselhorst, Thomas; Muenster-Kuehnel, Anja K.; Oschlies, Melanie; Tiralongo, Joe; Gerardy-Schahn, Rita; Itzstein, Mark von

    2007-01-01

    We report an easy and direct application of 'Saturation Transfer Double Difference' (STDD) NMR spectroscopy to identify ligands that bind to a Sepharose-immobilised target protein. The model protein, cytidine 5'-monophosphate sialic acid (CMP-Sia) synthetase, was expressed as a Strep-Tag II fusion protein and immobilised on Strep-Tactin Sepharose. STD NMR experiments of the protein-enriched Sepharose matrix in the presence of a binding ligand (cytidine 5'-triphosphate, CTP) and a non-binding ligand (α/β-glucose) clearly show that CTP binds to the immobilised enzyme, whereas glucose has no affinity. This approach has three major advantages: (a) only low quantities of protein are required, (b) no specialised NMR technology or the application of additional data analysis by non-routine methods is required, and (c) easy multiple use of the immobilised protein is available

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

    DEFF Research Database (Denmark)

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

    2011-01-01

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

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

    NARCIS (Netherlands)

    Koppelman, S.J.

    1995-01-01

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

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

    International Nuclear Information System (INIS)

    Hughes, M.F.

    1986-01-01

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

  11. Drosophila DNA-Binding Proteins in Polycomb Repression

    Directory of Open Access Journals (Sweden)

    Maksim Erokhin

    2018-01-01

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

  12. Characterization of cap binding proteins associated with the nucleus

    International Nuclear Information System (INIS)

    Patzelt, E.

    1986-04-01

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

  13. Molecular interactions between chondroitin-dermatan sulfate and growth factors/receptors/matrix proteins.

    Science.gov (United States)

    Mizumoto, Shuji; Yamada, Shuhei; Sugahara, Kazuyuki

    2015-10-01

    Recent functional studies on chondroitin sulfate-dermatan sulfate (CS-DS) demonstrated its indispensable roles in various biological events including brain development and cancer. CS-DS proteoglycans exert their physiological activity through interactions with specific proteins including growth factors, cell surface receptors, and matrix proteins. The characterization of these interactions is essential for regulating the biological functions of CS-DS proteoglycans. Although amino acid sequences on the bioactive proteins required for these interactions have already been elucidated, the specific saccharide sequences involved in the binding of CS-DS to target proteins have not yet been sufficiently identified. In this review, recent findings are described on the interaction between CS-DS and some proteins which are especially involved in the central nervous system and cancer development/metastasis. Copyright © 2015. Published by Elsevier Ltd.

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

    Science.gov (United States)

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

    2006-11-28

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

  15. A major protein component of the Bacillus subtilis biofilm matrix.

    Science.gov (United States)

    Branda, Steven S; Chu, Frances; Kearns, Daniel B; Losick, Richard; Kolter, Roberto

    2006-02-01

    Microbes construct structurally complex multicellular communities (biofilms) through production of an extracellular matrix. Here we present evidence from scanning electron microscopy showing that a wild strain of the Gram positive bacterium Bacillus subtilis builds such a matrix. Genetic, biochemical and cytological evidence indicates that the matrix is composed predominantly of a protein component, TasA, and an exopolysaccharide component. The absence of TasA or the exopolysaccharide resulted in a residual matrix, while the absence of both components led to complete failure to form complex multicellular communities. Extracellular complementation experiments revealed that a functional matrix can be assembled even when TasA and the exopolysaccharide are produced by different cells, reinforcing the view that the components contribute to matrix formation in an extracellular manner. Having defined the major components of the biofilm matrix and the control of their synthesis by the global regulator SinR, we present a working model for how B. subtilis switches between nomadic and sedentary lifestyles.

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

    Science.gov (United States)

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

    2016-01-29

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

  17. Mixed-matrix membrane adsorbers for protein separation

    NARCIS (Netherlands)

    Avramescu, M.E.; Borneman, Z.; Wessling, M.

    2003-01-01

    The separation of two similarly sized proteins, bovine serum albumin (BSA) and bovine hemoglobin (Hb) was carried out using a new type of ion-exchange mixed-matrix adsorber membranes. The adsorber membranes were prepared by incorporation of various types of Lewatit ion-exchange resins into an

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

    Science.gov (United States)

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

    2015-01-01

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

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

    International Nuclear Information System (INIS)

    Yamamoto, Osamu

    1979-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1979-12-01

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

  1. Visualizing the dental biofilm matrix by means of fluorescence lectin-binding analysis

    DEFF Research Database (Denmark)

    Tawakoli, Pune Nina; Neu, Thomas R; Busck, Mette Marie

    2017-01-01

    lectins to visualize and quantify extracellular glycoconjugates in dental biofilms. Lectin binding was screened on pooled supragingival biofilm samples collected from 76 subjects using confocal microscopy. FLBA was then performed with 10 selected lectins on biofilms grown in situ for 48 h in the absence......The extracellular matrix is a poorly studied, yet important component of dental biofilms. Fluorescence lectin-binding analysis (FLBA) is a powerful tool to characterize glycoconjugates in the biofilm matrix. This study aimed to systematically investigate the ability of 75 fluorescently labeled......-biofilms: Aleuria aurantia (AAL), Calystega sepiem (Calsepa), Lycopersicon esculentum (LEA), Morniga-G (MNA-G) and Helix pomatia (HPA). No significant correlation between the binding of specific lectins and bacterial composition was found. Fluorescently labeled lectins enable the visualization of glycoconjugates...

  2. Species specificity for HBsAg binding protein endonexin II

    NARCIS (Netherlands)

    deBruin, WCC; Leenders, WPJ; Moshage, H; vanHaelst, UJGM

    Background/Aims: Hepatitis B virus displays a distinct species and tissue tropism, Previously we have demonstrated that a human liver plasma membrane protein,vith a molecular weight of approximately 34 kiloDalton specifically binds to HBsAg. This protein was identified as endonexin II, a Ca2+

  3. Small GTP-binding proteins in human endothelial cells

    NARCIS (Netherlands)

    de Leeuw, H. P.; Koster, P. M.; Calafat, J.; Janssen, H.; van Zonneveld, A. J.; van Mourik, J. A.; Voorberg, J.

    1998-01-01

    Small GTP-binding proteins of the Ras superfamily control an extensive number of intracellular events by alternating between GDP- and GTP-bound conformation. The presence of members of this protein family was examined in human umbilical vein endothelial cells employing RT-PCR. Sequence analysis of

  4. Immuno-histochemical localization of cholesterol binding proteins in ...

    African Journals Online (AJOL)

    This manuscript aims to investigate immunocytochemical localization of cholesterol binding proteins (CBPs) in semi-thin sections of midgut of Schistocerca gregaria (Forskal). For this purpose ... Further, same protein was also localized in other tissues like fat body, testis, and ovary of male and female insects of S. gregaria.

  5. Expression, purification and crystallization of a lyssavirus matrix (M) protein

    Energy Technology Data Exchange (ETDEWEB)

    Assenberg, René [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Delmas, Olivier [UPRE Lyssavirus Dynamics and Host Adaptation, WHO Collaborating Centre for Reference and Research on Rabies, Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris CEDEX 15 (France); Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J. [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Bourhy, Hervé [UPRE Lyssavirus Dynamics and Host Adaptation, WHO Collaborating Centre for Reference and Research on Rabies, Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris CEDEX 15 (France); Grimes, Jonathan M., E-mail: jonathan@strubi.ox.ac.uk [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom)

    2008-04-01

    The expression, purification and crystallization of the full-length matrix protein from three lyssaviruses is described. The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress.

  6. Expression, purification and crystallization of a lyssavirus matrix (M) protein

    International Nuclear Information System (INIS)

    Assenberg, René; Delmas, Olivier; Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J.; Bourhy, Hervé; Grimes, Jonathan M.

    2008-01-01

    The expression, purification and crystallization of the full-length matrix protein from three lyssaviruses is described. The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6 1 22 or P6 5 22, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress

  7. Conformational plasticity of the Ebola virus matrix protein.

    Science.gov (United States)

    Radzimanowski, Jens; Effantin, Gregory; Weissenhorn, Winfried

    2014-11-01

    Filoviruses are the causative agents of a severe and often fatal hemorrhagic fever with repeated outbreaks in Africa. They are negative sense single stranded enveloped viruses that can cross species barriers from its natural host bats to primates including humans. The small size of the genome poses limits to viral adaption, which may be partially overcome by conformational plasticity. Here we review the different conformational states of the Ebola virus (EBOV) matrix protein VP40 that range from monomers, to dimers, hexamers, and RNA-bound octamers. This conformational plasticity that is required for the viral life cycle poses a unique opportunity for development of VP40 specific drugs. Furthermore, we compare the structure to homologous matrix protein structures from Paramyxoviruses and Bornaviruses and we predict that they do not only share the fold but also the conformational flexibility of EBOV VP40. © 2014 The Protein Society.

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

    NARCIS (Netherlands)

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

    1993-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Yoon Sup Choi

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

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

    Science.gov (United States)

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

    2008-04-01

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

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

    Science.gov (United States)

    Khorshid, Mohsen; Rodak, Christoph; Zavolan, Mihaela

    2011-01-01

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

  12. Structural properties of the intrinsically disordered, multiple calcium ion-binding otolith matrix macromolecule-64 (OMM-64).

    Science.gov (United States)

    Poznar, Monika; Hołubowicz, Rafał; Wojtas, Magdalena; Gapiński, Jacek; Banachowicz, Ewa; Patkowski, Adam; Ożyhar, Andrzej; Dobryszycki, Piotr

    2017-11-01

    Fish otoliths are calcium carbonate biominerals that are involved in hearing and balance sensing. An organic matrix plays a crucial role in their formation. Otolith matrix macromolecule-64 (OMM-64) is a highly acidic, calcium-binding protein (CBP) found in rainbow trout otoliths. It is a component of high-molecular-weight aggregates, which influence the size, shape and polymorph of calcium carbonate in vitro. In this study, a protocol for the efficient expression and purification of OMM-64 was developed. For the first time, the complete structural characteristics of OMM-64 were described. Various biophysical methods were combined to show that OMM-64 occurs as an intrinsically disordered monomer. Under denaturing conditions (pH, temperature) OMM-64 exhibits folding propensity. It was determined that OMM-64 binds approximately 61 calcium ions with millimolar affinity. The folding-unfolding experiments showed that calcium ions induced the collapse of OMM-64. The effect of other counter ions present in trout endolymph on OMM-64 conformational changes was studied. The significance of disordered properties of OMM-64 and the possible function of this protein is discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2017-01-01

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

  14. Three-dimensional structure of the human immunodeficiency virus type 1 matrix protein.

    Science.gov (United States)

    Massiah, M A; Starich, M R; Paschall, C; Summers, M F; Christensen, A M; Sundquist, W I

    1994-11-25

    The HIV-1 matrix protein forms an icosahedral shell associated with the inner membrane of the mature virus. Genetic analyses have indicated that the protein performs important functions throughout the viral life-cycle, including anchoring the transmembrane envelope protein on the surface of the virus, assisting in viral penetration, transporting the proviral integration complex across the nuclear envelope, and localizing the assembling virion to the cell membrane. We now report the three-dimensional structure of recombinant HIV-1 matrix protein, determined at high resolution by nuclear magnetic resonance (NMR) methods. The HIV-1 matrix protein is the first retroviral matrix protein to be characterized structurally and only the fourth HIV-1 protein of known structure. NMR signal assignments required recently developed triple-resonance (1H, 13C, 15N) NMR methodologies because signals for 91% of 132 assigned H alpha protons and 74% of the 129 assignable backbone amide protons resonate within chemical shift ranges of 0.8 p.p.m. and 1 p.p.m., respectively. A total of 636 nuclear Overhauser effect-derived distance restraints were employed for distance geometry-based structure calculations, affording an average of 13.0 NMR-derived distance restraints per residue for the experimentally constrained amino acids. An ensemble of 25 refined distance geometry structures with penalties (sum of the squares of the distance violations) of 0.32 A2 or less and individual distance violations under 0.06 A was generated; best-fit superposition of ordered backbone heavy atoms relative to mean atom positions afforded root-mean-square deviations of 0.50 (+/- 0.08) A. The folded HIV-1 matrix protein structure is composed of five alpha-helices, a short 3(10) helical stretch, and a three-strand mixed beta-sheet. Helices I to III and the 3(10) helix pack about a central helix (IV) to form a compact globular domain that is capped by the beta-sheet. The C-terminal helix (helix V) projects away

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

    Directory of Open Access Journals (Sweden)

    Stormo Gary D

    2005-07-01

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

  16. Metal binding proteins, recombinant host cells and methods

    Science.gov (United States)

    Summers, Anne O.; Caguiat, Jonathan J.

    2004-06-15

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

  17. HIV gp120 binds to mannose receptor on vaginal epithelial cells and induces production of matrix metalloproteinases.

    Directory of Open Access Journals (Sweden)

    Sashaina E Fanibunda

    Full Text Available BACKGROUND: During sexual transmission of HIV in women, the virus breaches the multi-layered CD4 negative stratified squamous epithelial barrier of the vagina, to infect the sub-epithelial CD4 positive immune cells. However the mechanisms by which HIV gains entry into the sub-epithelial zone is hitherto unknown. We have previously reported human mannose receptor (hMR as a CD4 independent receptor playing a role in HIV transmission on human spermatozoa. The current study was undertaken to investigate the expression of hMR in vaginal epithelial cells, its HIV gp120 binding potential, affinity constants and the induction of matrix metalloproteinases (MMPs downstream of HIV gp120 binding to hMR. PRINCIPAL FINDINGS: Human vaginal epithelial cells and the immortalized vaginal epithelial cell line Vk2/E6E7 were used in this study. hMR mRNA and protein were expressed in vaginal epithelial cells and cell line, with a molecular weight of 155 kDa. HIV gp120 bound to vaginal proteins with high affinity, (Kd = 1.2±0.2 nM for vaginal cells, 1.4±0.2 nM for cell line and the hMR antagonist mannan dose dependently inhibited this binding. Both HIV gp120 binding and hMR exhibited identical patterns of localization in the epithelial cells by immunofluorescence. HIV gp120 bound to immunopurified hMR and affinity constants were 2.9±0.4 nM and 3.2±0.6 nM for vaginal cells and Vk2/E6E7 cell line respectively. HIV gp120 induced an increase in MMP-9 mRNA expression and activity by zymography, which could be inhibited by an anti-hMR antibody. CONCLUSION: hMR expressed by vaginal epithelial cells has high affinity for HIV gp120 and this binding induces production of MMPs. We propose that the induction of MMPs in response to HIV gp120 may lead to degradation of tight junction proteins and the extracellular matrix proteins in the vaginal epithelium and basement membrane, leading to weakening of the epithelial barrier; thereby facilitating transport of HIV across the

  18. Transduction proteins of olfactory receptor cells: identification of guanine nucleotide binding proteins and protein kinase C

    International Nuclear Information System (INIS)

    Anholt, R.R.H.; Mumby, S.M.; Stoffers, D.A.; Girard, P.R.; Kuo, J.F.; Snyder, S.H.

    1987-01-01

    The authors have analyzed guanine nucleotide binding proteins (G-proteins) in the olfactory epithelium of Rana catesbeiana using subunit-specific antisera. The olfactory epithelium contained the α subunits of three G-proteins, migrating on polyacrylamide gels in SDS with apparent molecular weights of 45,000, 42,000, and 40,000, corresponding to G/sub s/, G/sub i/, and G/sub o/, respectively. A single β subunit with an apparent molecular weight of 36,000 was detected. An antiserum against the α subunit of retinal transducin failed to detect immunoreactive proteins in olfactory cilia detached from the epithelium. The olfactory cilia appeared to be enriched in immunoreactive G/sub sα/ relative to G/sub ichemical bond/ and G/sub ochemical bond/ when compared to membranes prepared from the olfactory epithelium after detachment of the cilia. Bound antibody was detected by autoradiography after incubation with [ 125 I]protein. Immunohistochemical studies using an antiserum against the β subunit of G-proteins revealed intense staining of the ciliary surface of the olfactory epithelium and of the axon bundles in the lamina propria. In contrast, an antiserum against a common sequence of the α subunits preferentially stained the cell membranes of the olfactory receptor cells and the acinar cells of Bowman's glands and the deep submucosal glands. In addition to G-proteins, they have identified protein kinase C in olfactory cilia via a protein kinase C specific antiserum and via phorbol ester binding. However, in contrast to the G-proteins, protein kinase C occurred also in cilia isolated from respiratory epithelium

  19. Cartilage oligomeric matrix protein enhances matrix assembly during chondrogenesis of human mesenchymal stem cells.

    Science.gov (United States)

    Haleem-Smith, Hana; Calderon, Raul; Song, Yingjie; Tuan, Rocky S; Chen, Faye H

    2012-04-01

    Cartilage oligomeric matrix protein/thrombospondin-5 (COMP/TSP5) is an abundant cartilage extracellular matrix (ECM) protein that interacts with major cartilage ECM components, including aggrecan and collagens. To test our hypothesis that COMP/TSP5 functions in the assembly of the ECM during cartilage morphogenesis, we have employed mesenchymal stem cell (MSC) chondrogenesis in vitro as a model to examine the effects of COMP over-expression on neo-cartilage formation. Human bone marrow-derived MSCs were transfected with either full-length COMP cDNA or control plasmid, followed by chondrogenic induction in three-dimensional pellet or alginate hydrogel culture. MSC chondrogenesis and ECM production was estimated based on quantitation of sulfated glycosaminoglycan (sGAG) accumulation, immunohistochemistry of the presence and distribution of cartilage ECM proteins, and real-time RT-PCR analyis of mRNA expression of cartilage markers. Our results showed that COMP over-expression resulted in increased total sGAG content during the early phase of MSC chondrogenesis, and increased immuno-detectable levels of aggrecan and collagen type II in the ECM of COMP-transfected pellet and alginate cultures, indicating more abundant cartilaginous matrix. COMP transfection did not significantly increase the transcript levels of the early chondrogenic marker, Sox9, or aggrecan, suggesting that enhancement of MSC cartilage ECM was effected at post-transcriptional levels. These findings strongly suggest that COMP functions in mesenchymal chondrogenesis by enhancing cartilage ECM organization and assembly. The action of COMP is most likely mediated not via direct changes in cartilage matrix gene expression but via interactions of COMP with other cartilage ECM proteins, such as aggrecan and collagens, that result in enhanced assembly and retention.

  20. CARTILAGE OLIGOMERIC MATRIX PROTEIN ENHANCES MATRIX ASSEMBLY DURING CHONDROGENESIS OF HUMAN MESENCHYMAL STEM CELLS

    Science.gov (United States)

    Haleem-Smith, Hana; Calderon, Raul; Song, Yingjie; Tuan, Rocky S.; Chen, Faye H.

    2011-01-01

    Cartilage oligomeric matrix protein/thrombospondin-5 (COMP/TSP5) is an abundant cartilage extracellular matrix (ECM) protein that interacts with major cartilage ECM components, including aggrecan and collagens. To test our hypothesis that COMP/TSP5 functions in the assembly of the ECM during cartilage morphogenesis, we have employed mesenchymal stem cell (MSC) chondrogenesis in vitro as a model to examine the effects of COMP over-expression on neo-cartilage formation. Human bone marrow-derived MSCs were transfected with either full-length COMP cDNA or control plasmid, followed by chondrogenic induction in three-dimensional pellet or alginate-hydrogel culture. MSC chondrogenesis and ECM production was estimated based on quantitation of sulfated glycosaminoglycan (sGAG) accumulation, immunohistochemistry of the presence and distribution of cartilage ECM proteins, and real-time RT-PCR analyis of mRNA expression of cartilage markers. Our results showed that COMP over-expression resulted in increased total sGAG content during the early phase of MSC chondrogenesis, and increased immuno-detectable levels of aggrecan and collagen type II in the ECM of COMP-transfected pellet and alginate cultures, indicating more abundant cartilaginous matrix. COMP transfection did not significantly increase the transcript levels of the early chondrogenic marker, Sox9, or aggrecan, suggesting that enhancement of MSC cartilage ECM was effected at post-transcriptional levels. These findings strongly suggest that COMP functions in mesenchymal chondrogenesis by enhancing cartilage ECM organization and assembly. The action of COMP is most likely mediated not via direct changes in cartilage matrix gene expression but via interactions of COMP with other cartilage ECM proteins, such as aggrecan and collagens, that result in enhanced assembly and retention. PMID:22095699

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

    Directory of Open Access Journals (Sweden)

    Carson M Andorf

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

  2. Echinococcus granulosus fatty acid binding proteins subcellular localization.

    Science.gov (United States)

    Alvite, Gabriela; Esteves, Adriana

    2016-05-01

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

  3. Differential plasma protein binding to metal oxide nanoparticles

    International Nuclear Information System (INIS)

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

    2009-01-01

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

  4. Improved gel electrophoresis matrix for hydrophobic protein separation and identification.

    Science.gov (United States)

    Tokarski, Caroline; Fillet, Marianne; Rolando, Christian

    2011-03-01

    We propose an improved acrylamide gel for the separation of hydrophobic proteins. The separation strategy is based on the incorporation of N-alkylated and N,N'-dialkylated acrylamide monomers in the gel composition in order to increase hydrophobic interactions between the gel matrix and the membrane proteins. Focusing on the most efficient monomer, N,N'-dimethylacrylamide, the potentiality of the new matrix was evaluated on membrane proteins of the human colon HCT-116 cell line. Protein analysis was performed using an adapted analytical strategy based on FT-ICR tandem mass spectrometry. As a result of this comparative study, including advanced reproducibility experiments, more hydrophobic proteins were identified in the new gel (average GRAVY: -0.085) than in the classical gel (average GRAVY: -0.411). Highly hydrophobic peptides were identified reaching a GRAVY value up to 1.450, therefore indicating their probable locations in the membrane. Focusing on predicted transmembrane domains, it can be pointed out that 27 proteins were identified in the hydrophobic gel containing up to 11 transmembrane domains; in the classical gel, only 5 proteins containing 1 transmembrane domain were successfully identified. For example, multiple ionic channels and receptors were characterized in the hydrophobic gel such as the sodium/potassium channel and the glutamate or the transferrin receptors whereas they are traditionally detected using specific enrichment techniques such as immunoprecipitation. In total, membrane proteins identified in the classical gel are well documented in the literature, while most of the membrane proteins only identified on the hydrophobic gel have rarely or never been described using a proteomic-based approach. 2010 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2018-04-17

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

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

    Science.gov (United States)

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

    2017-04-04

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

  7. An odorant-binding protein as a new allergen from Siberian hamster (Phodopus sungorus).

    Science.gov (United States)

    Torres, J A; Pastor-Vargas, C; de las Heras, M; Vivanco, F; Cuesta, Javier; Sastre, J

    2012-01-01

    A case of anaphylaxis following a bite from a Siberian hamster (SH; Phodopus sungorus) is described. Skin prick tests with hair, urine and salivary gland extracts from SH were positive, while the tests were negative for hair extracts from other rodents. IgE immunoblotting with the patient serum revealed 3 IgE-binding bands of about 18, 21 and 23 kDa. When the patient's serum was preincubated with rabbit, mouse and gerbil hair extracts, no inhibition of the 3 SH IgE-binding bands was demonstrated. Proteins extracted from the 3 bands were analyzed by N-terminal sequencing and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry, and peptides were sequenced. IgE-binding bands were identified as being an odorant-binding protein belonging to the lipocalin family. Analysis of the 3 IgE-binding bands found in the hair, urine and salivary glands of SH showed a new allergenic protein lacking cross-reactivity with allergens from other rodents. The 3 bands likely correspond to isoforms of a single allergen. Copyright © 2011 S. Karger AG, Basel.

  8. Trans-acting translational regulatory RNA binding proteins.

    Science.gov (United States)

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

    2018-05-01

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

  9. Phosphorus Binding Sites in Proteins: Structural Preorganization and Coordination

    DEFF Research Database (Denmark)

    Gruber, Mathias Felix; Greisen, Per Junior; Junker, Märta Caroline

    2014-01-01

    to individual structures that bind to phosphate groups; here, we investigate a total of 8307 structures obtained from the RCSB Protein Data Bank (PDB). An analysis of the binding site amino acid propensities reveals very characteristic first shell residue distributions, which are found to be influenced...... by the characteristics of the phosphorus compound and by the presence of cobound cations. The second shell, which supports the coordinating residues in the first shell, is found to consist mainly of protein backbone groups. Our results show how the second shell residue distribution is dictated mainly by the first shell...

  10. Acyl-CoA binding protein and epidermal barrier function

    DEFF Research Database (Denmark)

    Bloksgaard, Maria; Neess, Ditte; Færgeman, Nils J

    2014-01-01

    The acyl-CoA binding protein (ACBP) is a 10kDa intracellular protein expressed in all eukaryotic species and mammalian tissues investigated. It binds acyl-CoA esters with high specificity and affinity and is thought to act as an intracellular transporter of acyl-CoA esters between different...... includes tousled and greasy fur, development of alopecia and scaling of the skin with age. Furthermore, epidermal barrier function is compromised causing a ~50% increase in transepidermal water loss relative to that of wild type mice. Lipidomic analyses indicate that this is due to significantly reduced...

  11. Pooled-matrix protein interaction screens using Barcode Fusion Genetics.

    Science.gov (United States)

    Yachie, Nozomu; Petsalaki, Evangelia; Mellor, Joseph C; Weile, Jochen; Jacob, Yves; Verby, Marta; Ozturk, Sedide B; Li, Siyang; Cote, Atina G; Mosca, Roberto; Knapp, Jennifer J; Ko, Minjeong; Yu, Analyn; Gebbia, Marinella; Sahni, Nidhi; Yi, Song; Tyagi, Tanya; Sheykhkarimli, Dayag; Roth, Jonathan F; Wong, Cassandra; Musa, Louai; Snider, Jamie; Liu, Yi-Chun; Yu, Haiyuan; Braun, Pascal; Stagljar, Igor; Hao, Tong; Calderwood, Michael A; Pelletier, Laurence; Aloy, Patrick; Hill, David E; Vidal, Marc; Roth, Frederick P

    2016-04-22

    High-throughput binary protein interaction mapping is continuing to extend our understanding of cellular function and disease mechanisms. However, we remain one or two orders of magnitude away from a complete interaction map for humans and other major model organisms. Completion will require screening at substantially larger scales with many complementary assays, requiring further efficiency gains in proteome-scale interaction mapping. Here, we report Barcode Fusion Genetics-Yeast Two-Hybrid (BFG-Y2H), by which a full matrix of protein pairs can be screened in a single multiplexed strain pool. BFG-Y2H uses Cre recombination to fuse DNA barcodes from distinct plasmids, generating chimeric protein-pair barcodes that can be quantified via next-generation sequencing. We applied BFG-Y2H to four different matrices ranging in scale from ~25 K to 2.5 M protein pairs. The results show that BFG-Y2H increases the efficiency of protein matrix screening, with quality that is on par with state-of-the-art Y2H methods. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  12. Binding energies of double-Λ hypernuclei and ΛΛ G-matrix

    International Nuclear Information System (INIS)

    Himeno, Hiroyuki; Sakuda, Toshimi; Nagata, Sinobu; Yamamoto, Yasuo.

    1993-01-01

    Binding energies of double-Λ hypernuclei ΛΛ 10 Be, ΛΛ 13 B and ΛΛ 6 He are calculated on the basis of G-matrix theory in finite nuclei. The core + Λ + Λ three-body model is adopted and the G-matrix for ΛΛ interaction is treated consistently with the model space. As the bare interaction the Nijmegen model D and model F are used. It is discussed that the consistency of the interaction with the model space is very important to calculate reliably the binding energies. It is shown that if the new event of double-Λ hypernuclei is interpreted as ΛΛ 13 B, model D reproduces the experimental data very well, whereas model F does not. (author)

  13. Temperature-dependent binding of cyclosporine to an erythrocyte protein

    International Nuclear Information System (INIS)

    Agarwal, R.P.; Threatte, G.A.; McPherson, R.A.

    1987-01-01

    In this competitive binding assay to measure endogenous binding capacity for cyclosporine (CsA) in erythrocyte lysates, a fixed amount of [ 3 H]CsA plus various concentrations of unlabeled CsA is incubated with aliquots of a test hemolysate. Free CsA is then adsorbed onto charcoal and removed by centrifugation; CsA complexed with a cyclosporine-binding protein (CsBP) remains in the supernate. We confirmed the validity of this charcoal-separation mode of binding analysis by comparison with equilibrium dialysis. Scatchard plot analysis of the results at 4 degrees C yielded a straight line with slope corresponding to a binding constant of 1.9 X 10(7) L/mol and a saturation capacity of approximately 4 mumol per liter of packed erythrocytes. Similar analysis of binding data at 24 degrees C and 37 degrees C showed that the binding constant decreased with increasing temperature, but the saturation capacity did not change. CsBP was not membrane bound but appeared to be freely distributed within erythrocytes. 125 I-labeled CsA did not complex with the erythrocyte CsBP. Several antibiotics and other drugs did not inhibit binding between CsA and CsBP. These findings may explain the temperature-dependent uptake of CsA by erythrocytes in whole blood and suggest that measurement of CsBP in erythrocytes or lymphocytes may help predict therapeutic response or toxicity after administration of CsA

  14. Interaction of bovine gallbladder mucin and calcium-binding protein: effects on calcium phosphate precipitation.

    Science.gov (United States)

    Afdhal, N H; Ostrow, J D; Koehler, R; Niu, N; Groen, A K; Veis, A; Nunes, D P; Offner, G D

    1995-11-01

    Gallstones consist of calcium salts and cholesterol crystals, arrayed on a matrix of gallbladder mucin (GBM), and regulatory proteins like calcium-binding protein (CBP). To determine if interactions between CBP and GBM follow a biomineralization scheme, their mutual binding and effects on CaHPO4 precipitation were studied. Binding of CBP to GBM was assessed by inhibition of the fluorescence of the complex of GBM with bis-1,8-anilinonaphthalene sulfonic acid (bis-ANS). The effects of the proteins on precipitation of CaHPO4 were assessed by nephelometry and gravimetry. Precipitates were analyzed for calcium, phosphate, and protein. CBP and bis-ANS competitively displaced each other from 30 binding sites on mucin, with a 1:1 stoichiometry and similar affinity. The rate of precipitation of CaHPO4 was retarded by mucin and CBP. Precipitate mass was unaffected by GBM alone but decreased with the addition of CBP. Complexing CBP with GBM abolished or moderated this latter effect, altered precipitate morphology, and changed the stoichiometric ratios of Ca to PO4 in the precipitates from 1:1 to 3:2. Mucin and CBP were incorporated into the precipitates. These studies suggest that the formation of calcium-containing gallstones is a biomineralization process regulated by both GBM and CBP.

  15. Fusicoccin-Binding Proteins in Arabidopsis thaliana (L.) Heynh. 1

    Science.gov (United States)

    Meyer, Christiane; Feyerabend, Martin; Weiler, Elmar W.

    1989-01-01

    Using the novel radioligand, [3H]-9′-nor-fusicoccin-8′-alcohol, high affinity binding sites for fusicoccin were characterized in preparations from leaves of Arabidopsis thaliana (L.) Heynh. The binding site copartitioned with the plasmalemma marker, vanadate-sensitive K+, Mg2+-ATPase, when microsomal fractions were further purified by aqueous two-phase partitioning in polyethylene glycol-dextran phase systems and sedimented at an equilibrium density of 1.17 grams per cubic centimeter in continuous sucrose density gradients, as did the ATPase marker. The binding of [3H]-9′-nor-fusicoccin-8′-alcohol was saturable and Scatchard analysis revealed a biphasic plot with two apparent dissociation constants (KD), KD1 = 1.5 nanomolar and KD2 = 42 nanomolar, for the radioligand. Binding was optimal at pH 6, thermolabile, and was reduced by 70% when the membrane vesicles were pretreated with trypsin. The data are consistent with the presence of one or several binding proteins for fusicoccin at the plasma membrane of A. thaliana. Binding of the radioligand was unaffected by pretreatment of the sites with various alkylating and reducing agents, but was reduced by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, diethylpyrocarbonate, chloramine T, and periodate. A number of detergents were tested to find optimum conditions for solubilization. Nonanoyl-N-methylglucamide (50 millimolar) solubilized 70% of the radioligand-binding protein complex in undissociated form. Photoaffinity labeling of membrane preparations with a tritiated azido analog of fusicoccin resulted in the labeling of a 34 ± 1 kilodalton polypeptide. Labeling of this polypeptide, presumably the fusicoccin-binding protein, was severely reduced in the presence of unlabeled fusicoccin. PMID:16666603

  16. The RNA-binding protein repertoire of Arabidopsis thaliana

    KAUST Repository

    Marondedze, Claudius

    2016-07-11

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

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

    Science.gov (United States)

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

    2018-04-29

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

  18. Annexin A2 Mediates the Localization of Measles Virus Matrix Protein at the Plasma Membrane.

    Science.gov (United States)

    Koga, Ritsuko; Kubota, Marie; Hashiguchi, Takao; Yanagi, Yusuke; Ohno, Shinji

    2018-02-28

    Annexins are a family of structurally related proteins that bind negatively charged membrane phospholipids in a Ca 2+ -dependent manner. Annexin A2 (AnxA2), a member of the family, has been implicated in a variety of cellular functions including the organization of membrane domains, vesicular trafficking and cell-cell adhesion. AnxA2 generally forms the heterotetrameric complex with a small Ca 2+ -binding protein S100A10. Measles virus (MV), a member of the family Paramyxoviridae , is an enveloped virus with a nonsegmented negative strand RNA genome. Knockdown of AnxA2 greatly reduced MV growth in cells, without affecting its entry and viral RNA production. In MV-infected, AnxA2-knockdown cells, the expression level of the matrix (M) protein, but not other viral proteins, was reduced compared with that in control cells, and the distribution of the M protein at the plasma membrane was decreased. The M protein lines the inner surface of the envelope and plays an important role in virus assembly by connecting the nucleocapsid to the envelope proteins. The M protein bound to AnxA2 independently of AnxA2's phosphorylation or its association with S100A10, and was co-localized with AnxA2 within cells. Truncation of the N-terminal 10 amino acid residues, but not the N-terminal 5 residues, compromised the ability of the M protein to interact with AnxA2 and localize at the plasma membrane. These results indicate that AnxA2 mediates the localization of the MV M protein at the plasma membrane by interacting with its N-terminal region (especially residues at positions 6-10), thereby aiding in MV assembly. IMPORTANCE Measles virus (MV) is an important human pathogen, still claiming ∼ 100,000 lives per year despite the presence of effective vaccines, and causes occasional outbreaks even in developed countries. Replication of viruses largely relies on the functions of host cells. Our study revealed that the reduction of the host protein annexin A2 compromises the replication of

  19. Neuronal differentiation modulates the dystrophin Dp71d binding to the nuclear matrix

    International Nuclear Information System (INIS)

    Rodriguez-Munoz, Rafael; Villarreal-Silva, Marcela; Gonzalez-Ramirez, Ricardo; Garcia-Sierra, Francisco; Mondragon, Monica; Mondragon, Ricardo; Cerna, Joel; Cisneros, Bulmaro

    2008-01-01

    The function of dystrophin Dp71 in neuronal cells remains unknown. To approach this issue, we have selected the PC12 neuronal cell line. These cells express both a Dp71f cytoplasmic variant and a Dp71d nuclear isoform. In this study, we demonstrated by electron and confocal microscopy analyses of in situ nuclear matrices and Western blotting evaluation of cell extracts that Dp71d associates with the nuclear matrix. Interestingly, this binding is modulated during NGF-induced neuronal differentiation of PC12 cells with a twofold increment in the differentiated cells, compared to control cells. Also, distribution of Dp71d along the periphery of the nuclear matrix observed in the undifferentiated cells is replaced by intense fluorescent foci localized in Center of the nucleoskeletal structure. In summary, we revealed that Dp71d is a dynamic component of nuclear matrix that might participate in the nuclear modeling occurring during neuronal differentiation

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

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Z.H.

    1988-01-01

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

  1. Binding of rare earths to serum proteins and DNA

    International Nuclear Information System (INIS)

    Rosoff, B.; Spencer, H.

    1979-01-01

    In order to investigate further the physiological behavior of rare earths and rare earth chelates, studies of the binding of 46 Sc, 91 Y, and 140 La to serum proteins and to nucleic acids were performed using the methods of equilibrium dialysis and ultrafiltration. The binding of lanthanum and yttrium as the chlorides to α-globulin increased as the free rare earth concentration increased. When scandium and lanthanum were chelated in nitrilotriacetate (NTA) the binding to α-globulin was considerably less and there was no binding to albumin. The binding of 46 Sc chelated to ethylenediamine di(O-hydroxyphenylacetate) (EDDHA) was five times greater than of 46 Sc chloride. When the free scandium concentration was increased, the moles bound per mole of protein increased proportionally and the binding was reversible. Scandium was 100% filterable from a mixture of human serum and from the scandium chelates with high stability constants scandium diethylenetriaminepentaacetate (ScDTPA), scandium ethylenediaminetetraacetate (ScEDTA) and scandium cyclohexane trans-1,2-diaminetetraacetate (ScCDTA) respectively. In contrast, only 2% of the scandium was filterable when scandium nitrilotriacetate, a scandium chelate of low stability constant, was used. (Auth.)

  2. Myristoylated α subunits of guanine nucleotide-binding regulatory proteins

    International Nuclear Information System (INIS)

    Buss, J.E.; Mumby, S.M.; Casey, P.J.; Gilman, A.G.; Sefton, B.M.

    1987-01-01

    Antisera directed against specific subunits of guanine nucleotide-binding regulatory proteins (G proteins) were used to immunoprecipitate these polypeptides from metabolically labeled cells. This technique detects, in extracts of a human astrocytoma cell line, the α subunits of G/sub s/ (stimulatory) (α 45 and α 52 ), a 41-kDa subunit of G/sub i/ (inhibitory) (α 41 ), a 40-kDa protein (α 40 ), and the 36-kDa β subunit. No protein that comigrated with the α subunit of G 0 (unknown function) (α 39 ) was detected. In cells grown in the presence of [ 3 H]myristic acid, α 41 and α 40 contained 3 H label, while the β subunit did not. Chemical analysis of lipids attached covalently to purified α 41 and α 39 from bovine brain also revealed myristic acid. Similar analysis of brain G protein β and γ subunits and of G/sub t/ (Transducin) subunits (α, β, and γ) failed to reveal fatty acids. The fatty acid associated with α 41 , α 40 , and α 39 was stable to treatment with base, suggesting that the lipid is linked to the polypeptide via an amide bond. These GTP binding proteins are thus identified as members of a select group of proteins that contains myristic acid covalently attached to the peptide backbone. Myristate may play an important role in stabilizing interactions of G proteins with phospholipid or with membrane-bound proteins

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

    International Nuclear Information System (INIS)

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

    1991-01-01

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

  4. Expression, purification and crystallization of a lyssavirus matrix (M) protein

    Science.gov (United States)

    Assenberg, René; Delmas, Olivier; Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J.; Bourhy, Hervé; Grimes, Jonathan M.

    2008-01-01

    The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6122 or P6522, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress. PMID:18391421

  5. Distance matrix-based approach to protein structure prediction.

    Science.gov (United States)

    Kloczkowski, Andrzej; Jernigan, Robert L; Wu, Zhijun; Song, Guang; Yang, Lei; Kolinski, Andrzej; Pokarowski, Piotr

    2009-03-01

    Much structural information is encoded in the internal distances; a distance matrix-based approach can be used to predict protein structure and dynamics, and for structural refinement. Our approach is based on the square distance matrix D = [r(ij)(2)] containing all square distances between residues in proteins. This distance matrix contains more information than the contact matrix C, that has elements of either 0 or 1 depending on whether the distance r (ij) is greater or less than a cutoff value r (cutoff). We have performed spectral decomposition of the distance matrices D = sigma lambda(k)V(k)V(kT), in terms of eigenvalues lambda kappa and the corresponding eigenvectors v kappa and found that it contains at most five nonzero terms. A dominant eigenvector is proportional to r (2)--the square distance of points from the center of mass, with the next three being the principal components of the system of points. By predicting r (2) from the sequence we can approximate a distance matrix of a protein with an expected RMSD value of about 7.3 A, and by combining it with the prediction of the first principal component we can improve this approximation to 4.0 A. We can also explain the role of hydrophobic interactions for the protein structure, because r is highly correlated with the hydrophobic profile of the sequence. Moreover, r is highly correlated with several sequence profiles which are useful in protein structure prediction, such as contact number, the residue-wise contact order (RWCO) or mean square fluctuations (i.e. crystallographic temperature factors). We have also shown that the next three components are related to spatial directionality of the secondary structure elements, and they may be also predicted from the sequence, improving overall structure prediction. We have also shown that the large number of available HIV-1 protease structures provides a remarkable sampling of conformations, which can be viewed as direct structural information about the

  6. Targeting Human Cancer by a Glycosaminoglycan Binding Malaria Protein

    DEFF Research Database (Denmark)

    Salanti, Ali; Clausen, Thomas M.; Agerbæk, Mette Ø.

    2015-01-01

    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...

  7. Co-suppression of sterol-regulatory element binding protein ...

    African Journals Online (AJOL)

    Administrator

    2011-06-22

    Jun 22, 2011 ... In Arabidopsis,. At5g35220 gene being sterol regulatory element-binding protein site 2, protease and metalloendopeptidase activity were required for chloroplast development and play a role in regulation of endodermal plastid size and number that are involved in ethylene-dependent gravitropism of light-.

  8. Methods of use of cellulose binding domain proteins

    Science.gov (United States)

    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.

  9. Immunoglobulin classes, metal binding proteins, and trace metals in ...

    African Journals Online (AJOL)

    , IgA and IgM), metal binding proteins (Transferrin, Caeruloplasmin, Alpha-2- Macroglobulin and Haptoglobin) and nutritionally essential trace metals/heavy metals (Zn, Fe, Se, Cu, Mg, Cd and Pb) in Nigerian cassava processors using single ...

  10. Proteomic analysis of heparin-binding proteins from human seminal ...

    Indian Academy of Sciences (India)

    Prakash

    (MALDI TOF/MS) for protein analysis of human HBPs. We resolved 70 ... Thus, the combined effects of seminal plasma components support the survival of ...... The BBXB motif of RANTES is the principal site for heparin binding and controls ...

  11. Enhanced binding by dextran-grafting to Protein A affinity chromatographic media.

    Science.gov (United States)

    Zhao, Lan; Zhu, Kai; Huang, Yongdong; Li, Qiang; Li, Xiunan; Zhang, Rongyue; Su, Zhiguo; Wang, Qibao; Ma, Guanghui

    2017-04-01

    Dextran-grafted Protein A affinity chromatographic medium was prepared by grafting dextran to agarose-based matrix, followed by epoxy-activation and Protein A coupling site-directed to sulfhydryl groups of cysteine molecules. An enhancement of both the binding performance and the stability was achieved for this dextran-grafted Protein A chromatographic medium. Its dynamic binding capacity was 61 mg immunoglobulin G/mL suction-dried gel, increased by 24% compared with that of the non-grafted medium. The binding capacity of dextran-grafted medium decreased about 7% after 40 cleaning-in-place cycles, much lower than that of the non-grafted medium as decreased about 15%. Confocal laser scanning microscopy results showed that immunoglobulin G was bound to both the outside and the inside of dextran-grafted medium faster than that of non-grafted one. Atomic force microscopy showed that this dextran-grafted Protein A medium had much rougher surface with a vertical coordinate range of ±80 nm, while that of non-grafted one was ±10 nm. Grafted dextran provided a more stereo surface morphology and immunoglobulin G molecules were more easily to be bound. This high-performance dextran-grafted Protein A affinity chromatographic medium has promising applications in large-scale antibody purification. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. RBPmap: a web server for mapping binding sites of RNA-binding proteins.

    Science.gov (United States)

    Paz, Inbal; Kosti, Idit; Ares, Manuel; Cline, Melissa; Mandel-Gutfreund, Yael

    2014-07-01

    Regulation of gene expression is executed in many cases by RNA-binding proteins (RBPs) that bind to mRNAs as well as to non-coding RNAs. RBPs recognize their RNA target via specific binding sites on the RNA. Predicting the binding sites of RBPs is known to be a major challenge. We present a new webserver, RBPmap, freely accessible through the website http://rbpmap.technion.ac.il/ for accurate prediction and mapping of RBP binding sites. RBPmap has been developed specifically for mapping RBPs in human, mouse and Drosophila melanogaster genomes, though it supports other organisms too. RBPmap enables the users to select motifs from a large database of experimentally defined motifs. In addition, users can provide any motif of interest, given as either a consensus or a PSSM. The algorithm for mapping the motifs is based on a Weighted-Rank approach, which considers the clustering propensity of the binding sites and the overall tendency of regulatory regions to be conserved. In addition, RBPmap incorporates a position-specific background model, designed uniquely for different genomic regions, such as splice sites, 5' and 3' UTRs, non-coding RNA and intergenic regions. RBPmap was tested on high-throughput RNA-binding experiments and was proved to be highly accurate. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Sampling and energy evaluation challenges in ligand binding protein design.

    Science.gov (United States)

    Dou, Jiayi; Doyle, Lindsey; Jr Greisen, Per; Schena, Alberto; Park, Hahnbeom; Johnsson, Kai; Stoddard, Barry L; Baker, David

    2017-12-01

    The steroid hormone 17α-hydroxylprogesterone (17-OHP) is a biomarker for congenital adrenal hyperplasia and hence there is considerable interest in development of sensors for this compound. We used computational protein design to generate protein models with binding sites for 17-OHP containing an extended, nonpolar, shape-complementary binding pocket for the four-ring core of the compound, and hydrogen bonding residues at the base of the pocket to interact with carbonyl and hydroxyl groups at the more polar end of the ligand. Eight of 16 designed proteins experimentally tested bind 17-OHP with micromolar affinity. A co-crystal structure of one of the designs revealed that 17-OHP is rotated 180° around a pseudo-two-fold axis in the compound and displays multiple binding modes within the pocket, while still interacting with all of the designed residues in the engineered site. Subsequent rounds of mutagenesis and binding selection improved the ligand affinity to nanomolar range, while appearing to constrain the ligand to a single bound conformation that maintains the same "flipped" orientation relative to the original design. We trace the discrepancy in the design calculations to two sources: first, a failure to model subtle backbone changes which alter the distribution of sidechain rotameric states and second, an underestimation of the energetic cost of desolvating the carbonyl and hydroxyl groups of the ligand. The difference between design model and crystal structure thus arises from both sampling limitations and energy function inaccuracies that are exacerbated by the near two-fold symmetry of the molecule. © 2017 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

  14. Structure-guided design of an engineered streptavidin with reusability to purify streptavidin-binding peptide tagged proteins or biotinylated proteins.

    Directory of Open Access Journals (Sweden)

    Sau-Ching Wu

    Full Text Available Development of a high-affinity streptavidin-binding peptide (SBP tag allows the tagged recombinant proteins to be affinity purified using the streptavidin matrix without the need of biotinylation. The major limitation of this powerful technology is the requirement to use biotin to elute the SBP-tagged proteins from the streptavidin matrix. Tight biotin binding by streptavidin essentially allows the matrix to be used only once. To address this problem, differences in interactions of biotin and SBP with streptavidin were explored. Loop3-4 which serves as a mobile lid for the biotin binding pocket in streptavidin is in the closed state with biotin binding. In contrast, this loop is in the open state with SBP binding. Replacement of glycine-48 with a bulkier residue (threonine in this loop selectively reduces the biotin binding affinity (Kd from 4 × 10(-14 M to 4.45 × 10(-10 M without affecting the SBP binding affinity. Introduction of a second mutation (S27A to the first mutein (G48T results in the development of a novel engineered streptavidin SAVSBPM18 which could be recombinantly produced in the functional form from Bacillus subtilis via secretion. To form an intact binding pocket for tight binding of SBP, two diagonally oriented subunits in a tetrameric streptavidin are required. It is vital for SAVSBPM18 to be stably in the tetrameric state in solution. This was confirmed using an HPLC/Laser light scattering system. SAVSBPM18 retains high binding affinity to SBP but has reversible biotin binding capability. The SAVSBPM18 matrix can be applied to affinity purify SBP-tagged proteins or biotinylated molecules to homogeneity with high recovery in a reusable manner. A mild washing step is sufficient to regenerate the matrix which can be reused for multiple rounds. Other applications including development of automated protein purification systems, lab-on-a-chip micro-devices, reusable biosensors, bioreactors and microarrays, and strippable

  15. Structure-guided design of an engineered streptavidin with reusability to purify streptavidin-binding peptide tagged proteins or biotinylated proteins.

    Science.gov (United States)

    Wu, Sau-Ching; Wong, Sui-Lam

    2013-01-01

    Development of a high-affinity streptavidin-binding peptide (SBP) tag allows the tagged recombinant proteins to be affinity purified using the streptavidin matrix without the need of biotinylation. The major limitation of this powerful technology is the requirement to use biotin to elute the SBP-tagged proteins from the streptavidin matrix. Tight biotin binding by streptavidin essentially allows the matrix to be used only once. To address this problem, differences in interactions of biotin and SBP with streptavidin were explored. Loop3-4 which serves as a mobile lid for the biotin binding pocket in streptavidin is in the closed state with biotin binding. In contrast, this loop is in the open state with SBP binding. Replacement of glycine-48 with a bulkier residue (threonine) in this loop selectively reduces the biotin binding affinity (Kd) from 4 × 10(-14) M to 4.45 × 10(-10) M without affecting the SBP binding affinity. Introduction of a second mutation (S27A) to the first mutein (G48T) results in the development of a novel engineered streptavidin SAVSBPM18 which could be recombinantly produced in the functional form from Bacillus subtilis via secretion. To form an intact binding pocket for tight binding of SBP, two diagonally oriented subunits in a tetrameric streptavidin are required. It is vital for SAVSBPM18 to be stably in the tetrameric state in solution. This was confirmed using an HPLC/Laser light scattering system. SAVSBPM18 retains high binding affinity to SBP but has reversible biotin binding capability. The SAVSBPM18 matrix can be applied to affinity purify SBP-tagged proteins or biotinylated molecules to homogeneity with high recovery in a reusable manner. A mild washing step is sufficient to regenerate the matrix which can be reused for multiple rounds. Other applications including development of automated protein purification systems, lab-on-a-chip micro-devices, reusable biosensors, bioreactors and microarrays, and strippable detection agents for

  16. RSAT matrix-clustering: dynamic exploration and redundancy reduction of transcription factor binding motif collections.

    Science.gov (United States)

    Castro-Mondragon, Jaime Abraham; Jaeger, Sébastien; Thieffry, Denis; Thomas-Chollier, Morgane; van Helden, Jacques

    2017-07-27

    Transcription factor (TF) databases contain multitudes of binding motifs (TFBMs) from various sources, from which non-redundant collections are derived by manual curation. The advent of high-throughput methods stimulated the production of novel collections with increasing numbers of motifs. Meta-databases, built by merging these collections, contain redundant versions, because available tools are not suited to automatically identify and explore biologically relevant clusters among thousands of motifs. Motif discovery from genome-scale data sets (e.g. ChIP-seq) also produces redundant motifs, hampering the interpretation of results. We present matrix-clustering, a versatile tool that clusters similar TFBMs into multiple trees, and automatically creates non-redundant TFBM collections. A feature unique to matrix-clustering is its dynamic visualisation of aligned TFBMs, and its capability to simultaneously treat multiple collections from various sources. We demonstrate that matrix-clustering considerably simplifies the interpretation of combined results from multiple motif discovery tools, and highlights biologically relevant variations of similar motifs. We also ran a large-scale application to cluster ∼11 000 motifs from 24 entire databases, showing that matrix-clustering correctly groups motifs belonging to the same TF families, and drastically reduced motif redundancy. matrix-clustering is integrated within the RSAT suite (http://rsat.eu/), accessible through a user-friendly web interface or command-line for its integration in pipelines. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. Thermal unfolding of a Ca- and Lanthanide-binding protein

    Energy Technology Data Exchange (ETDEWEB)

    Fahmy, Karim [Helmholtz-Zentrum Dresden-Rossendorf e.V., Dresden (Germany). Biophysics; Goettfert, M. [Technische Univ. Dresden (Germany); Knoeppel, J.

    2017-06-01

    The MIIA (metal ion-induced autocleavage)-domain of the protein Vic001052 from the pathogen Vibrio coralliilyticus, comprises 173 amino acids and exhibits Ca-dependent autoproteolytic activity. It shows homology to nodulation proteins which are secreted by Rhizobiacea into plant host cells where they exert Ca-dependent functions. We have studied the structural and energetic aspects of metal protein interactions of the MIIA domain which appear attractive for engineering metal-binding synthetic peptides. Using a non-cleavable MIIA domain construct, we detected very similar structural changes upon binding to Ca{sup 2+} and Eu{sup 3+}. The thermal denaturation of the Ca-bound state was studied by circular dichroism spectroscopy. The metal-bound folded state unfolds reversibly into an unstructured metal-free state similar to the metal-free state at room temperature.

  18. Predicting protein-protein interactions from multimodal biological data sources via nonnegative matrix tri-factorization.

    Science.gov (United States)

    Wang, Hua; Huang, Heng; Ding, Chris; Nie, Feiping

    2013-04-01

    Protein interactions are central to all the biological processes and structural scaffolds in living organisms, because they orchestrate a number of cellular processes such as metabolic pathways and immunological recognition. Several high-throughput methods, for example, yeast two-hybrid system and mass spectrometry method, can help determine protein interactions, which, however, suffer from high false-positive rates. Moreover, many protein interactions predicted by one method are not supported by another. Therefore, computational methods are necessary and crucial to complete the interactome expeditiously. In this work, we formulate the problem of predicting protein interactions from a new mathematical perspective--sparse matrix completion, and propose a novel nonnegative matrix factorization (NMF)-based matrix completion approach to predict new protein interactions from existing protein interaction networks. Through using manifold regularization, we further develop our method to integrate different biological data sources, such as protein sequences, gene expressions, protein structure information, etc. Extensive experimental results on four species, Saccharomyces cerevisiae, Drosophila melanogaster, Homo sapiens, and Caenorhabditis elegans, have shown that our new methods outperform related state-of-the-art protein interaction prediction methods.

  19. Collagen and related extracellular matrix proteins in atherosclerotic plaque development.

    Science.gov (United States)

    Shami, Annelie; Gonçalves, Isabel; Hultgårdh-Nilsson, Anna

    2014-10-01

    The structure, composition and turnover of the extracellular matrix (ECM) as well as cell-matrix interactions are crucial in the developing atherosclerotic plaque. There is a need for further insight into specific proteins in the ECM and their functions in the developing plaque, and during the last few years a number of publications have highlighted this very important field of research. These novel findings will be addressed in the present review. This review covers literature focused on collagen and ECM proteins interacting with collagen, and what their roles may be in plaque development. Acute myocardial infarction and stroke are common diseases that cause disability and mortality, and the underlying mechanism is often the rupture of a vulnerable atherosclerotic plaque. The vascular ECM and the tissue repair in the atherosclerotic lesion are important players in plaque progression. Understanding how specific proteins in the ECM interact with cells in the plaque and affect the fate of the plaque can lead to new treatments for cardiovascular disease.

  20. Crystal structure of an orthomyxovirus matrix protein reveals mechanisms for self-polymerization and membrane association.

    Science.gov (United States)

    Zhang, Wenting; Zheng, Wenjie; Toh, Yukimatsu; Betancourt-Solis, Miguel A; Tu, Jiagang; Fan, Yanlin; Vakharia, Vikram N; Liu, Jun; McNew, James A; Jin, Meilin; Tao, Yizhi J

    2017-08-08

    Many enveloped viruses encode a matrix protein. In the influenza A virus, the matrix protein M1 polymerizes into a rigid protein layer underneath the viral envelope to help enforce the shape and structural integrity of intact viruses. The influenza virus M1 is also known to mediate virus budding as well as the nuclear export of the viral nucleocapsids and their subsequent packaging into nascent viral particles. Despite extensive studies on the influenza A virus M1 (FLUA-M1), only crystal structures of its N-terminal domain are available. Here we report the crystal structure of the full-length M1 from another orthomyxovirus that infects fish, the infectious salmon anemia virus (ISAV). The structure of ISAV-M1 assumes the shape of an elbow, with its N domain closely resembling that of the FLUA-M1. The C domain, which is connected to the N domain through a flexible linker, is made of four α-helices packed as a tight bundle. In the crystal, ISAV-M1 monomers form infinite 2D arrays with a network of interactions involving both the N and C domains. Results from liposome flotation assays indicated that ISAV-M1 binds membrane via electrostatic interactions that are primarily mediated by a positively charged surface loop from the N domain. Cryoelectron tomography reconstruction of intact ISA virions identified a matrix protein layer adjacent to the inner leaflet of the viral membrane. The physical dimensions of the virion-associated matrix layer are consistent with the 2D ISAV-M1 crystal lattice, suggesting that the crystal lattice is a valid model for studying M1-M1, M1-membrane, and M1-RNP interactions in the virion.

  1. The Cobalamin-binding Protein in Zebrafish is an Intermediate Between the Three Cobalamin-binding Proteins in Human

    DEFF Research Database (Denmark)

    Greibe, Eva Holm; Fedosov, Sergey; Nexø, Ebba

    2012-01-01

    are the oldest evolutionary derivatives followed by IF and HC (the latter being present only in reptiles and most but not all mammals). Our findings suggest that the only cobalamin-binding protein in zebrafish is an intermediate between the three human cobalamin binders. These findings support the hypothesis...

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

    Science.gov (United States)

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

    1987-08-01

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

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

    Science.gov (United States)

    Takemura, Kazuhiro; Matubayasi, Nobuyuki; Kitao, Akio

    2018-03-14

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

  4. Polyamine binding to proteins in oat and Petunia protoplasts

    Science.gov (United States)

    Mizrahi, Y.; Applewhite, P. B.; Galston, A. W.

    1989-01-01

    Previous work (A Apelbaum et al. [1988] Plant Physiol 88: 996-998) has demonstrated binding of labeled spermidine (Spd) to a developmentally regulated 18 kilodalton protein in tobacco tissue cultures derived from thin surface layer explants. To assess the general importance of such Spd-protein complexes, we attempted bulk isolation from protoplasts of Petunia and oat (Avena sativa). In Petunia, as in tobacco, fed radioactive Spd is bound to protein, but in oat, Spd is first converted to 1,3,-diaminopropane (DAP), probably by polyamine oxidase action. In oat, binding of DAP to protein depends on age of donor leaf and conditions of illumination and temperature, and the extraction of the DAP-protein complex depends upon buffer and pH. The yield of the DAP-protein complex was maximized by extraction of frozen-thawed protoplasts with a pH 8.8 carbonate buffer containing SDS. Its molecular size, based on Sephacryl column fractionation of ammonium sulfate precipitated material, exceeded 45 kilodaltons. Bound Spd or DAP can be released from their complexes by the action of Pronase, but not DNAse, RNAse, or strong salt solutions, indicating covalent attachment to protein.

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

    Directory of Open Access Journals (Sweden)

    Hosoi A

    2016-03-01

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

  6. Sampling protein motion and solvent effect during ligand binding

    Science.gov (United States)

    Limongelli, Vittorio; Marinelli, Luciana; Cosconati, Sandro; La Motta, Concettina; Sartini, Stefania; Mugnaini, Laura; Da Settimo, Federico; Novellino, Ettore; Parrinello, Michele

    2012-01-01

    An exhaustive description of the molecular recognition mechanism between a ligand and its biological target is of great value because it provides the opportunity for an exogenous control of the related process. Very often this aim can be pursued using high resolution structures of the complex in combination with inexpensive computational protocols such as docking algorithms. Unfortunately, in many other cases a number of factors, like protein flexibility or solvent effects, increase the degree of complexity of ligand/protein interaction and these standard techniques are no longer sufficient to describe the binding event. We have experienced and tested these limits in the present study in which we have developed and revealed the mechanism of binding of a new series of potent inhibitors of Adenosine Deaminase. We have first performed a large number of docking calculations, which unfortunately failed to yield reliable results due to the dynamical character of the enzyme and the complex role of the solvent. Thus, we have stepped up the computational strategy using a protocol based on metadynamics. Our approach has allowed dealing with protein motion and solvation during ligand binding and finally identifying the lowest energy binding modes of the most potent compound of the series, 4-decyl-pyrazolo[1,5-a]pyrimidin-7-one. PMID:22238423

  7. Cartilage oligomeric matrix protein specific antibodies are pathogenic

    DEFF Research Database (Denmark)

    Geng, Hui; Nandakumar, Kutty Selva; Pramhed, Anna

    2012-01-01

    -specific monoclonal antibodies (mAbs). METHODS: B cell immunodominant regions on the COMP molecule were measured with a novel enzyme-linked immunosorbent assay using mammalian expressed full-length mouse COMP as well as a panel of recombinant mouse COMP fragments. 18 mAbs specific to COMP were generated......ABSTRACT: INTRODUCTION: Cartilage oligomeric matrix protein (COMP) is a major non-collagenous component of cartilage. Earlier, we developed a new mouse model for rheumatoid arthritis using COMP. This study was undertaken to investigate the epitope specificity and immunopathogenicity of COMP...

  8. Thermodynamics of protein folding: a random matrix formulation.

    Science.gov (United States)

    Shukla, Pragya

    2010-10-20

    The process of protein folding from an unfolded state to a biologically active, folded conformation is governed by many parameters, e.g. the sequence of amino acids, intermolecular interactions, the solvent, temperature and chaperon molecules. Our study, based on random matrix modeling of the interactions, shows, however, that the evolution of the statistical measures, e.g. Gibbs free energy, heat capacity, and entropy, is single parametric. The information can explain the selection of specific folding pathways from an infinite number of possible ways as well as other folding characteristics observed in computer simulation studies. © 2010 IOP Publishing Ltd

  9. Photoaffinity labelling of high affinity dopamine binding proteins

    International Nuclear Information System (INIS)

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

    1986-01-01

    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 ( 3 H)-SCH 23390 (a D 1 specific antagonist), was then performed to evaluate the loss of receptor density in the photolyzed preparation. AzB-AP-ADTN irreversibly blocked ( 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 ( 3 H)-SCH 23390 binding. Compounds which compete for D 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 1 (adenylate cyclase linked) dopamine receptor

  10. Guanylate kinase domains of the MAGUK family scaffold proteins as specific phospho-protein-binding modules

    OpenAIRE

    Zhu, Jinwei; Shang, Yuan; Xia, Caihao; Wang, Wenning; Wen, Wenyu; Zhang, Mingjie

    2011-01-01

    Membrane-associated guanylate kinases (MAGUK) family proteins contain an inactive guanylate kinase (GK) domain, whose function has been elusive. Here, this domain is revealed as a new type of phospho-peptide-binding module, in which the GMP-binding site has evolved to accommodate phospho-serines or -threonines.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-07-27

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

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

    Science.gov (United States)

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

    2017-09-15

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

  13. Deoxyribonucleic-binding homeobox proteins are augmented in human cancer

    DEFF Research Database (Denmark)

    Wewer, U M; Mercurio, A M; Chung, S Y

    1990-01-01

    Homeobox genes encode sequence-specific DNA-binding proteins that are involved in the regulation of gene expression during embryonic development. In this study, we examined the expression of homeobox proteins in human cancer. Antiserum was obtained against a synthetic peptide derived from...... was then isolated and used to elicit a rabbit antiserum. In immunostaining, both antisera reacted with the nuclei of cultured tumor cells. In tissue sections of human carcinoma, nuclear immunoreactivity was observed in the tumor cells in 40 of 42 cases examined. Adjacent normal epithelial tissue obtained from......, the presence of the homeobox transcript in human carcinoma was documented by in situ hybridization and RNase protection mapping. These results demonstrate that human cancer is associated with the expression of homeobox proteins. Such homeobox proteins, as well as other regulatory proteins, could be involved...

  14. Membrane and inclusion body targeting of lyssavirus matrix proteins.

    Science.gov (United States)

    Pollin, Reiko; Granzow, Harald; Köllner, Bernd; Conzelmann, Karl-Klaus; Finke, Stefan

    2013-02-01

    Lyssavirus matrix proteins (M) support virus budding and have accessory functions that may contribute to host cell manipulation and adaptation to specific hosts. Here, we show that rabies virus (RABV) and European Bat Lyssavirus Type 1 (EBLV-1) M proteins differ in targeting and accumulation at cellular membranes. In contrast to RABV M, EBLV-1 M expressed from authentic EBLV-1 or chimeric RABV accumulated at the Golgi apparatus. Chimeric M proteins revealed that Golgi association depends on the integrity of the entire EBLV-1 M protein. Since RABV and EBLV-1 M differ in the use of cellular membranes for particle formation, differential membrane targeting and transport of M might determine the site of virus production. Moreover, both RABV and EBLV-1 M were for the first time detected within the nucleus and in Negri body-like inclusions bodies. Whereas nuclear M may imply hitherto unknown functions of lyssavirus M in host cell manipulation, the presence of M in inclusion bodies may correlate with regulatory functions of M in virus RNA synthesis. The data strongly support a model in which targeting of lyssavirus M proteins to distinctintracellular sites is a key determinant of diverse features in lyssavirus replication, host adaptation and pathogenesis. © 2012 Blackwell Publishing Ltd.

  15. Milk proteins interact with goat Binder of SPerm (BSP) proteins and decrease their binding to sperm.

    Science.gov (United States)

    de Menezes, Erika Bezerra; van Tilburg, Mauricio; Plante, Geneviève; de Oliveira, Rodrigo V; Moura, Arlindo A; Manjunath, Puttaswamy

    2016-11-01

    Seminal plasma Binder of SPerm (BSP) proteins bind to sperm at ejaculation and promote capacitation. When in excess, however, BSP proteins damage the sperm membrane. It has been suggested that milk components of semen extenders associate with BSP proteins, potentially protecting sperm. Thus, this study was conducted to investigate if milk proteins interact with BSP proteins and reduce BSP binding to goat sperm. Using gel filtration chromatography, milk was incubated with goat seminal plasma proteins and loaded onto columns with and without calcium. Milk was also fractionated into parts containing mostly whey proteins or mostly caseins, incubated with seminal plasma proteins and subjected to gel filtration. Eluted fractions were evaluated by immunoblot using anti-goat BSP antibodies, confirming milk protein-BSP protein interactions. As determined by ELISA, milk proteins coated on polystyrene wells bound to increasing of goat BSP proteins. Far-western dot blots confirmed that BSP proteins bound to caseins and β-lactoglobulin in a concentration-dependent manner. Then, cauda epididymal sperm from five goats was incubated with seminal plasma; seminal plasma followed by milk; and milk followed by seminal plasma. Sperm membrane proteins were extracted and evaluated by immunoblotting. The pattern of BSP binding to sperm membrane proteins was reduced by 59.3 % when epididymal sperm were incubated with seminal plasma and then with skimmed milk (p  0.05). In conclusion, goat BSP proteins have an affinity for caseins and whey proteins. Milk reduces BSP binding to goat sperm, depending whether or not sperm had been previously exposed to seminal plasma. Such events may explain the protective effect of milk during goat sperm preservation.

  16. OmpL1 is an extracellular matrix- and plasminogen-interacting protein of Leptospira spp.

    Science.gov (United States)

    Fernandes, Luis G V; Vieira, Monica L; Kirchgatter, Karin; Alves, Ivy J; de Morais, Zenaide M; Vasconcellos, Silvio A; Romero, Eliete C; Nascimento, Ana L T O

    2012-10-01

    Leptospirosis is a zoonosis with multisystem involvement caused by pathogenic strains of the genus Leptospira. OmpL1 is an outer membrane protein of Leptospira spp. that is expressed during infection. In this work, we investigated novel features of this protein. We describe that OmpL1 is a novel leptospiral extracellular matrix (ECM)-binding protein and a plasminogen (PLG) receptor. The recombinant protein was expressed in Escherichia coli BL21(DE3) Star/pLysS as inclusion bodies, refolded, and purified by metal-chelating chromatography. The protein presented a typical β-strand secondary structure, as evaluated by circular dichroism spectroscopy. The recombinant protein reacted with antibodies in serum samples from convalescent leptospirosis patients with a high specificity compared to serum samples from individuals with unrelated diseases. These data strengthen the usefulness of OmpL1 as a diagnostic marker of leptospirosis. The characterization of the immunogenicity of recombinant OmpL1 in inoculated BALB/c mice showed that the protein has the capacity to elicit humoral and cellular immune responses, as denoted by high antibody titers and the proliferation of lymphocytes. We demonstrate that OmpL1 has the ability to mediate attachment to laminin and plasma fibronectin, with K(D) (equilibrium dissociation constant) values of 2,099.93 ± 871.03 nM and 1,239.23 ± 506.85 nM, respectively. OmpL1 is also a PLG receptor, with a K(D) of 368.63 ± 121.23 nM, capable of generating enzymatically active plasmin. This is the first report that shows and characterizes OmpL1 as an ECM-interacting and a PLG-binding protein of Leptospira spp. that may play a role in bacterial pathogenesis when expressed during infection.

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

    Directory of Open Access Journals (Sweden)

    Huiying Zhao

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

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

    Science.gov (United States)

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

    2013-08-20

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

  19. Phosphatidylserine Lateral Organization Influences the Interaction of Influenza Virus Matrix Protein 1 with Lipid Membranes.

    Science.gov (United States)

    Bobone, Sara; Hilsch, Malte; Storm, Julian; Dunsing, Valentin; Herrmann, Andreas; Chiantia, Salvatore

    2017-06-15

    Influenza A virus matrix protein 1 (M1) is an essential component involved in the structural stability of the virus and in the budding of new virions from infected cells. A deeper understanding of the molecular basis of virion formation and the budding process is required in order to devise new therapeutic approaches. We performed a detailed investigation of the interaction between M1 and phosphatidylserine (PS) (i.e., its main binding target at the plasma membrane [PM]), as well as the distribution of PS itself, both in model membranes and in living cells. To this end, we used a combination of techniques, including Förster resonance energy transfer (FRET), confocal microscopy imaging, raster image correlation spectroscopy, and number and brightness (N&B) analysis. Our results show that PS can cluster in segregated regions in the plane of the lipid bilayer, both in model bilayers constituted of PS and phosphatidylcholine and in living cells. The viral protein M1 interacts specifically with PS-enriched domains, and such interaction in turn affects its oligomerization process. Furthermore, M1 can stabilize PS domains, as observed in model membranes. For living cells, the presence of PS clusters is suggested by N&B experiments monitoring the clustering of the PS sensor lactadherin. Also, colocalization between M1 and a fluorescent PS probe suggest that, in infected cells, the matrix protein can specifically bind to the regions of PM in which PS is clustered. Taken together, our observations provide novel evidence regarding the role of PS-rich domains in tuning M1-lipid and M1-M1 interactions at the PM of infected cells. IMPORTANCE Influenza virus particles assemble at the plasma membranes (PM) of infected cells. This process is orchestrated by the matrix protein M1, which interacts with membrane lipids while binding to the other proteins and genetic material of the virus. Despite its importance, the initial step in virus assembly (i.e., M1-lipid interaction) is still

  20. Insulin-like growth factors and insulin-like growth factor binding proteins in mammary gland function

    International Nuclear Information System (INIS)

    Marshman, Emma; Streuli, Charles H

    2002-01-01

    Insulin-like growth factor (IGF)-mediated proliferation and survival are essential for normal development in the mammary gland during puberty and pregnancy. IGFs interact with IGF-binding proteins and regulate their function. The present review focuses on the role of IGFs and IGF-binding proteins in the mammary gland and describes how modulation of their actions occurs by association with hormones, other growth factors and the extracellular matrix. The review will also highlight the involvement of the IGF axis in breast cancer

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

    Science.gov (United States)

    Ma, Xin; Guo, Jing; Sun, Xiao

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Vesa Kirjavainen

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

  3. Characterization of the proteins comprising the integral matrix of Strongylocentrotus purpuratus embryonic spicules

    Science.gov (United States)

    Killian, C. E.; Wilt, F. H.

    1996-01-01

    In the present study, we enumerate and characterize the proteins that comprise the integral spicule matrix of the Strongylocentrotus purpuratus embryo. Two-dimensional gel electrophoresis of [35S]methionine radiolabeled spicule matrix proteins reveals that there are 12 strongly radiolabeled spicule matrix proteins and approximately three dozen less strongly radiolabeled spicule matrix proteins. The majority of the proteins have acidic isoelectric points; however, there are several spicule matrix proteins that have more alkaline isoelectric points. Western blotting analysis indicates that SM50 is the spicule matrix protein with the most alkaline isoelectric point. In addition, two distinct SM30 proteins are identified in embryonic spicules, and they have apparent molecular masses of approximately 43 and 46 kDa. Comparisons between embryonic spicule matrix proteins and adult spine integral matrix proteins suggest that the embryonic 43-kDa SM30 protein is an embryonic isoform of SM30. An adult 49-kDa spine matrix protein is also identified as a possible adult isoform of SM30. Analysis of the SM30 amino acid sequences indicates that a portion of SM30 proteins is very similar to the carbohydrate recognition domain of C-type lectin proteins.

  4. Platelet activation by extracellular matrix proteins in haemostasis and thrombosis.

    Science.gov (United States)

    Watson, Steve P

    2009-01-01

    The prevention of excessive blood loss to avoid fatal haemorrhage is a pivotal process for all organisms possessing a circulatory system. Increased circulating blood volume and pressure, as required in larger animals, make this process all the more important and challenging. It is essential to have a powerful and rapid system to detect damage and generate an effective seal, and which is also exquisitely regulated to prevent unwanted, excessive or systemic activation so as to avoid blockage of vessels. Thus, a highly specialised and efficient haemostatic system has evolved that consists of cellular (platelets) and protein (coagulation factors) components. Importantly, this is able to support haemostasis in both the low shear environment of the venous system and the high shear environment of the arterial system. Endothelial cells, lining the entire circulation system, play a crucial role in the delicate balance between activation and inhibition of the haemostatic system. An intact and healthy endothelium supports blood flow by preventing attachment of cells and proteins which is required for initiation of coagulation and platelet activation. Endothelial cells produce and release the two powerful soluble inhibitors of platelet activation, nitric oxide and prostacyclin, and express high levels of CD39 which rapidly metabolises the major platelet feedback agonist, ADP. This antithrombotic environment however can rapidly change following activation or removal of endothelial cells through injury or rupture of atherosclerotic plaques. Loss of endothelial cells exposes the subendothelial extracellular matrix which creates strong signals for activation of the haemostatic system including powerful platelet adhesion and activation. Quantitative and qualitative changes in the composition of the subendothelial extracellular matrix influence these prothrombotic characteristics with life threatening thrombotic and bleeding complications, as illustrated by formation of

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

    Science.gov (United States)

    Chen, Eileen; Joseph, Simpson

    2015-07-01

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

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

    Directory of Open Access Journals (Sweden)

    Daniel M Dupont

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

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

    Science.gov (United States)

    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. © 2014 John Wiley & Sons A/S.

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

    International Nuclear Information System (INIS)

    Kalinina, S.N.

    1986-01-01

    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 α- and β-subunits of GTP-binding proteins from bovine retinal photoreceptor membranes

  9. NMR Structure of the Myristylated Feline Immunodeficiency Virus Matrix Protein

    Directory of Open Access Journals (Sweden)

    Lola A. Brown

    2015-04-01

    Full Text Available Membrane targeting by the Gag proteins of the human immunodeficiency viruses (HIV types-1 and -2 is mediated by Gag’s N-terminally myristylated matrix (MA domain and is dependent on cellular phosphatidylinositol-4,5-bisphosphate [PI(4,5P2]. To determine if other lentiviruses employ a similar membrane targeting mechanism, we initiated studies of the feline immunodeficiency virus (FIV, a widespread feline pathogen with potential utility for development of human therapeutics. Bacterial co-translational myristylation was facilitated by mutation of two amino acids near the amino-terminus of the protein (Q5A/G6S; myrMAQ5A/G6S. These substitutions did not affect virus assembly or release from transfected cells. NMR studies revealed that the myristyl group is buried within a hydrophobic pocket in a manner that is structurally similar to that observed for the myristylated HIV-1 protein. Comparisons with a recent crystal structure of the unmyristylated FIV protein [myr(-MA] indicate that only small changes in helix orientation are required to accommodate the sequestered myr group. Depletion of PI(4,5P2 from the plasma membrane of FIV-infected CRFK cells inhibited production of FIV particles, indicating that, like HIV, FIV hijacks the PI(4,5P2 cellular signaling system to direct intracellular Gag trafficking during virus assembly.

  10. NMR structure of the myristylated feline immunodeficiency virus matrix protein.

    Science.gov (United States)

    Brown, Lola A; Cox, Cassiah; Baptiste, Janae; Summers, Holly; Button, Ryan; Bahlow, Kennedy; Spurrier, Vaughn; Kyser, Jenna; Luttge, Benjamin G; Kuo, Lillian; Freed, Eric O; Summers, Michael F

    2015-04-30

    Membrane targeting by the Gag proteins of the human immunodeficiency viruses (HIV types-1 and -2) is mediated by Gag's N-terminally myristylated matrix (MA) domain and is dependent on cellular phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. To determine if other lentiviruses employ a similar membrane targeting mechanism, we initiated studies of the feline immunodeficiency virus (FIV), a widespread feline pathogen with potential utility for development of human therapeutics. Bacterial co-translational myristylation was facilitated by mutation of two amino acids near the amino-terminus of the protein (Q5A/G6S; myrMAQ5A/G6S). These substitutions did not affect virus assembly or release from transfected cells. NMR studies revealed that the myristyl group is buried within a hydrophobic pocket in a manner that is structurally similar to that observed for the myristylated HIV-1 protein. Comparisons with a recent crystal structure of the unmyristylated FIV protein [myr(-)MA] indicate that only small changes in helix orientation are required to accommodate the sequestered myr group. Depletion of PI(4,5)P2 from the plasma membrane of FIV-infected CRFK cells inhibited production of FIV particles, indicating that, like HIV, FIV hijacks the PI(4,5)P2 cellular signaling system to direct intracellular Gag trafficking during virus assembly.

  11. Processing of the glycosomal matrix-protein import receptor PEX5 of Trypanosoma brucei

    International Nuclear Information System (INIS)

    Gualdrón-López, Melisa; Michels, Paul A.M.

    2013-01-01

    Highlights: ► Most eukaryotic cells have a single gene for the peroxin PEX5. ► PEX5 is sensitive to in vitro proteolysis in distantly related organisms. ► TbPEX5 undergoes N-terminal truncation in vitro and possibly in vivo. ► Truncated TbPEX5 is still capable of binding PTS1-containing proteins. ► PEX5 truncation is physiologically relevant or an evolutionary conserved artifact. -- Abstract: Glycolysis in kinetoplastid protists such as Trypanosoma brucei is compartmentalized in peroxisome-like organelles called glycosomes. Glycosomal matrix-protein import involves a cytosolic receptor, PEX5, which recognizes the peroxisomal-targeting signal type 1 (PTS1) present at the C-terminus of the majority of matrix proteins. PEX5 appears generally susceptible to in vitro proteolytic processing. On western blots of T. brucei, two PEX5 forms are detected with apparent M r of 100 kDa and 72 kDa. 5′-RACE-PCR showed that TbPEX5 is encoded by a unique transcript that can be translated into a protein of maximally 72 kDa. However, recombinant PEX5 migrates aberrantly in SDS–PAGE with an apparent M r of 100 kDa, similarly as observed for the native peroxin. In vitro protease susceptibility analysis of native and 35 S-labelled PEX5 showed truncation of the 100 kDa form at the N-terminal side by unknown parasite proteases, giving rise to the 72 kDa form which remains functional for PTS1 binding. The relevance of these observations is discussed

  12. Processing of the glycosomal matrix-protein import receptor PEX5 of Trypanosoma brucei

    Energy Technology Data Exchange (ETDEWEB)

    Gualdrón-López, Melisa [Research Unit for Tropical Diseases, de Duve Institute, Université catholique de Louvain, Brussels (Belgium); Michels, Paul A.M., E-mail: paul.michels@uclouvain.be [Research Unit for Tropical Diseases, de Duve Institute, Université catholique de Louvain, Brussels (Belgium)

    2013-02-01

    Highlights: ► Most eukaryotic cells have a single gene for the peroxin PEX5. ► PEX5 is sensitive to in vitro proteolysis in distantly related organisms. ► TbPEX5 undergoes N-terminal truncation in vitro and possibly in vivo. ► Truncated TbPEX5 is still capable of binding PTS1-containing proteins. ► PEX5 truncation is physiologically relevant or an evolutionary conserved artifact. -- Abstract: Glycolysis in kinetoplastid protists such as Trypanosoma brucei is compartmentalized in peroxisome-like organelles called glycosomes. Glycosomal matrix-protein import involves a cytosolic receptor, PEX5, which recognizes the peroxisomal-targeting signal type 1 (PTS1) present at the C-terminus of the majority of matrix proteins. PEX5 appears generally susceptible to in vitro proteolytic processing. On western blots of T. brucei, two PEX5 forms are detected with apparent M{sub r} of 100 kDa and 72 kDa. 5′-RACE-PCR showed that TbPEX5 is encoded by a unique transcript that can be translated into a protein of maximally 72 kDa. However, recombinant PEX5 migrates aberrantly in SDS–PAGE with an apparent M{sub r} of 100 kDa, similarly as observed for the native peroxin. In vitro protease susceptibility analysis of native and {sup 35}S-labelled PEX5 showed truncation of the 100 kDa form at the N-terminal side by unknown parasite proteases, giving rise to the 72 kDa form which remains functional for PTS1 binding. The relevance of these observations is discussed.

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

    Science.gov (United States)

    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.

  14. Mannan-binding proteins from boar seminal plasma

    Czech Academy of Sciences Publication Activity Database

    Jelínková-Slavíčková, Petra; Liberda, J.; Maňásková, Pavla; Ryšlavá, H.; Jonáková, Věra; Tichá, M.

    2004-01-01

    Roč. 62, 1-2 (2004), s. 167-182 ISSN 0165-0378. [Congress of the European Society for Reproductive & Developmental Immunology /4./. Rhodes, 04.06.2003-06.06.2003] R&D Projects: GA ČR GA303/02/0433; GA ČR GP303/02/P069; GA MŠk VS96141; GA MZd NJ7463 Institutional research plan: CEZ:AV0Z5052915 Keywords : boar seminal plasma proteins * mannan-binding proteins * oviductal epithelium Subject RIV: CE - Biochemistry Impact factor: 2.726, year: 2004

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

    Science.gov (United States)

    Hicks, G. R.; Rice, M. S.; Lomax, T. L.

    1993-01-01

    We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948-4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or multimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may

  16. A chimeric protein composed of NuMA fused to the DNA binding domain of LANA is sufficient for the ori-P-dependent DNA replication

    International Nuclear Information System (INIS)

    Ohsaki, Eriko; Ueda, Keiji

    2017-01-01

    The Kaposi's sarcoma-associated herpesvirus (KSHV) genome is stably maintained in KSHV-infected PEL cell lines during cell division. We previously showed that accumulation of LANA in the nuclear matrix fraction could be important for the latent DNA replication, and that the functional significance of LANA should be its recruitment of ori-P to the nuclear matrix. Here, we investigated whether the forced localization of the LANA-DNA binding domain (DBD) to the nuclear matrix facilitated ori-P-containing plasmid replication. We demonstrated that chimeric proteins constructed by fusion of LANA DBD with the nuclear mitotic apparatus protein (NuMA), which is one of the components of the nuclear matrix, could bind with ori-P and enhance replication of an ori-P-containing plasmid, compared with that in the presence of DBD alone. These results further suggested that the ori-P recruitment to the nuclear matrix through the binding with DBD is important for latent viral DNA replication. - Highlights: •KSHV replication in latency depends on LANA localization to the nuclear matrix. •LANA DBD was fused with NuMA, a nuclear matrix protein, at the N- and C-terminus. •NuMA-DBD was in the nuclear matrix and supported the ori-P dependent replication. •LANA in the nuclear matrix should be important for the KSHV replication in latency.

  17. A chimeric protein composed of NuMA fused to the DNA binding domain of LANA is sufficient for the ori-P-dependent DNA replication

    Energy Technology Data Exchange (ETDEWEB)

    Ohsaki, Eriko; Ueda, Keiji, E-mail: kueda@virus.med.osaka-u.ac.jp

    2017-01-15

    The Kaposi's sarcoma-associated herpesvirus (KSHV) genome is stably maintained in KSHV-infected PEL cell lines during cell division. We previously showed that accumulation of LANA in the nuclear matrix fraction could be important for the latent DNA replication, and that the functional significance of LANA should be its recruitment of ori-P to the nuclear matrix. Here, we investigated whether the forced localization of the LANA-DNA binding domain (DBD) to the nuclear matrix facilitated ori-P-containing plasmid replication. We demonstrated that chimeric proteins constructed by fusion of LANA DBD with the nuclear mitotic apparatus protein (NuMA), which is one of the components of the nuclear matrix, could bind with ori-P and enhance replication of an ori-P-containing plasmid, compared with that in the presence of DBD alone. These results further suggested that the ori-P recruitment to the nuclear matrix through the binding with DBD is important for latent viral DNA replication. - Highlights: •KSHV replication in latency depends on LANA localization to the nuclear matrix. •LANA DBD was fused with NuMA, a nuclear matrix protein, at the N- and C-terminus. •NuMA-DBD was in the nuclear matrix and supported the ori-P dependent replication. •LANA in the nuclear matrix should be important for the KSHV replication in latency.

  18. Glycation of extracellular matrix proteins and its role in atherosclerosis 

    Directory of Open Access Journals (Sweden)

    Aleksandra Kuzan

    2012-10-01

    Full Text Available Glycation consists in formation of advanced glycation end-products (AGE during non-enzymatic reaction between reducing sugars and proteins, lipids or nucleic acids. This review is focused mainly on glycation of collagen and its role in acceleration of vascular disease. Collagen is an extracellular matrix protein characterized by unique structure forming fibrils with great anti-tensile and anti-breaking strength. The protein builds the connective tissue and is responsible for biomechanical properties of blood vessels. It is reported that higher content of glycated collagen correlates with lower elasticity and greater toughness of the vessel walls and, as a consequence, a faster rate of atherosclerosis development. Numerous mechanisms connected with AGE formation are involved in atherogenesis, among others: receptor-mediated production of free radicals, triggering an inflammatory process, activation of leukocytes and thrombocytes, facilitation of LDL binding, change in level of growth factors, adhesion molecules, MMP and some other proteins’ expression. The coverages allow the development of therapeutic strategies to prevent or slow down the pathological processes connected with glycation of collagen and other proteins in the artery wall. The main strategies are based on limitation of exogenous AGE, consumption of products which contain rutin, treatment with drugs which inhibit AGE formation, such aspyridoxamine, and chemicals which are able to cleave already formed AGE protein-protein crosslinks, such as ALT-711.

  19. Planar cell polarity proteins differentially regulate extracellular matrix organization and assembly during zebrafish gastrulation.

    Science.gov (United States)

    Dohn, Michael R; Mundell, Nathan A; Sawyer, Leah M; Dunlap, Julie A; Jessen, Jason R

    2013-11-01

    Zebrafish gastrulation cell movements occur in the context of dynamic changes in extracellular matrix (ECM) organization and require the concerted action of planar cell polarity (PCP) proteins that regulate cell elongation and mediolateral alignment. Data obtained using Xenopus laevis gastrulae have shown that integrin-fibronectin interactions underlie the formation of polarized cell protrusions necessary for PCP and have implicated PCP proteins themselves as regulators of ECM. By contrast, the relationship between establishment of PCP and ECM assembly/remodeling during zebrafish gastrulation is unclear. We previously showed that zebrafish embryos carrying a null mutation in the four-pass transmembrane PCP protein vang-like 2 (vangl2) exhibit increased matrix metalloproteinase activity and decreased immunolabeling of fibronectin. These data implicated for the first time a core PCP protein in the regulation of pericellular proteolysis of ECM substrates and raised the question of whether other zebrafish PCP proteins also impact ECM organization. In Drosophila melanogaster, the cytoplasmic PCP protein Prickle binds Van Gogh and regulates its function. Here we report that similar to vangl2, loss of zebrafish prickle1a decreases fibronectin protein levels in gastrula embryos. We further show that Prickle1a physically binds Vangl2 and regulates both the subcellular distribution and total protein level of Vangl2. These data suggest that the ability of Prickle1a to impact fibronectin organization is at least partly due to effects on Vangl2. In contrast to loss of either Vangl2 or Prickle1a function, we find that glypican4 (a Wnt co-receptor) and frizzled7 mutant gastrula embryos with disrupted non-canonical Wnt signaling exhibit the opposite phenotype, namely increased fibronectin assembly. Our data show that glypican4 mutants do not have decreased proteolysis of ECM substrates, but instead have increased cell surface cadherin protein expression and increased intercellular

  20. Functional interaction between Smad, CREB binding protein, and p68 RNA helicase

    International Nuclear Information System (INIS)

    Warner, Dennis R.; Bhattacherjee, Vasker; Yin, Xiaolong; Singh, Saurabh; Mukhopadhyay, Partha; Pisano, M. Michele; Greene, Robert M.

    2004-01-01

    The transforming growth factors β control a diversity of biological processes including cellular proliferation, differentiation, apoptosis, and extracellular matrix production, and are critical effectors of embryonic patterning and development, including that of the orofacial region. TGFβ superfamily members signal through specific cell surface receptors that phosphorylate the cytoplasmic Smad proteins, resulting in their translocation to the nucleus and interaction with promoters of TGFβ-responsive genes. Subsequent alterations in transcription are cell type-specific and dependent on recruitment to the Smad/transcription factor complex of coactivators, such as CBP and p300, or corepressors, such as c-ski and SnoN. Since the affinity of Smads for DNA is generally low, additional accessory proteins that facilitate Smad/DNA binding are required, and are often cell- and tissue-specific. In order to identify novel Smad 3 binding proteins in developing orofacial tissue, a yeast two hybrid assay was employed in which the MH2 domain of Smad 3 was used to screen an expression library derived from mouse embryonic orofacial tissue. The RNA helicase, p68, was identified as a unique Smad binding protein, and the specificity of the interaction was confirmed through various in vitro and in vivo assays. Co-expression of Smad 3 and a CBP-Gal4 DNA binding domain fusion protein in a Gal4-luciferase reporter assay resulted in increased TGFβ-stimulated reporter gene transcription. Moreover, co-expression of p68 RNA helicase along with Smad 3 and CBP-Gal4 resulted in synergistic activation of Gal4-luciferase reporter expression. Collectively, these data indicate that the RNA helicase, p68, can directly interact with Smad 3 resulting in formation of a transcriptionally active ternary complex containing Smad 3, p68, and CBP. This offers a means of enhancing TGFβ-mediated cellular responses in developing orofacial tissue

  1. Matrix Gla Protein is Involved in Crystal Formation in Kidney of Hyperoxaluric Rats

    Directory of Open Access Journals (Sweden)

    Xiuli Lu

    2013-02-01

    Full Text Available Background: Matrix Gla protein (MGP is a molecular determinant regulating vascular calcification of the extracellular matrix. However, it is still unclear how MGP may be invovled in crystal formation in the kidney of hyperoxaluric rats. Methods: Male Sprague-Dawley rats were divided into the hyperoxaluric group and control group. Hyperoxaluric rats were administrated by 0.75% ethylene glycol (EG for up to 8 weeks. Renal MGP expression was detected by the standard avidin-biotin complex (ABC method. Renal crystal deposition was observed by a polarizing microscope. Total RNA and protein from the rat kidney tissue were extracted. The levels of MGP mRNA and protein expression were analyzed by the real-time polymerase chain reaction (RT-PCR and Western blot. Results: Hyperoxaluria was induced successfully in rats. The MGP was polarly distributed, on the apical membrane of renal tubular epithelial cells, and was found in the ascending thick limbs of Henle's loop (cTAL and the distal convoluted tubule (DCT in hyperoxaluric rats, its expression however, was present in the medullary collecting duct (MCD in stone-forming rats. Crystals with multilaminated structure formed in the injurious renal tubules with lack of MGP expression.MGP mRNA expression was significantly upregulated by the crystals' stimulations. Conclusion: Our results suggested that the MGP was involved in crystals formation by the continuous expression, distributing it polarly in the renal tubular cells and binding directly to the crystals.

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

    DEFF Research Database (Denmark)

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

    2006-01-01

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

  3. PRODIGY : a web server for predicting the binding affinity of protein-protein complexes

    NARCIS (Netherlands)

    Xue, Li; Garcia Lopes Maia Rodrigues, João; Kastritis, Panagiotis L; Bonvin, Alexandre Mjj; Vangone, Anna

    2016-01-01

    Gaining insights into the structural determinants of protein-protein interactions holds the key for a deeper understanding of biological functions, diseases and development of therapeutics. An important aspect of this is the ability to accurately predict the binding strength for a given

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

    African Journals Online (AJOL)

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

  5. Surface displaced alfa-enolase of Lactobacillus plantarum is a fibronectin binding protein

    Directory of Open Access Journals (Sweden)

    Muscariello Lidia

    2009-02-01

    Full Text Available Abstract Background Lactic acid bacteria of the genus Lactobacillus and Bifidobacterium are one of the most important health promoting groups of the human intestinal microbiota. Their protective role within the gut consists in out competing invading pathogens for ecological niches and metabolic substrates. Among the features necessary to provide health benefits, commensal microorganisms must have the ability to adhere to human intestinal cells and consequently to colonize the gut. Studies on mechanisms mediating adhesion of lactobacilli to human intestinal cells showed that factors involved in the interaction vary mostly among different species and strains, mainly regarding interaction between bacterial adhesins and extracellular matrix or mucus proteins. We have investigated the adhesive properties of Lactobacillus plantarum, a member of the human microbiota of healthy individuals. Results We show the identification of a Lactobacillus plantarum LM3 cell surface protein (48 kDa, which specifically binds to human fibronectin (Fn, an extracellular matrix protein. By means of mass spectrometric analysis this protein was identified as the product of the L. plantarum enoA1 gene, coding the EnoA1 alfa-enolase. Surface localization of EnoA1 was proved by immune electron microscopy. In the mutant strain LM3-CC1, carrying the enoA1 null mutation, the 48 kDa adhesin was not anymore detectable neither by anti-enolase Western blot nor by Fn-overlay immunoblotting assay. Moreover, by an adhesion assay we show that LM3-CC1 cells bind to fibronectin-coated surfaces less efficiently than wild type cells, thus demonstrating the significance of the surface displaced EnoA1 protein for the L. plantarum LM3 adhesion to fibronectin. Conclusion Adhesion to host tissues represents a crucial early step in the colonization process of either pathogens or commensal bacteria. We demonstrated the involvement of the L. plantarum Eno A1 alfa-enolase in Fn-binding, by studying

  6. Biophysical characterization and crystal structure of the Feline Immunodeficiency Virus p15 matrix protein.

    Science.gov (United States)

    Serrière, Jennifer; Robert, Xavier; Perez, Magali; Gouet, Patrice; Guillon, Christophe

    2013-06-24

    Feline Immunodeficiency Virus (FIV) is a viral pathogen that infects domestic cats and wild felids. During the viral replication cycle, the FIV p15 matrix protein oligomerizes to form a closed matrix that underlies the lipidic envelope of the virion. Because of its crucial role in the early and late stages of viral morphogenesis, especially in viral assembly, FIV p15 is an interesting target in the development of potential new therapeutic strategies. Our biochemical study of FIV p15 revealed that it forms a stable dimer in solution under acidic conditions and at high concentration, unlike other retroviral matrix proteins. We determined the crystal structure of full-length FIV p15 to 2 Å resolution and observed a helical organization of the protein, typical for retroviral matrix proteins. A hydrophobic pocket that could accommodate a myristoyl group was identified, and the C-terminal end of FIV p15, which is mainly unstructured, was visible in electron density maps. As FIV p15 crystallizes in acidic conditions but with one monomer in the asymmetric unit, we searched for the presence of a biological dimer in the crystal. No biological assembly was detected by the PISA server, but the three most buried crystallographic interfaces have interesting features: the first one displays a highly conserved tryptophan acting as a binding platform, the second one is located along a 2-fold symmetry axis and the third one resembles the dimeric interface of EIAV p15. Because the C-terminal end of p15 is involved in two of these three interfaces, we investigated the structure and assembly of a C-terminal-truncated form of p15 lacking 14 residues. The truncated FIV p15 dimerizes in solution at a lower concentration and crystallizes with two molecules in the asymmetric unit. The EIAV-like dimeric interface is the only one to be retained in the new crystal form. The dimeric form of FIV p15 in solution and its extended C-terminal end are characteristic among lentiviral matrix proteins

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

    OpenAIRE

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

    1987-01-01

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

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

    Science.gov (United States)

    Chitin-binding proteins (CBPs) existed in various species and involved in different biology processes. In the present study, we cloned a full length cDNA of chitin-binding protein-like (PpCBP-like) from Pteromalus puparum, a pupal endoparasitoid of Pieris rapae. PpCBP-like encoded a 96 putative amin...

  9. Inhibition of Cyclic Adenosine Monophosphate (cAMP-response Element-binding Protein (CREB-binding Protein (CBP/β-Catenin Reduces Liver Fibrosis in Mice

    Directory of Open Access Journals (Sweden)

    Yosuke Osawa

    2015-11-01

    Full Text Available Wnt/β-catenin is involved in every aspect of embryonic development and in the pathogenesis of many human diseases, and is also implicated in organ fibrosis. However, the role of β-catenin-mediated signaling on liver fibrosis remains unclear. To explore this issue, the effects of PRI-724, a selective inhibitor of the cAMP-response element-binding protein-binding protein (CBP/β-catenin interaction, on liver fibrosis were examined using carbon tetrachloride (CCl4- or bile duct ligation (BDL-induced mouse liver fibrosis models. Following repetitive CCl4 administrations, the nuclear translocation of β-catenin was observed only in the non-parenchymal cells in the liver. PRI-724 treatment reduced the fibrosis induced by CCl4 or BDL. C-82, an active form of PRI-724, inhibited the activation of isolated primary mouse quiescent hepatic stellate cells (HSCs and promoted cell death in culture-activated HSCs. During the fibrosis resolution period, an increase in F4/80+ CD11b+ and Ly6Clow CD11b+ macrophages was induced by CCl4 and was sustained for two weeks thereafter, even after having stopped CCl4 treatment. PRI-724 accelerated the resolution of CCl4-induced liver fibrosis, and this was accompanied by increased matrix metalloproteinase (MMP-9, MMP-2, and MMP-8 expression in intrahepatic leukocytes. In conclusion, targeting the CBP/β-catenin interaction may become a new therapeutic strategy in treating liver fibrosis.

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

    Science.gov (United States)

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

    2017-03-24

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

  11. The BRCT domain is a phospho-protein binding domain.

    Science.gov (United States)

    Yu, Xiaochun; Chini, Claudia Christiano Silva; He, Miao; Mer, Georges; Chen, Junjie

    2003-10-24

    The carboxyl-terminal domain (BRCT) of the Breast Cancer Gene 1 (BRCA1) protein is an evolutionarily conserved module that exists in a large number of proteins from prokaryotes to eukaryotes. Although most BRCT domain-containing proteins participate in DNA-damage checkpoint or DNA-repair pathways, or both, the function of the BRCT domain is not fully understood. We show that the BRCA1 BRCT domain directly interacts with phosphorylated BRCA1-Associated Carboxyl-terminal Helicase (BACH1). This specific interaction between BRCA1 and phosphorylated BACH1 is cell cycle regulated and is required for DNA damage-induced checkpoint control during the transition from G2 to M phase of the cell cycle. Further, we show that two other BRCT domains interact with their respective physiological partners in a phosphorylation-dependent manner. Thirteen additional BRCT domains also preferentially bind phospho-peptides rather than nonphosphorylated control peptides. These data imply that the BRCT domain is a phospho-protein binding domain involved in cell cycle control.

  12. Porcine bladder acellular matrix (ACM): protein expression, mechanical properties

    Energy Technology Data Exchange (ETDEWEB)

    Farhat, Walid A [Department of Surgery, Division of Urology, University of Toronto and Hospital for Sick Children, Toronto, ON M5G 1X8 (Canada); Chen Jun; Haig, Jennifer; Antoon, Roula; Litman, Jessica; Yeger, Herman [Department of Developmental and Stem Cell Biology, Research Institute, Hospital for Sick Children, Toronto, ON M5G 1X8 (Canada); Sherman, Christopher [Department of Anatomic Pathology, Sunnybrook and Women' s College Health Sciences Centre, Toronto, ON (Canada); Derwin, Kathleen [Department of Biomedical Engineering, Lerner Research Institute and Orthopaedic Research Center, Cleveland Clinic Foundation, Cleveland, OH (United States)], E-mail: walid.farhat@sickkids.ca

    2008-06-01

    Experimentally, porcine bladder acellular matrix (ACM) that mimics extracellular matrix has excellent potential as a bladder substitute. Herein we investigated the spatial localization and expression of different key cellular and extracellular proteins in the ACM; furthermore, we evaluated the inherent mechanical properties of the resultant ACM prior to implantation. Using a proprietary decellularization method, the DNA contents in both ACM and normal bladder were measured; in addition we used immunohistochemistry and western blots to quantify and localize the different cellular and extracellular components, and finally the mechanical testing was performed using a uniaxial mechanical testing machine. The mean DNA content in the ACM was significantly lower in the ACM compared to the bladder. Furthermore, the immunohistochemical and western blot analyses showed that collagen I and IV were preserved in the ACM, but possibly denatured collagen III in the ACM. Furthermore, elastin, laminin and fibronectin were mildly reduced in the ACM. Although the ACM did not exhibit nucleated cells, residual cellular components (actin, myosin, vimentin and others) were still present. There was, on the other hand, no significant difference in the mean stiffness between the ACM and the bladder. Although our decellularization method is effective in removing nuclear material from the bladder while maintaining its inherent mechanical properties, further work is mandatory to determine whether these residual DNA and cellular remnants would lead to any immune reaction, or if the mechanical properties of the ACM are preserved upon implantation and cellularization.

  13. Porcine bladder acellular matrix (ACM): protein expression, mechanical properties

    International Nuclear Information System (INIS)

    Farhat, Walid A; Chen Jun; Haig, Jennifer; Antoon, Roula; Litman, Jessica; Yeger, Herman; Sherman, Christopher; Derwin, Kathleen

    2008-01-01

    Experimentally, porcine bladder acellular matrix (ACM) that mimics extracellular matrix has excellent potential as a bladder substitute. Herein we investigated the spatial localization and expression of different key cellular and extracellular proteins in the ACM; furthermore, we evaluated the inherent mechanical properties of the resultant ACM prior to implantation. Using a proprietary decellularization method, the DNA contents in both ACM and normal bladder were measured; in addition we used immunohistochemistry and western blots to quantify and localize the different cellular and extracellular components, and finally the mechanical testing was performed using a uniaxial mechanical testing machine. The mean DNA content in the ACM was significantly lower in the ACM compared to the bladder. Furthermore, the immunohistochemical and western blot analyses showed that collagen I and IV were preserved in the ACM, but possibly denatured collagen III in the ACM. Furthermore, elastin, laminin and fibronectin were mildly reduced in the ACM. Although the ACM did not exhibit nucleated cells, residual cellular components (actin, myosin, vimentin and others) were still present. There was, on the other hand, no significant difference in the mean stiffness between the ACM and the bladder. Although our decellularization method is effective in removing nuclear material from the bladder while maintaining its inherent mechanical properties, further work is mandatory to determine whether these residual DNA and cellular remnants would lead to any immune reaction, or if the mechanical properties of the ACM are preserved upon implantation and cellularization

  14. Porcine bladder acellular matrix (ACM): protein expression, mechanical properties.

    Science.gov (United States)

    Farhat, Walid A; Chen, Jun; Haig, Jennifer; Antoon, Roula; Litman, Jessica; Sherman, Christopher; Derwin, Kathleen; Yeger, Herman

    2008-06-01

    Experimentally, porcine bladder acellular matrix (ACM) that mimics extracellular matrix has excellent potential as a bladder substitute. Herein we investigated the spatial localization and expression of different key cellular and extracellular proteins in the ACM; furthermore, we evaluated the inherent mechanical properties of the resultant ACM prior to implantation. Using a proprietary decellularization method, the DNA contents in both ACM and normal bladder were measured; in addition we used immunohistochemistry and western blots to quantify and localize the different cellular and extracellular components, and finally the mechanical testing was performed using a uniaxial mechanical testing machine. The mean DNA content in the ACM was significantly lower in the ACM compared to the bladder. Furthermore, the immunohistochemical and western blot analyses showed that collagen I and IV were preserved in the ACM, but possibly denatured collagen III in the ACM. Furthermore, elastin, laminin and fibronectin were mildly reduced in the ACM. Although the ACM did not exhibit nucleated cells, residual cellular components (actin, myosin, vimentin and others) were still present. There was, on the other hand, no significant difference in the mean stiffness between the ACM and the bladder. Although our decellularization method is effective in removing nuclear material from the bladder while maintaining its inherent mechanical properties, further work is mandatory to determine whether these residual DNA and cellular remnants would lead to any immune reaction, or if the mechanical properties of the ACM are preserved upon implantation and cellularization.

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

    Lifescience Database Archive (English)

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

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

    Science.gov (United States)

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

    2012-08-27

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

  17. The clinical significance of fatty acid binding proteins

    Directory of Open Access Journals (Sweden)

    Barbara Choromańska

    2011-11-01

    Full Text Available Excessive levels of free fatty acids are toxic to cells. The human body has evolved a defense mechanism in the form of small cytoplasmic proteins called fatty acid binding proteins (FABPs that bind long-chain fatty acids (LCFA, and then refer them to appropriate intracellular disposal sites (oxidation in mitochondria and peroxisomes or storage in the endoplasmic reticulum. So far, nine types of these proteins have been described, and their name refers to the place in which they were first identified or where they can be found in the greatest concentration. The most important FABPs were isolated from the liver (L-FABP, heart (H-FABP, intestine (I-FABP, brain (B-FABP, epidermis (E-FABP and adipocytes (A-FABP. Determination of H-FABP is used in the diagnosis of myocardial infarction, and L-FABP in kidney lesions of different etiologies. It is postulated that FABPs play an important role in the pathogenesis of metabolic diseases. Elevated levels of A-FABP have been found in the pericardial fat tissue and were associated with cardiac dysfunction in obese people. A rise in A-FABP has been observed in patients with type II diabetes. I-FABP is known as a marker of cell damage in the small intestine. Increased concentration of B-FABP has been associated with human brain tumors such as glioblastoma and astrocytoma, as well as with neurodegenerative diseases (Alzheimer’s, Parkinson’s and other disorders of cognitive function. The aim of this work was to present current data on the clinical significance of fatty acid binding proteins.

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

    Science.gov (United States)

    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. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  19. The Collagen Binding Proteins of Streptococcus mutans and Related Streptococci

    Science.gov (United States)

    Avilés-Reyes, Alejandro; Miller, James H.; Lemos, José A.; Abranches, Jacqueline

    2016-01-01

    Summary 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 utilized by S. mutans and related streptococci to colonize collagenous tissues, and the possible contribution of CBPs to infections in different sites of the host. PMID:26991416

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

    Science.gov (United States)

    Hung, Che-Lun; Hua, Guan-Jie

    2013-01-01

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

  1. Structure of Drosophila Oskar reveals a novel RNA binding protein

    Science.gov (United States)

    Yang, Na; Yu, Zhenyu; Hu, Menglong; Wang, Mingzhu; Lehmann, Ruth; Xu, Rui-Ming

    2015-01-01

    Oskar (Osk) protein plays critical roles during Drosophila germ cell development, yet its functions in germ-line formation and body patterning remain poorly understood. This situation contrasts sharply with the vast knowledge about the function and mechanism of osk mRNA localization. Osk is predicted to have an N-terminal LOTUS domain (Osk-N), which has been suggested to bind RNA, and a C-terminal hydrolase-like domain (Osk-C) of unknown function. Here, we report the crystal structures of Osk-N and Osk-C. Osk-N shows a homodimer of winged-helix–fold modules, but without detectable RNA-binding activity. Osk-C has a lipase-fold structure but lacks critical catalytic residues at the putative active site. Surprisingly, we found that Osk-C binds the 3′UTRs of osk and nanos mRNA in vitro. Mutational studies identified a region of Osk-C important for mRNA binding. These results suggest possible functions of Osk in the regulation of stability, regulation of translation, and localization of relevant mRNAs through direct interaction with their 3′UTRs, and provide structural insights into a novel protein–RNA interaction motif involving a hydrolase-related domain. PMID:26324911

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

    Science.gov (United States)

    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.

  3. Integrating protein structures and precomputed genealogies in the Magnum database: Examples with cellular retinoid binding proteins

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    Bradley Michael E

    2006-02-01

    Full Text Available Abstract Background When accurate models for the divergent evolution of protein sequences are integrated with complementary biological information, such as folded protein structures, analyses of the combined data often lead to new hypotheses about molecular physiology. This represents an excellent example of how bioinformatics can be used to guide experimental research. However, progress in this direction has been slowed by the lack of a publicly available resource suitable for general use. Results The precomputed Magnum database offers a solution to this problem for ca. 1,800 full-length protein families with at least one crystal structure. The Magnum deliverables include 1 multiple sequence alignments, 2 mapping of alignment sites to crystal structure sites, 3 phylogenetic trees, 4 inferred ancestral sequences at internal tree nodes, and 5 amino acid replacements along tree branches. Comprehensive evaluations revealed that the automated procedures used to construct Magnum produced accurate models of how proteins divergently evolve, or genealogies, and correctly integrated these with the structural data. To demonstrate Magnum's capabilities, we asked for amino acid replacements requiring three nucleotide substitutions, located at internal protein structure sites, and occurring on short phylogenetic tree branches. In the cellular retinoid binding protein family a site that potentially modulates ligand binding affinity was discovered. Recruitment of cellular retinol binding protein to function as a lens crystallin in the diurnal gecko afforded another opportunity to showcase the predictive value of a browsable database containing branch replacement patterns integrated with protein structures. Conclusion We integrated two areas of protein science, evolution and structure, on a large scale and created a precomputed database, known as Magnum, which is the first freely available resource of its kind. Magnum provides evolutionary and structural

  4. A unique bivalent binding and inhibition mechanism by the yatapoxvirus interleukin 18 binding protein.

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    Brian Krumm

    Full Text Available Interleukin 18 (IL18 is a cytokine that plays an important role in inflammation as well as host defense against microbes. Mammals encode a soluble inhibitor of IL18 termed IL18 binding protein (IL18BP that modulates IL18 activity through a negative feedback mechanism. Many poxviruses encode homologous IL18BPs, which contribute to virulence. Previous structural and functional studies on IL18 and IL18BPs revealed an essential binding hot spot involving a lysine on IL18 and two aromatic residues on IL18BPs. The aromatic residues are conserved among the very diverse mammalian and poxviruses IL18BPs with the notable exception of yatapoxvirus IL18BPs, which lack a critical phenylalanine residue. To understand the mechanism by which yatapoxvirus IL18BPs neutralize IL18, we solved the crystal structure of the Yaba-Like Disease Virus (YLDV IL18BP and IL18 complex at 1.75 Å resolution. YLDV-IL18BP forms a disulfide bonded homo-dimer engaging IL18 in a 2∶2 stoichiometry, in contrast to the 1∶1 complex of ectromelia virus (ECTV IL18BP and IL18. Disruption of the dimer interface resulted in a functional monomer, however with a 3-fold decrease in binding affinity. The overall architecture of the YLDV-IL18BP:IL18 complex is similar to that observed in the ECTV-IL18BP:IL18 complex, despite lacking the critical lysine-phenylalanine interaction. Through structural and mutagenesis studies, contact residues that are unique to the YLDV-IL18BP:IL18 binding interface were identified, including Q67, P116 of YLDV-IL18BP and Y1, S105 and D110 of IL18. Overall, our studies show that YLDV-IL18BP is unique among the diverse family of mammalian and poxvirus IL-18BPs in that it uses a bivalent binding mode and a unique set of interacting residues for binding IL18. However, despite this extensive divergence, YLDV-IL18BP binds to the same surface of IL18 used by other IL18BPs, suggesting that all IL18BPs use a conserved inhibitory mechanism by blocking a putative receptor-binding

  5. The nucleolar phosphoprotein B23 targets Newcastle disease virus matrix protein to the nucleoli and facilitates viral replication.

    Science.gov (United States)

    Duan, Zhiqiang; Chen, Jian; Xu, Haixu; Zhu, Jie; Li, Qunhui; He, Liang; Liu, Huimou; Hu, Shunlin; Liu, Xiufan

    2014-03-01

    The cellular nucleolar proteins are reported to facilitate the replication cycles of some human and animal viruses by interaction with viral proteins. In this study, a nucleolar phosphoprotein B23 was identified to interact with Newcastle disease virus (NDV) matrix (M) protein. We found that NDV M protein accumulated in the nucleolus by binding B23 early in infection, but resulted in the redistribution of B23 from the nucleoli to the nucleoplasm later in infection. In vitro binding studies utilizing deletion mutants indicated that amino acids 30-60 of M and amino acids 188-245 of B23 were required for binding. Furthermore, knockdown of B23 by siRNA or overexpression of B23 or M-binding B23-derived polypeptides remarkably reduced cytopathic effect and inhibited NDV replication. Collectively, we show that B23 facilitates NDV replication by targeting M to the nucleolus, demonstrating for the first time a direct role for nucleolar protein B23 in a paramyxovirus replication process. Copyright © 2014 Elsevier Inc. All rights reserved.

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

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    Shubhadip Das

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

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

    Science.gov (United States)

    Miao, Yinglong; McCammon, J Andrew

    2018-03-20

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

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

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    Kamala Boonyodying

    2012-06-01

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

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

    International Nuclear Information System (INIS)

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

    2006-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Margarita Salas

    2016-08-01

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

  11. Tailoring odorant-binding protein coatings characteristics for surface acoustic wave biosensor development

    Energy Technology Data Exchange (ETDEWEB)

    Di Pietrantonio, F., E-mail: fabio.dp@idasc.cnr.it [Institute of Acoustics and Sensors “O. M. Corbino”, National Research Council of Italy, Via del Fosso del Cavaliere 100, 00133 Rome (Italy); Benetti, M. [Institute of Acoustics and Sensors “O. M. Corbino”, National Research Council of Italy, Via del Fosso del Cavaliere 100, 00133 Rome (Italy); Dinca, V. [National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, PO Box MG-16, 077125 Magurele (Romania); Cannatà, D. [Institute of Acoustics and Sensors “O. M. Corbino”, National Research Council of Italy, Via del Fosso del Cavaliere 100, 00133 Rome (Italy); Verona, E. [Institute for Photonics and Nanotechnologies, National Research Council of Italy, Via del Cineto Romano 42, 00156 Rome (Italy); D’Auria, S. [Institute of Protein Biochemistry, National Research Council of Italy, Via Pietro Castellino 111, 80131 Naples (Italy); Dinescu, M. [National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, PO Box MG-16, 077125 Magurele (Romania)

    2014-05-01

    In this study, wild type bovine odorant-binding proteins (wtbOBPs) were deposited by matrix-assisted pulsed laser evaporation (MAPLE) and utilized as active material on surface acoustic wave (SAW) biosensors. Fourier transform infrared spectroscopy (FTIR), and atomic force microscopy (AFM) were used to determine the chemical, morphological characteristics of the protein thin films. The FTIR data demonstrates that the functional groups of wtbOBPs do not suffer significant changes in the MAPLE-deposited films when compared to the reference one. The topographical studies show that the homogeneity, density and the roughness of the coatings are related mainly to the laser parameters (fluence and number of pulses). SAW biosensor responses to different concentrations of R-(–)-1-octen-3-ol (octenol) and R-(–)-carvone (carvone) were evaluated. The obtained sensitivities, achieved through the optimization of deposition parameters, demonstrated that MAPLE is a promising deposition technique for SAW biosensor implementation.

  12. Structural and binding properties of two paralogous fatty acid binding proteins of Taenia solium metacestode.

    Directory of Open Access Journals (Sweden)

    Seon-Hee Kim

    Full Text Available BACKGROUND: Fatty acid (FA binding proteins (FABPs of helminths are implicated in acquisition and utilization of host-derived hydrophobic substances, as well as in signaling and cellular interactions. We previously demonstrated that secretory hydrophobic ligand binding proteins (HLBPs of Taenia solium metacestode (TsM, a causative agent of neurocysticercosis (NC, shuttle FAs in the surrounding host tissues and inwardly transport the FAs across the parasite syncytial membrane. However, the protein molecules responsible for the intracellular trafficking and assimilation of FAs have remained elusive. METHODOLOGY/PRINCIPAL FINDINGS: We isolated two novel TsMFABP genes (TsMFABP1 and TsMFABP2, which encoded 133- and 136-amino acid polypeptides with predicted molecular masses of 14.3 and 14.8 kDa, respectively. They shared 45% sequence identity with each other and 15-95% with other related-members. Homology modeling demonstrated a characteristic β-barrel composed of 10 anti-parallel β-strands and two α-helices. TsMFABP2 harbored two additional loops between β-strands two and three, and β-strands six and seven, respectively. TsMFABP1 was secreted into cyst fluid and surrounding environments, whereas TsMFABP2 was intracellularly confined. Partially purified native proteins migrated to 15 kDa with different isoelectric points of 9.2 (TsMFABP1 and 8.4 (TsMFABP2. Both native and recombinant proteins bound to 11-([5-dimethylaminonaphthalene-1-sulfonyl]aminoundecannoic acid, dansyl-DL-α-amino-caprylic acid, cis-parinaric acid and retinol, which were competitively inhibited by oleic acid. TsMFABP1 exhibited high affinity toward FA analogs. TsMFABPs showed weak binding activity to retinol, but TsMFABP2 showed relatively high affinity. Isolation of two distinct genes from an individual genome strongly suggested their paralogous nature. Abundant expression of TsMFABP1 and TsMFABP2 in the canal region of worm matched well with the histological distributions

  13. Comparison of two methods forecasting binding rate of plasma protein.

    Science.gov (United States)

    Hongjiu, Liu; Yanrong, Hu

    2014-01-01

    By introducing the descriptors calculated from the molecular structure, the binding rates of plasma protein (BRPP) with seventy diverse drugs are modeled by a quantitative structure-activity relationship (QSAR) technique. Two algorithms, heuristic algorithm (HA) and support vector machine (SVM), are used to establish linear and nonlinear models to forecast BRPP. Empirical analysis shows that there are good performances for HA and SVM with cross-validation correlation coefficients Rcv(2) of 0.80 and 0.83. Comparing HA with SVM, it was found that SVM has more stability and more robustness to forecast BRPP.

  14. Prediction of RNA-Binding Proteins by Voting Systems

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    C. R. Peng

    2011-01-01

    Full Text Available It is important to identify which proteins can interact with RNA for the purpose of protein annotation, since interactions between RNA and proteins influence the structure of the ribosome and play important roles in gene expression. This paper tries to identify proteins that can interact with RNA using voting systems. Firstly through Weka, 34 learning algorithms are chosen for investigation. Then simple majority voting system (SMVS is used for the prediction of RNA-binding proteins, achieving average ACC (overall prediction accuracy value of 79.72% and MCC (Matthew’s correlation coefficient value of 59.77% for the independent testing dataset. Then mRMR (minimum redundancy maximum relevance strategy is used, which is transferred into algorithm selection. In addition, the MCC value of each classifier is assigned to be the weight of the classifier’s vote. As a result, best average MCC values are attained when 22 algorithms are selected and integrated through weighted votes, which are 64.70% for the independent testing dataset, and ACC value is 82.04% at this moment.

  15. Prion Fragment Peptides Are Digested with Membrane Type Matrix Metalloproteinases and Acquire Enzyme Resistance through Cu2+-Binding

    Directory of Open Access Journals (Sweden)

    Aya Kojima

    2014-05-01

    Full Text Available Prions are the cause of neurodegenerative disease in humans and other mammals. The structural conversion of the prion protein (PrP from a normal cellular protein (PrPC to a protease-resistant isoform (PrPSc is thought to relate to Cu2+ binding to histidine residues. In this study, we focused on the membrane-type matrix metalloproteinases (MT-MMPs such as MT1-MMP and MT3-MMP, which are expressed in the brain as PrPC-degrading proteases. We synthesized 21 prion fragment peptides. Each purified peptide was individually incubated with recombinant MT1-MMP or MT3-MMP in the presence or absence of Cu2+ and the cleavage sites determined by LC-ESI-MS analysis. Recombinant MMP-7 and human serum (HS were also tested as control. hPrP61-90, from the octapeptide-repeat region, was cleaved by HS but not by the MMPs tested here. On the other hand, hPrP92-168 from the central region was cleaved by MT1-MMP and MT3-MMP at various sites. These cleavages were inhibited by treatment with Cu2+. The C-terminal peptides had higher resistance than the central region. The data obtained from this study suggest that MT-MMPs expressed in the brain might possess PrPC-degrading activity.

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

    Science.gov (United States)

    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.

  17. Ice cream structure modification by ice-binding proteins.

    Science.gov (United States)

    Kaleda, Aleksei; Tsanev, Robert; Klesment, Tiina; Vilu, Raivo; Laos, Katrin

    2018-04-25

    Ice-binding proteins (IBPs), also known as antifreeze proteins, were added to ice cream to investigate their effect on structure and texture. Ice recrystallization inhibition was assessed in the ice cream mixes using a novel accelerated microscope assay and the ice cream microstructure was studied using an ice crystal dispersion method. It was found that adding recombinantly produced fish type III IBPs at a concentration 3 mg·L -1 made ice cream hard and crystalline with improved shape preservation during melting. Ice creams made with IBPs (both from winter rye, and type III IBP) had aggregates of ice crystals that entrapped pockets of the ice cream mixture in a rigid network. Larger individual ice crystals and no entrapment in control ice creams was observed. Based on these results a model of ice crystals aggregates formation in the presence of IBPs was proposed. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Prediction of vitamin interacting residues in a vitamin binding protein using evolutionary information.

    Science.gov (United States)

    Panwar, Bharat; Gupta, Sudheer; Raghava, Gajendra P S

    2013-02-07

    The vitamins are important cofactors in various enzymatic-reactions. In past, many inhibitors have been designed against vitamin binding pockets in order to inhibit vitamin-protein interactions. Thus, it is important to identify vitamin interacting residues in a protein. It is possible to detect vitamin-binding pockets on a protein, if its tertiary structure is known. Unfortunately tertiary structures of limited proteins are available. Therefore, it is important to develop in-silico models for predicting vitamin interacting residues in protein from its primary structure. In this study, first we compared protein-interacting residues of vitamins with other ligands using Two Sample Logo (TSL). It was observed that ATP, GTP, NAD, FAD and mannose preferred {G,R,K,S,H}, {G,K,T,S,D,N}, {T,G,Y}, {G,Y,W} and {Y,D,W,N,E} residues respectively, whereas vitamins preferred {Y,F,S,W,T,G,H} residues for the interaction with proteins. Furthermore, compositional information of preferred and non-preferred residues along with patterns-specificity was also observed within different vitamin-classes. Vitamins A, B and B6 preferred {F,I,W,Y,L,V}, {S,Y,G,T,H,W,N,E} and {S,T,G,H,Y,N} interacting residues respectively. It suggested that protein-binding patterns of vitamins are different from other ligands, and motivated us to develop separate predictor for vitamins and their sub-classes. The four different prediction modules, (i) vitamin interacting residues (VIRs), (ii) vitamin-A interacting residues (VAIRs), (iii) vitamin-B interacting residues (VBIRs) and (iv) pyridoxal-5-phosphate (vitamin B6) interacting residues (PLPIRs) have been developed. We applied various classifiers of SVM, BayesNet, NaiveBayes, ComplementNaiveBayes, NaiveBayesMultinomial, RandomForest and IBk etc., as machine learning techniques, using binary and Position-Specific Scoring Matrix (PSSM) features of protein sequences. Finally, we selected best performing SVM modules and obtained highest MCC of 0.53, 0.48, 0.61, 0

  19. Immunochemical characterization of the brain glutamate binding protein

    International Nuclear Information System (INIS)

    Roy, S.

    1986-01-01

    A glutamate binding protein (GBP) was purified from bovine and rat brain to near homogeneity. Polyclonal antibodies were raised against this protein. An enzyme-linked-immunosorbent-assay was used to quantify and determine the specificity of the antibody response. The antibodies were shown to strongly react with bovine brain GBP and the analogous protein from rat brain. The antibodies did not show any crossreactivity with the glutamate metabolizing enzymes, glutamate dehydrogenase, glutamine synthetase and glutamyl transpeptidase, however it crossreacted moderately with glutamate decarboxylase. The antibodies were also used to define the possible physiologic activity of GBP in synaptic membranes. The antibodies were shown: (i) to inhibit the excitatory amino-acid stimulation of thiocyanate (SCN)flux, (ii) had no effect on transport of L-Glutamic acid across the synaptic membrane, and (iii) had no effect on the depolarization-induced release of L-glutamate. When the anti-GBP antibodies were used to localize and quantify the GBP distribution in various subcellular fractions and in brain tissue samples, it was found that the hippocampus had the highest immunoreactivity followed by the cerebral cortex, cerebellar cortex and caudate-putamen. The distribution of immunoreactivity in the subcellular fraction were as follows: synaptic membranes > crude mitochondrial fraction > homogenate > myelin. In conclusion these studies suggest that: (a) the rat brain GBP and the bovine brain GBP are immunologically homologous protein, (b) there are no structural similarities between the GBP and the glutamate metabolizing enzymes with the exception of glutamate decarboxylase and (c) the subcellular and regional distribution of the GBP immunoreactivity followed a similar pattern as observed for L-[ 3 H]-binding

  20. Investigation of the binding of dioxin selective pentapeptides to a polyaniline matrix

    KAUST Repository

    Archibong, Edikan; Wang, Ling; Ivanov, Ivan; Lita, Adrian; Redda, Kinfe; Mateeva, Nelly

    2012-01-01

    Polyaniline in form of emeraldine salt and emeraldine base was used as a matrix to attach several labeled and non-labeled dioxin selective pentapeptides both directly to the polymer and using glutaraldehyde as a linker. The peptides have been selected as a model to study the binding process due to their smaller size, lower sensitivity to the environment and potential application as solid state extraction reagents for chlorinated toxins. The composition and the properties of the compounds were investigated by means of elemental analysis, XPS, FTIR, UV/vis, and fluorescence spectroscopy. The results have shown that 3.30-7.76% peptides were attached to the emeraldine base both with and without a linker. Glutaraldehyde and the peptides were connected to the matrix via chemical bond resulting in formation of compounds whit similar composition and stability in a broad pH range. The influence of the linker and the peptides on the electronic properties and composition of the polymer have been investigated by principal component analysis. © 2012 Elsevier B.V. All rights reserved.

  1. Investigation of the binding of dioxin selective pentapeptides to a polyaniline matrix

    KAUST Repository

    Archibong, Edikan

    2012-08-01

    Polyaniline in form of emeraldine salt and emeraldine base was used as a matrix to attach several labeled and non-labeled dioxin selective pentapeptides both directly to the polymer and using glutaraldehyde as a linker. The peptides have been selected as a model to study the binding process due to their smaller size, lower sensitivity to the environment and potential application as solid state extraction reagents for chlorinated toxins. The composition and the properties of the compounds were investigated by means of elemental analysis, XPS, FTIR, UV/vis, and fluorescence spectroscopy. The results have shown that 3.30-7.76% peptides were attached to the emeraldine base both with and without a linker. Glutaraldehyde and the peptides were connected to the matrix via chemical bond resulting in formation of compounds whit similar composition and stability in a broad pH range. The influence of the linker and the peptides on the electronic properties and composition of the polymer have been investigated by principal component analysis. © 2012 Elsevier B.V. All rights reserved.

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

    KAUST Repository

    Chen, Peng

    2015-12-03

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

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

    KAUST Repository

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

    2015-01-01

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

  4. Isolation and functional characterization of CE1 binding proteins

    Directory of Open Access Journals (Sweden)

    Yu Ji-hyun

    2010-12-01

    Full Text Available Abstract Background Abscisic acid (ABA is a plant hormone that controls seed germination, protective responses to various abiotic stresses and seed maturation. The ABA-dependent processes entail changes in gene expression. Numerous genes are regulated by ABA, and promoter analyses of the genes revealed that cis-elements sharing the ACGTGGC consensus sequence are ubiquitous among ABA-regulated gene promoters. The importance of the core sequence, which is generally known as ABA response element (ABRE, has been demonstrated by various experiments, and its cognate transcription factors known as ABFs/AREBs have been identified. Although necessary, ABRE alone is not sufficient, and another cis-element known as "coupling element (CE" is required for full range ABA-regulation of gene expression. Several CEs are known. However, despite their importance, the cognate transcription factors mediating ABA response via CEs have not been reported to date. Here, we report the isolation of transcription factors that bind one of the coupling elements, CE1. Results To isolate CE1 binding proteins, we carried out yeast one-hybrid screens. Reporter genes containing a trimer of the CE1 element were prepared and introduced into a yeast strain. The yeast was transformed with library DNA that represents RNA isolated from ABA-treated Arabidopsis seedlings. From the screen of 3.6 million yeast transformants, we isolated 78 positive clones. Analysis of the clones revealed that a group of AP2/ERF domain proteins binds the CE1 element. We investigated their expression patterns and analyzed their overexpression lines to investigate the in vivo functions of the CE element binding factors (CEBFs. Here, we show that one of the CEBFs, AtERF13, confers ABA hypersensitivity in Arabidopsis, whereas two other CEBFs enhance sugar sensitivity. Conclusions Our results indicate that a group of AP2/ERF superfamily proteins interacts with CE1. Several CEBFs are known to mediate defense or

  5. Isolation and functional characterization of CE1 binding proteins.

    Science.gov (United States)

    Lee, Sun-ji; Park, Ji Hye; Lee, Mi Hun; Yu, Ji-hyun; Kim, Soo Young

    2010-12-16

    Abscisic acid (ABA) is a plant hormone that controls seed germination, protective responses to various abiotic stresses and seed maturation. The ABA-dependent processes entail changes in gene expression. Numerous genes are regulated by ABA, and promoter analyses of the genes revealed that cis-elements sharing the ACGTGGC consensus sequence are ubiquitous among ABA-regulated gene promoters. The importance of the core sequence, which is generally known as ABA response element (ABRE), has been demonstrated by various experiments, and its cognate transcription factors known as ABFs/AREBs have been identified. Although necessary, ABRE alone is not sufficient, and another cis-element known as "coupling element (CE)" is required for full range ABA-regulation of gene expression. Several CEs are known. However, despite their importance, the cognate transcription factors mediating ABA response via CEs have not been reported to date. Here, we report the isolation of transcription factors that bind one of the coupling elements, CE1. To isolate CE1 binding proteins, we carried out yeast one-hybrid screens. Reporter genes containing a trimer of the CE1 element were prepared and introduced into a yeast strain. The yeast was transformed with library DNA that represents RNA isolated from ABA-treated Arabidopsis seedlings. From the screen of 3.6 million yeast transformants, we isolated 78 positive clones. Analysis of the clones revealed that a group of AP2/ERF domain proteins binds the CE1 element. We investigated their expression patterns and analyzed their overexpression lines to investigate the in vivo functions of the CE element binding factors (CEBFs). Here, we show that one of the CEBFs, AtERF13, confers ABA hypersensitivity in Arabidopsis, whereas two other CEBFs enhance sugar sensitivity. Our results indicate that a group of AP2/ERF superfamily proteins interacts with CE1. Several CEBFs are known to mediate defense or abiotic stress response, but the physiological functions

  6. Evolving Transcription Factor Binding Site Models From Protein Binding Microarray Data

    KAUST Repository

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

    2016-01-01

    Protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner. In this paper, we describe the PBM motif model building problem. We apply several evolutionary computation methods and compare their performance with the interior point method, demonstrating their performance advantages. In addition, given the PBM domain knowledge, we propose and describe a novel method called kmerGA which makes domain-specific assumptions to exploit PBM data properties to build more accurate models than the other models built. The effectiveness and robustness of kmerGA is supported by comprehensive performance benchmarking on more than 200 datasets, time complexity analysis, convergence analysis, parameter analysis, and case studies. To demonstrate its utility further, kmerGA is applied to two real world applications: 1) PBM rotation testing and 2) ChIP-Seq peak sequence prediction. The results support the biological relevance of the models learned by kmerGA, and thus its real world applicability.

  7. Evolving Transcription Factor Binding Site Models From Protein Binding Microarray Data

    KAUST Repository

    Wong, Ka-Chun

    2016-02-02

    Protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner. In this paper, we describe the PBM motif model building problem. We apply several evolutionary computation methods and compare their performance with the interior point method, demonstrating their performance advantages. In addition, given the PBM domain knowledge, we propose and describe a novel method called kmerGA which makes domain-specific assumptions to exploit PBM data properties to build more accurate models than the other models built. The effectiveness and robustness of kmerGA is supported by comprehensive performance benchmarking on more than 200 datasets, time complexity analysis, convergence analysis, parameter analysis, and case studies. To demonstrate its utility further, kmerGA is applied to two real world applications: 1) PBM rotation testing and 2) ChIP-Seq peak sequence prediction. The results support the biological relevance of the models learned by kmerGA, and thus its real world applicability.

  8. Peeping into human renal calcium oxalate stone matrix: characterization of novel proteins involved in the intricate mechanism of urolithiasis.

    Directory of Open Access Journals (Sweden)

    Kanu Priya Aggarwal

    Full Text Available BACKGROUND: The increasing number of patients suffering from urolithiasis represents one of the major challenges which nephrologists face worldwide today. For enhancing therapeutic outcomes of this disease, the pathogenic basis for the formation of renal stones is the need of hour. Proteins are found as major component in human renal stone matrix and are considered to have a potential role in crystal-membrane interaction, crystal growth and stone formation but their role in urolithiasis still remains obscure. METHODS: Proteins were isolated from the matrix of human CaOx containing kidney stones. Proteins having MW>3 kDa were subjected to anion exchange chromatography followed by molecular-sieve chromatography. The effect of these purified proteins was tested against CaOx nucleation and growth and on oxalate injured Madin-Darby Canine Kidney (MDCK renal epithelial cells for their activity. Proteins were identified by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF MS followed by database search with MASCOT server. In silico molecular interaction studies with CaOx crystals were also investigated. RESULTS: Five proteins were identified from the matrix of calcium oxalate kidney stones by MALDI-TOF MS followed by database search with MASCOT server with the competence to control the stone formation process. Out of which two proteins were promoters, two were inhibitors and one protein had a dual activity of both inhibition and promotion towards CaOx nucleation and growth. Further molecular modelling calculations revealed the mode of interaction of these proteins with CaOx at the molecular level. CONCLUSIONS: We identified and characterized Ethanolamine-phosphate cytidylyltransferase, Ras GTPase-activating-like protein, UDP-glucose:glycoprotein glucosyltransferase 2, RIMS-binding protein 3A, Macrophage-capping protein as novel proteins from the matrix of human calcium oxalate stone which play a critical role in kidney stone

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

    International Nuclear Information System (INIS)

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

    1990-01-01

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

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

    NARCIS (Netherlands)

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

    1994-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-12-25

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

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

    Science.gov (United States)

    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. Copyright 2010 Elsevier Inc. All rights reserved.

  13. Isolation and Purification of Thiamine Binding Protein from Mung Bean

    Directory of Open Access Journals (Sweden)

    DWIRINI RETNO GUNARTI

    2013-03-01

    Full Text Available Thiamine has fundamental role in energy metabolism. The organs mostly sensitive to the lack of thiamine levels in the body are the nervous system and the heart. Thiamine deficiency causes symptoms of polyneuritis and cardiovascular diseases. Because of its importance in the metabolism of carbohydrates, we need to measure the levels of thiamine in the body fluids by using an easy and inexpensive way without compromising the sensitivity and selectivity. An option to it is thiamine measurement based on the principle of which is analogous to ELISA, in which a thiamine binding protein (TBP act by replacing antibodies. The presence of TBP in several seeds have been reported by previous researchers, but the presence of TBP in mung beans has not been studied. This study was aimed to isolate and purify TBP from mung bean. The protein was isolated from mung bean through salting out by ammonium sulphate of 40, 70, and 90% (w/v. TBP has a negative charge as shown by cellulose acetate electrophoresis. The result obtained after salting out by ammonium sulphate was further purified bymeans of DEAE-cellulose chromatography and affinity chromatography. In precipitation of 90% of salting out method, one peak protein was obtained by using affinity chromatography. The protein was analyzed by SDS PAGE electrophoresis. The result of SDS PAGE electrophoresis showed that TBP has a molecular weight of 72.63 kDa.

  14. Fanconi anemia proteins localize to chromatin and the nuclear matrix in a DNA damage- and cell cycle-regulated manner.

    Science.gov (United States)

    Qiao, F; Moss, A; Kupfer, G M

    2001-06-29

    Fanconi anemia (FA) is a genetic disease characterized by congenital defects, bone marrow failure, and cancer susceptibility. Cells from patients with FA exhibit genomic instability and hypersensitivity to DNA cross linking agents such as mitomycin C. Despite the identification of seven complementation groups and the cloning of six genes, the function of the encoded gene products remains elusive. The FancA (Fanconi anemia complementation group A), FancC, and FancG proteins have been detected within a nuclear complex, but no change in level, binding, or localization has been reported as a result of drug treatment or cell cycle. We show that in immunofluorescence studies, FancA appears as a non-nucleolar nuclear protein that is excluded from condensed, mitotic chromosomes. Biochemical fractionation reveals that the FA proteins are found in nuclear matrix and chromatin and that treatment with mitomycin C results in increase of the FA proteins in nuclear matrix and chromatin fractions. This induction occurs in wild-type cells and mutant FA-D (Fanconi complementation group D) cells but not in mutant FA-A cells. Immunoprecipitation of FancA protein in chromatin demonstrates the coprecipitation of FancA, FancC, and FancG, showing that the FA proteins move together as a complex. Also, fractionation of mitotic cells confirms the lack of FA proteins in chromatin or the nuclear matrix. Furthermore, phosphorylation of FancG was found to be temporally correlated with exit of the FA complex from chromosomes at mitosis. Taken together, these findings suggest a role for FA proteins in chromatin and nuclear matrix.

  15. Porcine circovirus type 2 ORF4 protein binds heavy chain ferritin

    Indian Academy of Sciences (India)

    Porcine circovirus type 2 ORF4 protein binds heavy chain ferritin. Qizhuang Lv Kangkang Guo Tao Wang ... Keywords. Cellular protein; FHC; ORF4 protein; porcine circovirus type 2 (PCV2); yeast two-hybrid ... Journal of Biosciences | News ...

  16. Acyl-CoA binding protein is an essential protein in mammalian cell lines

    DEFF Research Database (Denmark)

    Knudsen, Jens; Færgeman, Nils J.

    2002-01-01

    In the present work, small interference RNA was used to knock-down acyl-CoA binding protein (ACBP) in HeLa, HepG2 and Chang cells. Transfection with ACBP-specific siRNA stopped growth, detached cells from the growth surface and blocked thymidine and acetate incorporation. The results show...

  17. Differential dissociation micromethod for the investigation of binding of metandrostenolone (Nerobol) to plasma proteins

    International Nuclear Information System (INIS)

    Bojadzsieva, Milka; Kocsar, Laszlo; Kremmer, Tibor

    1985-01-01

    A micromethod was developed to determine the binding of anabolic streoids to plasma proteins. The new procedure combines precipitation with ammonium sulphate and differential dissociation. The binding parameters (association constant, specific binding capacity) are calculated on the basis of dissociation curves of sup(3)H-metandrostenolone from the precipitated sexual binding globuline. (L.E.)

  18. Differential dissociation micromethod for the investigation of binding of metandrostenolone (Nerobol) to plasma proteins

    Energy Technology Data Exchange (ETDEWEB)

    Bojadzsieva, M.; Kocsar, L. (Orszagos Frederic Joliot-Curie Sugarbiologiai es Sugaregeszseguegyi Kutato Intezet, Budapest (Hungary)); Kremmer, T. (Orszagos Onkologiai Intezet, Budapest (Hungary))

    1985-01-01

    A micromethod was developed to determine the binding of anabolic steroids to plasma proteins. The new procedure combines precipitation with ammonium sulphate and differential dissociation. The binding parameters (association constant, specific binding capacity) are calculated on the basis of dissociation curves of sup(3)H-metandrostenolone from the precipitated sexual binding globuline.

  19. Oligomerization and polymerization of the filovirus matrix protein VP40

    International Nuclear Information System (INIS)

    Timmins, Joanna; Schoehn, Guy; Kohlhaas, Christine; Klenk, Hans-Dieter; Ruigrok, Rob W.H.; Weissenhorn, Winfried

    2003-01-01

    The matrix protein VP40 from Ebola virus plays an important role in the assembly process of virus particles by interacting with cellular factors, cellular membranes, and the ribonuclearprotein particle complex. Here we show that the N-terminal domain of VP40 folds into a mixture of two different oligomeric states in vitro, namely hexameric and octameric ringlike structures, as detected by gel filtration chromatography, chemical cross-linking, and electron microscopy. Octamer formation depends largely on the interaction with nucleic acids, which in turn confers in vitro SDS resistance. Refolding experiments with a nucleic acid free N-terminal domain preparation reveal a mostly dimeric form of VP40, which is transformed into an SDS resistant octamer upon incubation with E. coli nucleic acids. In addition, we demonstrate that the N-terminal domain of Marburg virus VP40 also folds into ringlike structures, similar to Ebola virus VP40. Interestingly, Marburg virus VP40 rings reveal a high tendency to polymerize into rods composed of stacked rings. These results may suggest distinct roles for different oligomeric forms of VP40 in the filovirus life cycle

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

    Science.gov (United States)

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

    2016-03-14

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

  1. Expression of a fatty acid-binding protein in yeast

    International Nuclear Information System (INIS)

    Scholz, H.

    1991-06-01

    The unicellular eukaryotic microorganism, Saccharomyces cerevisiae, transformed with a plasmid containing a cDNA fragment encoding bovine heart fatty acid-binding protein (H-FABP C ) under the control of the inducible yeast GAL10 promoter, expressed FABP during growth on galactose. The maximum level of immunoreactive FABP, identical in size and isoelectric point to native protein, was reached after approximately 16 hours of induction. In contrast, transcription of the gene was induced within half an hour. Both, protein and mRNA were unstable and degraded within 1 h after repression of transcription. Analysis of subcellular fractions showed that FABP was exclusively associated with the cytosol. FABP expressed in yeast cells was functional as was demonstrated by its capacity to bind long chain fatty acids in an in vitro assay. Growth of all transformants on galactose as the carbon source showed no phenotype at temperatures up to 37 deg C, but the growth of FABP-expressing cells at 37 deg C was significantly retarded. Among the biochemical effects of FABP expression on lipid metabolism is a marked reduction of chain elongation and desaturation of exogenously added 14 C-palmitic acid. This effect is most pronounced in triacylglycerols and phospholipids when cells grow at 30 deg C and 37 deg C, respectively. In an in vitro assay determining the desaturation of palmitoyl CoA by microsomal membranes cytosol with or without exo- or endogenous FABP showed the same stimulation of the reaction. The desaturation of exogenously added 14 C-stearic acid, the pattern of unlabelled fatty acids (saturated vs. unsaturated) and the distribution of exogenously added radioactive fatty acids (palmitic, stearic or oleic acid) among lipid classes was not significantly affected. Using high concentrations (1 mM) the uptake of fatty acids was first stimulated and then inhibited when FABP was expressed. (author)

  2. Computational analysis and prediction of the binding motif and protein interacting partners of the Abl SH3 domain.

    Directory of Open Access Journals (Sweden)

    Tingjun Hou

    2006-01-01

    Full Text Available Protein-protein interactions, particularly weak and transient ones, are often mediated by peptide recognition domains, such as Src Homology 2 and 3 (SH2 and SH3 domains, which bind to specific sequence and structural motifs. It is important but challenging to determine the binding specificity of these domains accurately and to predict their physiological interacting partners. In this study, the interactions between 35 peptide ligands (15 binders and 20 non-binders and the Abl SH3 domain were analyzed using molecular dynamics simulation and the Molecular Mechanics/Poisson-Boltzmann Solvent Area method. The calculated binding free energies correlated well with the rank order of the binding peptides and clearly distinguished binders from non-binders. Free energy component analysis revealed that the van der Waals interactions dictate the binding strength of peptides, whereas the binding specificity is determined by the electrostatic interaction and the polar contribution of desolvation. The binding motif of the Abl SH3 domain was then determined by a virtual mutagenesis method, which mutates the residue at each position of the template peptide relative to all other 19 amino acids and calculates the binding free energy difference between the template and the mutated peptides using the Molecular Mechanics/Poisson-Boltzmann Solvent Area method. A single position mutation free energy profile was thus established and used as a scoring matrix to search peptides recognized by the Abl SH3 domain in the human genome. Our approach successfully picked ten out of 13 experimentally determined binding partners of the Abl SH3 domain among the top 600 candidates from the 218,540 decapeptides with the PXXP motif in the SWISS-PROT database. We expect that this physical-principle based method can be applied to other protein domains as well.

  3. Identification and molecular properties of SUMO-binding proteins in arabidopsis

    KAUST Repository

    Park, Hyeongcheol

    2011-05-20

    Reversible conjugation of the small ubiquitin modifier (SUMO) peptide to proteins (SUMOylation) plays important roles in cellular processes in animals and yeasts. However, little is known about plant SUMO targets. To identify SUMO substrates in Arabidopsis and to probe for biological functions of SUMO proteins, we constructed 6xHis-3xFLAG fused AtSUMO1 (HFAtSUMO1) controlled by the CaMV35S promoter for transformation into Arabidopsis Col-0. After heat treatment, an increased sumoylation pattern was detected in the transgenic plants. SUMO1-modified proteins were selected after two-dimensional gel electrophoresis (2-DE) image analysis and identified using matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). We identified 27 proteins involved in a variety of processes such as nucleic acid metabolism, signaling, metabolism, and including proteins of unknown functions. Binding and sumoylation patterns were confirmed independently. Surprisingly, MCM3 (At5G46280), a DNA replication licensing factor, only interacted with and became sumoylated by AtSUMO1, but not by SUMO1ΔGG or AtSUMO3. The results suggest specific interactions between sumoylation targets and particular sumoylation enzymes. ©2011 KSMCB.

  4. A newly identified protein of Leptospira interrogans mediates binding to laminin.

    Science.gov (United States)

    Longhi, Mariana T; Oliveira, Tatiane R; Romero, Eliete C; Gonçales, Amane P; de Morais, Zenaide M; Vasconcellos, Silvio A; Nascimento, Ana L T O

    2009-10-01

    Pathogenic Leptospira is the aetiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. The search for novel antigens that could be relevant in host-pathogen interactions is being pursued. These antigens have the potential to elicit several activities, including adhesion. This study focused on a hypothetical predicted lipoprotein of Leptospira, encoded by the gene LIC12895, thought to mediate attachment to extracellular matrix (ECM) components. The gene was cloned and expressed in Escherichia coli BL21 Star (DE3)pLys by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and characterized by circular dichroism spectroscopy. The capacity of the protein to mediate attachment to ECM components was evaluated by binding assays. The leptospiral protein encoded by LIC12895, named Lsa27 (leptospiral surface adhesin, 27 kDa), bound strongly to laminin in a dose-dependent and saturable fashion. Moreover, Lsa27 was recognized by antibodies from serum samples of confirmed leptospirosis specimens in both the initial and the convalescent phases of the disease. Lsa27 is most likely a surface protein of Leptospira as revealed in liquid-phase immunofluorescence assays with living organisms. Taken together, these data indicate that this newly identified membrane protein is expressed during natural infection and may play a role in mediating adhesion of L. interrogans to its host.

  5. A relay network of extracellular heme-binding proteins drives C. albicans iron acquisition from hemoglobin.

    Science.gov (United States)

    Kuznets, Galit; Vigonsky, Elena; Weissman, Ziva; Lalli, Daniela; Gildor, Tsvia; Kauffman, Sarah J; Turano, Paola; Becker, Jeffrey; Lewinson, Oded; Kornitzer, Daniel

    2014-10-01

    Iron scavenging constitutes a crucial challenge for survival of pathogenic microorganisms in the iron-poor host environment. Candida albicans, like many microbial pathogens, is able to utilize iron from hemoglobin, the largest iron pool in the host's body. Rbt5 is an extracellular glycosylphosphatidylinositol (GPI)-anchored heme-binding protein of the CFEM family that facilitates heme-iron uptake by an unknown mechanism. Here, we characterize an additional C. albicans CFEM protein gene, PGA7, deletion of which elicits a more severe heme-iron utilization phenotype than deletion of RBT5. The virulence of the pga7-/- mutant is reduced in a mouse model of systemic infection, consistent with a requirement for heme-iron utilization for C. albicans pathogenicity. The Pga7 and Rbt5 proteins exhibit distinct cell wall attachment, and discrete localization within the cell envelope, with Rbt5 being more exposed than Pga7. Both proteins are shown here to efficiently extract heme from hemoglobin. Surprisingly, while Pga7 has a higher affinity for heme in vitro, we find that heme transfer can occur bi-directionally between Pga7 and Rbt5, supporting a model in which they cooperate in a heme-acquisition relay. Together, our data delineate the roles of Pga7 and Rbt5 in a cell surface protein network that transfers heme from extracellular hemoglobin to the endocytic pathway, and provide a paradigm for how receptors embedded in the cell wall matrix can mediate nutrient uptake across the fungal cell envelope.

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

    Science.gov (United States)

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

    2017-12-05

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

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

    Science.gov (United States)

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

    2014-06-01

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

  8. Novel Prostate Specific Antigen plastic antibody designed with charged binding sites for an improved protein binding and its application in a biosensor of potentiometric transduction

    International Nuclear Information System (INIS)

    Rebelo, Tânia S.C.R.; Santos, C.; Costa-Rodrigues, J.; Fernandes, M.H.; Noronha, João P.; Sales, M. Goreti F.

    2014-01-01

    Graphical abstract: EF13-201, Novel Prostate Specific Antigen plastic antibody designed with charged binding sites for an improved protein binding and its application in a biosensor of potentiometric transduction. - Abstract: This work shows that the synthesis of protein plastic antibodies tailored with selected charged monomers around the binding site enhances protein binding. These charged receptor sites are placed over a neutral polymeric matrix, thus inducing a suitable orientation the protein reception to its site. This is confirmed by preparing control materials with neutral monomers and also with non-imprinted template. This concept has been applied here to Prostate Specific Antigen (PSA), the protein of choice for screening prostate cancer throughout the population, with serum levels >10 ng/mL pointing out a high probability of associated cancer. Protein Imprinted Materials with charged binding sites (C/PIM) have been produced by surface imprinting over graphene layers to which the protein was first covalently attached. Vinylbenzyl(trimethylammonium chloride) and vinyl benzoate were introduced as charged monomers labelling the binding site and were allowed to self-organize around the protein. The subsequent polymerization was made by radical polymerization of vinylbenzene. Neutral PIM (N/PIM) prepared without oriented charges and non imprinted materials (NIM) obtained without template were used as controls. These materials were used to develop simple and inexpensive potentiometric sensor for PSA. They were included as ionophores in plasticized PVC membranes, and tested over electrodes of solid or liquid conductive contacts, made of conductive carbon over a syringe or of inner reference solution over micropipette tips. The electrodes with charged monomers showed a more stable and sensitive response, with an average slope of -44.2 mV/decade and a detection limit of 5.8 × 10 −11 mol/L (2 ng/mL). The corresponding non-imprinted sensors showed lower

  9. Mapping the nuclear localization signal in the matrix protein of potato yellow dwarf virus.

    Science.gov (United States)

    Anderson, Gavin; Jang, Chanyong; Wang, Renyuan; Goodin, Michael

    2018-05-01

    The ability of the matrix (M) protein of potato yellow dwarf virus (PYDV) to remodel nuclear membranes is controlled by a di-leucine motif located at residues 223 and 224 of its primary structure. This function can be uncoupled from that of its nuclear localization signal (NLS), which is controlled primarily by lysine and arginine residues immediately downstream of the LL motif. In planta localization of green fluorescent protein fusions, bimolecular fluorescence complementation assays with nuclear import receptor importin-α1 and yeast-based nuclear import assays provided three independent experimental approaches to validate the authenticity of the M-NLS. The carboxy terminus of M is predicted to contain a nuclear export signal, which is belived to be functional, given the ability of M to bind the Arabidopsis nuclear export receptor 1 (XPO1). The nuclear shuttle activity of M has implications for the cell-to-cell movement of PYDV nucleocapsids, based upon its interaction with the N and Y proteins.

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

    Science.gov (United States)

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

    2013-06-14

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

  11. A bioinformatic survey of RNA-binding proteins in Plasmodium.

    Science.gov (United States)

    Reddy, B P Niranjan; Shrestha, Sony; Hart, Kevin J; Liang, Xiaoying; Kemirembe, Karen; Cui, Liwang; Lindner, Scott E

    2015-11-02

    The malaria parasites in the genus Plasmodium have a very complicated life cycle involving an invertebrate vector and a vertebrate host. RNA-binding proteins (RBPs) are critical factors involved in every aspect of the development of these parasites. However, very few RBPs have been functionally characterized to date in the human parasite Plasmodium falciparum. Using different bioinformatic methods and tools we searched P. falciparum genome to list and annotate RBPs. A representative 3D models for each of the RBD domain identified in P. falciparum was created using I-TESSAR and SWISS-MODEL. Microarray and RNAseq data analysis pertaining PfRBPs was performed using MeV software. Finally, Cytoscape was used to create protein-protein interaction network for CITH-Dozi and Caf1-CCR4-Not complexes. We report the identification of 189 putative RBP genes belonging to 13 different families in Plasmodium, which comprise 3.5% of all annotated genes. Almost 90% (169/189) of these genes belong to six prominent RBP classes, namely RNA recognition motifs, DEAD/H-box RNA helicases, K homology, Zinc finger, Puf and Alba gene families. Interestingly, almost all of the identified RNA-binding helicases and KH genes have cognate homologs in model species, suggesting their evolutionary conservation. Exploration of the existing P. falciparum blood-stage transcriptomes revealed that most RBPs have peak mRNA expression levels early during the intraerythrocytic development cycle, which taper off in later stages. Nearly 27% of RBPs have elevated expression in gametocytes, while 47 and 24% have elevated mRNA expression in ookinete and asexual stages. Comparative interactome analyses using human and Plasmodium protein-protein interaction datasets suggest extensive conservation of the PfCITH/PfDOZI and PfCaf1-CCR4-NOT complexes. The Plasmodium parasites possess a large number of putative RBPs belonging to most of RBP families identified so far, suggesting the presence of extensive post

  12. Retrovirus-specific differences in matrix and nucleocapsid protein-nucleic acid interactions: implications for genomic RNA packaging.

    Science.gov (United States)

    Sun, Meng; Grigsby, Iwen F; Gorelick, Robert J; Mansky, Louis M; Musier-Forsyth, Karin

    2014-01-01

    Retroviral RNA encapsidation involves a recognition event between genomic RNA (gRNA) and one or more domains in Gag. In HIV-1, the nucleocapsid (NC) domain is involved in gRNA packaging and displays robust nucleic acid (NA) binding and chaperone functions. In comparison, NC of human T-cell leukemia virus type 1 (HTLV-1), a deltaretrovirus, displays weaker NA binding and chaperone activity. Mutation of conserved charged residues in the deltaretrovirus bovine leukemia virus (BLV) matrix (MA) and NC domains affects virus replication and gRNA packaging efficiency. Based on these observations, we hypothesized that the MA domain may generally contribute to NA binding and genome encapsidation in deltaretroviruses. Here, we examined the interaction between HTLV-2 and HIV-1 MA proteins and various NAs in vitro. HTLV-2 MA displays higher NA binding affinity and better chaperone activity than HIV-1 MA. HTLV-2 MA also binds NAs with higher affinity than HTLV-2 NC and displays more robust chaperone function. Mutation of two basic residues in HTLV-2 MA α-helix II, previously implicated in BLV gRNA packaging, reduces NA binding affinity. HTLV-2 MA binds with high affinity and specificity to RNA derived from the putative packaging signal of HTLV-2 relative to nonspecific NA. Furthermore, an HIV-1 MA triple mutant designed to mimic the basic character of HTLV-2 MA α-helix II dramatically improves binding affinity and chaperone activity of HIV-1 MA in vitro and restores RNA packaging to a ΔNC HIV-1 variant in cell-based assays. Taken together, these results are consistent with a role for deltaretrovirus MA proteins in viral RNA packaging.

  13. Guanylate kinase domains of the MAGUK family scaffold proteins as specific phospho-protein-binding modules.

    Science.gov (United States)

    Zhu, Jinwei; Shang, Yuan; Xia, Caihao; Wang, Wenning; Wen, Wenyu; Zhang, Mingjie

    2011-11-25

    Membrane-associated guanylate kinases (MAGUKs) are a large family of scaffold proteins that play essential roles in tissue developments, cell-cell communications, cell polarity control, and cellular signal transductions. Despite extensive studies over the past two decades, the functions of the signature guanylate kinase domain (GK) of MAGUKs are poorly understood. Here we show that the GK domain of DLG1/SAP97 binds to asymmetric cell division regulatory protein LGN in a phosphorylation-dependent manner. The structure of the DLG1 SH3-GK tandem in complex with a phospho-LGN peptide reveals that the GMP-binding site of GK has evolved into a specific pSer/pThr-binding pocket. Residues both N- and C-terminal to the pSer are also critical for the specific binding of the phospho-LGN peptide to GK. We further demonstrate that the previously reported GK domain-mediated interactions of DLGs with other targets, such as GKAP/DLGAP1/SAPAP1 and SPAR, are also phosphorylation dependent. Finally, we provide evidence that other MAGUK GKs also function as phospho-peptide-binding modules. The discovery of the phosphorylation-dependent MAGUK GK/target interactions indicates that MAGUK scaffold-mediated signalling complex organizations are dynamically regulated.

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

    Science.gov (United States)

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

    2016-04-06

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

  15. Enhancer of rudimentary homologue interacts with scaffold attachment factor B at the nuclear matrix to regulate SR protein phosphorylation.

    Science.gov (United States)

    Drakouli, Sotiria; Lyberopoulou, Aggeliki; Papathanassiou, Maria; Mylonis, Ilias; Georgatsou, Eleni

    2017-08-01

    Scaffold attachment factor B1 (SAFB1) is an integral component of the nuclear matrix of vertebrate cells. It binds to DNA on scaffold/matrix attachment region elements, as well as to RNA and a multitude of different proteins, affecting basic cellular activities such as transcription, splicing and DNA damage repair. In the present study, we show that enhancer of rudimentary homologue (ERH) is a new molecular partner of SAFB1 and its 70% homologous paralogue, scaffold attachment factor B2 (SAFB2). ERH interacts directly in the nucleus with the C-terminal Arg-Gly-rich region of SAFB1/2 and co-localizes with it in the insoluble nuclear fraction. ERH, a small ubiquitous protein with striking homology among species and a unique structure, has also been implicated in fundamental cellular mechanisms. Our functional analyses suggest that the SAFB/ERH interaction does not affect SAFB1/2 function in transcription (e.g. as oestrogen receptor α co-repressors), although it reverses the inhibition exerted by SAFB1/2 on the splicing kinase SR protein kinase 1 (SRPK1), which also binds on the C-terminus of SAFB1/2. Accordingly, ERH silencing decreases lamin B receptor and SR protein phosphorylation, which are major SRPK1 substrates, further substantiating the role of SAFB1 and SAFB2 in the co-ordination of nuclear function. © 2017 Federation of European Biochemical Societies.

  16. A computational model of the LGI1 protein suggests a common binding site for ADAM proteins.

    Directory of Open Access Journals (Sweden)

    Emanuela Leonardi

    Full Text Available Mutations of human leucine-rich glioma inactivated (LGI1 gene encoding the epitempin protein cause autosomal dominant temporal lateral epilepsy (ADTLE, a rare familial partial epileptic syndrome. The LGI1 gene seems to have a role on the transmission of neuronal messages but the exact molecular mechanism remains unclear. In contrast to other genes involved in epileptic disorders, epitempin shows no homology with known ion channel genes but contains two domains, composed of repeated structural units, known to mediate protein-protein interactions.A three dimensional in silico model of the two epitempin domains was built to predict the structure-function relationship and propose a functional model integrating previous experimental findings. Conserved and electrostatic charged regions of the model surface suggest a possible arrangement between the two domains and identifies a possible ADAM protein binding site in the β-propeller domain and another protein binding site in the leucine-rich repeat domain. The functional model indicates that epitempin could mediate the interaction between proteins localized to different synaptic sides in a static way, by forming a dimer, or in a dynamic way, by binding proteins at different times.The model was also used to predict effects of known disease-causing missense mutations. Most of the variants are predicted to alter protein folding while several other map to functional surface regions. In agreement with experimental evidence, this suggests that non-secreted LGI1 mutants could be retained within the cell by quality control mechanisms or by altering interactions required for the secretion process.

  17. Calculations of proton-binding thermodynamics in proteins.

    Science.gov (United States)

    Beroza, P; Case, D A

    1998-01-01

    Computational models of proton binding can range from the chemically complex and statistically simple (as in the quantum calculations) to the chemically simple and statistically complex. Much progress has been made in the multiple-site titration problem. Calculations have improved with the inclusion of more flexibility in regard to both the geometry of the proton binding and the larger scale protein motions associated with titration. This article concentrated on the principles of current calculations, but did not attempt to survey their quantitative performance. This is (1) because such comparisons are given in the cited papers and (2) because continued developments in understanding conformational flexibility and interaction energies will be needed to develop robust methods with strong predictive power. Nevertheless, the advances achieved over the past few years should not be underestimated: serious calculations of protonation behavior and its coupling to conformational change can now be confidently pursued against a backdrop of increasing understanding of the strengths and limitations of such models. It is hoped that such theoretical advances will also spur renewed experimental interest in measuring both overall titration curves and individual pKa values or pKa shifts. Exploration of the shapes of individual titration curves (as measured by Hill coefficients and other parameters) would also be useful in assessing the accuracy of computations and in drawing connections to functional behavior.

  18. Convolutional neural network architectures for predicting DNA–protein binding

    Science.gov (United States)

    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

  19. Glycosylation status of vitamin D binding protein in cancer patients.

    Science.gov (United States)

    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.

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

    KAUST Repository

    Gao, Xin

    2016-05-06

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

  1. N-Acetylgalactosaminyltransferase 14, a novel insulin-like growth factor binding protein-3 binding partner

    International Nuclear Information System (INIS)

    Wu, Chen; Yao, Guangyin; Zou, Minji; Chen, Guangyu; Wang, Min; Liu, Jingqian; Wang, Jiaxi; Xu, Donggang

    2007-01-01

    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. Absence of penicillin-binding protein 4 from an apparently normal strain of Bacillus subtilis.

    OpenAIRE

    Buchanan, C E

    1987-01-01

    The phenotype of a Bacillus subtilis 168 strain with no detectable penicillin-binding protein 4 was examined. Despite the fact that penicillin-binding protein 4 is one of the most penicillin-sensitive proteins in the species, its apparent loss had no obvious effect on the organism or its susceptibility to various beta-lactam antibiotics.

  3. Inhibition of the vitamin B12 binding capacity of proteins by the hydrolysis product of cyclophosphamide

    International Nuclear Information System (INIS)

    Fenrych, W.; Ignatowicz, E.; Szczodrowska, E.

    1993-01-01

    The inhibitory effect of cyclophosphamide hydrolysis product (CPHP) on vitamin B 12 binding ability to proteins has been established. The ester N-(2-chloroethyl)-N'-(3-phosphopropyl)-etheylenediamine hydrochloride is probably responsible, in vitro, for blocking the protein binding sites. Preincubation of proteins with vitamin B 12 prevents the inhibitory effect of CPHP. (au)

  4. Peptide microarrays to probe for competition for binding sites in a protein interaction network

    NARCIS (Netherlands)

    Sinzinger, M.D.S.; Ruttekolk, I.R.R.; Gloerich, J.; Wessels, H.; Chung, Y.D.; Adjobo-Hermans, M.J.W.; Brock, R.E.

    2013-01-01

    Cellular protein interaction networks are a result of the binding preferences of a particular protein and the entirety of interactors that mutually compete for binding sites. Therefore, the reconstruction of interaction networks by the accumulation of interaction networks for individual proteins

  5. Characterization of the retinoblastoma binding proteins RBP1 and RBP2

    DEFF Research Database (Denmark)

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

    1993-01-01

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

  6. Mannan-binding lectin in cerebrospinal fluid: a leptomeningeal protein

    Directory of Open Access Journals (Sweden)

    Reiber Hansotto

    2012-08-01

    Full Text Available Abstract Background Mannan-binding lectin (MBL, a protein of the innate immune response is attracting increasing clinical interest, in particularly in relation to its deficiency. Due to its involvement in brain diseases, identifying the source of MBL in CSF is important. Analysis of cerebrospinal fluid (CSF can provide data that discriminates between blood-, brain-, and leptomeninges-derived proteins. To detect the source of MBL in CSF we need to consider three variables: the molecular size-dependent concentration gradient between CSF and blood, the variation in transfer between blood and CSF, and the CSF MBL concentration correlation with the albumin CSF/serum quotient (QAlb, i.e., with CSF flow rate. Methods MBL was assayed in samples of CSF and serum with an ELISA, coated with anti MBL antibodies. Routine parameters such as albumin-, immunoglobulin- CSF/serum quotients, oligoclonal IgG and cell count were used to characterize the patient groups. Groups comprised firstly, control patients without organic brain disease with normal CSF and normal barrier function and secondly, patients without inflammatory diseases but with increased QAlb, i.e. with a blood CSF barrier dysfunction. Results MBL concentration in CSF was at least five-fold higher than expected for a molecular-size-dependent passage from blood. Secondly, in a QIgM/QAlb quotient diagram (Reibergram 9/13 cases showed an intrathecal fraction in some cases over 80% of total CSF MBL concentration 3 The smaller inter-individual variation of MBL concentrations in CSF of the control group (CV = 66% compared to the MBL concentrations in serum (CV = 146% indicate an independent source of MBL in CSF. 4 The absolute MBL concentration in CSF increases with increasing QAlb. Among brain-derived proteins in CSF only the leptomeningeal proteins showed a (linear increase with decreasing CSF flow rate, neuronal and glial proteins are invariant to changes of QAlb. Conclusions MBL in CSF is

  7. Preparation of mixed matrix adsorber membranes for protein recovery

    NARCIS (Netherlands)

    Avramescu, M.E.; Girones nogue, Miriam; Borneman, Zandrie; Wessling, Matthias

    2003-01-01

    This paper presents a generic technology allowing the incorporation of functional entities into a porous substrate. Various ion exchange particles were incorporated into an ethylene vinyl alcohol (EVAL) copolymer porous matrix by an immersion phase separation process and a heterogeneous matrix,

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

    Science.gov (United States)

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

    2013-02-01

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

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

    Science.gov (United States)

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

    2015-06-01

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

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

    Science.gov (United States)

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

    2016-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Hu Jing

    2009-08-01

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

  12. Negligible depletion solid-phase microextraction with radiolabeled analytes to study free concentrations and protein binding : an example with [3H]Estradiol

    NARCIS (Netherlands)

    Heringa, M.B.; Pastor, D.; Algra, J.; Vaes, W.H.J.; Hemmens, J.L.M.

    2002-01-01

    A new method is presented that enables sensitive measurement of free concentrations of radiolabeled ligands. Additionally, protein binding of radiochemicals in complex matrixes can be determined with this new technique that combines negligible depletion solid-phase microextraction (nd-SPME) with

  13. Heparan sulfate proteoglycan from the extracellular matrix of human lung fibroblasts. Isolation, purification, and core protein characterization

    International Nuclear Information System (INIS)

    Heremans, A.; Cassiman, J.J.; Van den Berghe, H.; David, G.

    1988-01-01

    Confluent cultured human lung fibroblasts were labeled with 35SO4(2-). After 48 h of labeling, the pericellular matrix was prepared by Triton X-100 and deoxycholate extraction of the monolayers. Heparan sulfate proteoglycan (HSPG) accounted for nearly 80% of the total matrix [35S]proteoglycans. After solubilization in 6 M guanidinium HCl and cesium chloride density gradient centrifugation, the majority (78%) of these [35S] HSPG equilibrated at an average buoyant density of 1.35 g/ml. This major HSPG fraction was purified by ion-exchange chromatography on Mono Q and by gel filtration on Sepharose CL-4B, and further characterized by gel electrophoresis and immunoblotting. Intact [35S]HSPG eluted with Kav 0.1 from Sepharose CL-4B, whereas the protein-free [35S]heparan sulfate chains, obtained by alkaline borohydride treatment of the proteoglycan fractions, eluted with Kav 0.45 (Mr approximately 72,000). When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, core (protein) preparations, obtained by heparitinase digestion of 125I-labeled HSPG fractions, yielded one major labeled band with apparent molecular mass of approximately 300 kDa. Reduction with beta-mercaptoethanol slightly increased the apparent Mr of the labeled band, suggesting a single polypeptide structure and the presence of intrachain disulfide bonds. Immunoadsorption experiments and immunostaining of electrophoretically separated heparitinase-digested core proteins with monoclonal antibodies raised against matrix and cell surface-associated HSPG suggested that the major matrix-associated HSPG of cultured human lung fibroblasts is distinct from the HSPG that are anchored in the membranes of these cells. Binding studies suggested that this matrix HSPG interacts with several matrix components, both through its glycosaminoglycan chains and through its heparitinase-resistant core. (Abstract Truncated)

  14. The effects of GH and hormone replacement therapy on serum concentrations of mannan-binding lectin, surfactant protein D and vitamin D binding protein in Turner syndrome

    DEFF Research Database (Denmark)

    Gravholt, Claus Højbjerg; Leth-Larsen, Rikke; Lauridsen, Anna Lis

    2004-01-01

    function. In the present study we examined whether GH or hormone replacement therapy (HRT) in Turner syndrome (TS) influence the serum concentrations of MBL and two other proteins partaking in the innate immune defence, surfactant protein D (SP-D) and vitamin D binding protein (DBP). DESIGN: Study 1...

  15. Diffusion and Binding of Laponite Clay Nanoparticles into Collagen Fibers for the Formation of Leather Matrix.

    Science.gov (United States)

    Shi, Jiabo; Wang, Chunhua; Ngai, To; Lin, Wei

    2018-06-13

    Understanding accessibility and interactions of clay nanoparticles with collagen fibers is an important fundamental issue for the conversion of collagen to leather matrix. In this study, we have investigated the diffusion and binding of Laponite into the collagen fiber network. Our results indicate that the diffusion behaviors of Laponite into the collagen exhibit the Langmuir adsorption, verifying its affinity for collagen. The introduction of Laponite leads to a shift in the isoelectric point of collagen from ∼6.8 to ∼4.5, indicating the ionic bonding between the positively charged amino groups of the collagen and negatively charged Laponite under the tanning conditions. Fluorescence microscopy, atomic force microscopy, field-emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, and wide-angle X-ray diffraction analyses reveal that Laponite nanoparticles can penetrate into collagen microstructure and evenly distributed onto collagen fibrils, not altering native D-periodic banding patterns of collagen fibrils. Attenuated total reflectance-Fourier transform infrared and Raman spectroscopy detections further demonstrate the presence of noncovalent interactions, namely, ionic and hydrogen bonding, between Laponite and collagen. These findings provide a theoretical basis for the use of Laponite as an emerging tanning agent in leather manufacture.

  16. Clearance kinetics and matrix binding partners of the receptor for advanced glycation end products.

    Directory of Open Access Journals (Sweden)

    Pavle S Milutinovic

    Full Text Available Elucidating the sites and mechanisms of sRAGE action in the healthy state is vital to better understand the biological importance of the receptor for advanced glycation end products (RAGE. Previous studies in animal models of disease have demonstrated that exogenous sRAGE has an anti-inflammatory effect, which has been reasoned to arise from sequestration of pro-inflammatory ligands away from membrane-bound RAGE isoforms. We show here that sRAGE exhibits in vitro binding with high affinity and reversibly to extracellular matrix components collagen I, collagen IV, and laminin. Soluble RAGE administered intratracheally, intravenously, or intraperitoneally, does not distribute in a specific fashion to any healthy mouse tissue, suggesting against the existence of accessible sRAGE sinks and receptors in the healthy mouse. Intratracheal administration is the only effective means of delivering exogenous sRAGE to the lung, the organ in which RAGE is most highly expressed; clearance of sRAGE from lung does not differ appreciably from that of albumin.

  17. Translation initiation mediated by nuclear cap-binding protein complex.

    Science.gov (United States)

    Ryu, Incheol; Kim, Yoon Ki

    2017-04-01

    In mammals, cap-dependent translation of mRNAs is initiated by two distinct mechanisms: cap-binding complex (CBC; a heterodimer of CBP80 and 20)-dependent translation (CT) and eIF4E-dependent translation (ET). Both translation initiation mechanisms share common features in driving cap- dependent translation; nevertheless, they can be distinguished from each other based on their molecular features and biological roles. CT is largely associated with mRNA surveillance such as nonsense-mediated mRNA decay (NMD), whereas ET is predominantly involved in the bulk of protein synthesis. However, several recent studies have demonstrated that CT and ET have similar roles in protein synthesis and mRNA surveillance. In a subset of mRNAs, CT preferentially drives the cap-dependent translation, as ET does, and ET is responsible for mRNA surveillance, as CT does. In this review, we summarize and compare the molecular features of CT and ET with a focus on the emerging roles of CT in translation. [BMB Reports 2017; 50(4): 186-193].

  18. Is vitamin D binding protein a novel predictor of labour?

    Directory of Open Access Journals (Sweden)

    Stella Liong

    Full Text Available Vitamin D binding protein (VDBP has previously been identified in the amniotic fluid and cervicovaginal fluid (CVF of pregnant women. The biological functions of VDBP include acting as a carrier protein for vitamin D metabolites, the clearance of actin that is released during tissue injury and the augmentation of the pro-inflammatory response. This longitudinal observational study was conducted on 221 healthy pregnant women who spontaneously laboured and delivered either at term or preterm. Serial CVF samples were collected and VDBP was measured by ELISA. Binary logistic regression analysis was performed to assess the utility of VDBP as a predictor of labour. VDBP in the CVF did not change between 20 and 35 weeks' gestation. VDBP measured in-labour was significantly increased 4.2 to 7.4-fold compared to 4-7, 8-14 and 15-28 days before labour (P<0.05. VDBP concentration was 4.3-fold significantly higher at 0-3 days compared to 15-28 days pre-labour (P<0.05. The efficacy of VDBP to predict spontaneous labour onset within 3 days provided a positive and negative predictive value of 82.8% and 95.3% respectively (area under receiver operator characteristic curve  = 0.974. This longitudinal study of pregnant women suggests that VDBP in the CVF may be a useful predictor of labour.

  19. QM/MM Molecular Dynamics Studies of Metal Binding Proteins

    Directory of Open Access Journals (Sweden)

    Pietro Vidossich

    2014-07-01

    Full Text Available 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.

  20. Serum protein inhibition of thyrotropin binding to human thyroid tissue

    International Nuclear Information System (INIS)

    Beall, G.N.; Chopra, I.J.; Solomon, D.H.; Kruger, S.R.

    1978-01-01

    We used a modificaton of the TSH radioreceptor assay to detect TSH-binding inhibition (TBI) activity in serum and serum fractions from normal subjects and patients with Graves' disease. TBI activity is present in normal IgG prepared by DEAE-Sephadex chromatography and in normal globulins prepared by precipitation at 1.6 M ammonium sulfate. Other normal serum proteins also had TBI activity when large concentrations were tested. Gel filtration chromatography and powder block electrophoresis were used to prepare fractions of normal and Graves' disease sera. In these fractions from normal serum, TBI activity was found in both γ-globulin and α-globulin-albumin fractions electrophoretically and in both 7S and 4S peaks from gel filtration. TBI activity from Graves' disease patients' sera was similarly distributed, but relatively more TBI accompanied the electrophoretic γ-globulins. Sepharose Protein-A and anti-IgG were used as immunoabsorbents to isolate and purify IgG from normal and Graves' disease sera. TBI activity in IgG was proportional to the IgG concentration, indicating that the TBI which migrates as a γ-globulin electrophoretically is an IgG and thus may possibly be an antibody. Inhibitory activity found in normal serum globulins and in the non-IgG fractions of both normal and abnormal sera seriously interferes with attempts to use the TSH radioreceptor assay to study the hypothesized anti-TSH receptor antibody in the serum of patients with Graves' disease

  1. Split green fluorescent protein as a modular binding partner for protein crystallization

    International Nuclear Information System (INIS)

    Nguyen, Hau B.; Hung, Li-Wei; Yeates, Todd O.; Terwilliger, Thomas C.; Waldo, Geoffrey S.

    2013-01-01

    A strategy using a new split green fluorescent protein (GFP) as a modular binding partner to form stable protein complexes with a target protein is presented. The modular split GFP may open the way to rapidly creating crystallization variants. A modular strategy for protein crystallization using split green fluorescent protein (GFP) as a crystallization partner is demonstrated. Insertion of a hairpin containing GFP β-strands 10 and 11 into a surface loop of a target protein provides two chain crossings between the target and the reconstituted GFP compared with the single connection afforded by terminal GFP fusions. This strategy was tested by inserting this hairpin into a loop of another fluorescent protein, sfCherry. The crystal structure of the sfCherry-GFP(10–11) hairpin in complex with GFP(1–9) was determined at a resolution of 2.6 Å. Analysis of the complex shows that the reconstituted GFP is attached to the target protein (sfCherry) in a structurally ordered way. This work opens the way to rapidly creating crystallization variants by reconstituting a target protein bearing the GFP(10–11) hairpin with a variety of GFP(1–9) mutants engineered for favorable crystallization

  2. Identification of pheromone components and their binding affinity to the odorant binding protein CcapOBP83a-2 of the Mediterranean fruit fly, Ceratitis capitata

    Czech Academy of Sciences Publication Activity Database

    Siciliano, P.; He, X. L.; Woodcock, C.; Pickett, J. A.; Field, L. M.; Birkett, M. A.; Kalinová, Blanka; Gomulski, L. M.; Scolari, F.; Gasperi, G.; Malacrida, A. R.; Zhou, J. J.

    2014-01-01

    Roč. 48, May (2014), s. 51-62 ISSN 0965-1748 Institutional support: RVO:61388963 Keywords : medfly * Ceratitis capitata * olfaction * odorant binding protein * pheromone binding protein * pheromone * binding studies * protein expression * electroantennography * GC-EAG * fluorescence displacement Subject RIV: CE - Biochemistry Impact factor: 3.450, year: 2014

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

    Science.gov (United States)

    Messina, Troy; Talaga, David

    2006-03-01

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

  4. GenProBiS: web server for mapping of sequence variants to protein binding sites.

    Science.gov (United States)

    Konc, Janez; Skrlj, Blaz; Erzen, Nika; Kunej, Tanja; Janezic, Dusanka

    2017-07-03

    Discovery of potentially deleterious sequence variants is important and has wide implications for research and generation of new hypotheses in human and veterinary medicine, and drug discovery. The GenProBiS web server maps sequence variants to protein structures from the Protein Data Bank (PDB), and further to protein-protein, protein-nucleic acid, protein-compound, and protein-metal ion binding sites. The concept of a protein-compound binding site is understood in the broadest sense, which includes glycosylation and other post-translational modification sites. Binding sites were defined by local structural comparisons of whole protein structures using the Protein Binding Sites (ProBiS) algorithm and transposition of ligands from the similar binding sites found to the query protein using the ProBiS-ligands approach with new improvements introduced in GenProBiS. Binding site surfaces were generated as three-dimensional grids encompassing the space occupied by predicted ligands. The server allows intuitive visual exploration of comprehensively mapped variants, such as human somatic mis-sense mutations related to cancer and non-synonymous single nucleotide polymorphisms from 21 species, within the predicted binding sites regions for about 80 000 PDB protein structures using fast WebGL graphics. The GenProBiS web server is open and free to all users at http://genprobis.insilab.org. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. Interaction of glutaric aciduria type 1-related glutaryl-CoA dehydrogenase with mitochondrial matrix proteins.

    Directory of Open Access Journals (Sweden)

    Jessica Schmiesing

    Full Text Available Glutaric aciduria type 1 (GA1 is an inherited neurometabolic disorder caused by mutations in the GCDH gene encoding glutaryl-CoA dehydrogenase (GCDH, which forms homo- and heteromeric complexes in the mitochondrial matrix. GA1 patients are prone to the development of encephalopathic crises which lead to an irreversible disabling dystonic movement disorder. The clinical and biochemical manifestations of GA1 vary considerably and lack correlations to the genotype. Using an affinity chromatography approach we report here for the first time on the identification of mitochondrial proteins interacting directly with GCDH. Among others, dihydrolipoamide S-succinyltransferase (DLST involved in the formation of glutaryl-CoA, and the β-subunit of the electron transfer flavoprotein (ETFB serving as electron acceptor, were identified as GCDH binding partners. We have adapted the yellow fluorescent protein-based fragment complementation assay and visualized the oligomerization of GCDH as well as its direct interaction with DLST and ETFB in mitochondria of living cells. These data suggest that GCDH is a constituent of multimeric mitochondrial dehydrogenase complexes, and the characterization of their interrelated functions may provide new insights into the regulation of lysine oxidation and the pathophysiology of GA1.

  6. Transcriptional factor DLX3 promotes the gene expression of enamel matrix proteins during amelogenesis.

    Science.gov (United States)

    Zhang, Zhichun; Tian, Hua; Lv, Ping; Wang, Weiping; Jia, Zhuqing; Wang, Sainan; Zhou, Chunyan; Gao, Xuejun

    2015-01-01

    Mutation of distal-less homeobox 3 (DLX3) is responsible for human tricho-dento-osseous syndrome (TDO) with amelogenesis imperfecta, indicating a crucial role of DLX3 in amelogenesis. However, the expression pattern of DLX3 and its specific function in amelogenesis remain largely unknown. The aim of this study was to investigate the effects of DLX3 on enamel matrix protein (EMP) genes. By immunohistochemistry assays of mouse tooth germs, stronger immunostaining of DLX3 protein was identified in ameloblasts in the secretory stage than in the pre-secretory and maturation stages, and the same pattern was found for Dlx3 mRNA using Realtime PCR. In a mouse ameloblast cell lineage, forced expression of DLX3 up-regulated the expression of the EMP genes Amelx, Enam, Klk4, and Odam, whereas knockdown of DLX3 down-regulated these four EMP genes. Further, bioinformatics, chromatin immunoprecipitation, and luciferase assays revealed that DLX3 transactivated Enam, Amelx, and Odam through direct binding to their enhancer regions. Particularly, over-expression of mutant-DLX3 (c.571_574delGGGG, responsible for TDO) inhibited the activation function of DLX3 on expression levels and promoter activities of the Enam, Amelx, and Odam genes. Together, our data show that DLX3 promotes the expression of the EMP genes Amelx, Enam, Klk4, and Odam in amelogenesis, while mutant-DLX3 disrupts this regulatory function, thus providing insights into the molecular mechanisms underlying the enamel defects of TDO disease.

  7. Smad3 binds Scleraxis and Mohawk and regulates tendon matrix organization.

    Science.gov (United States)

    Berthet, Ellora; Chen, Carol; Butcher, Kristin; Schneider, Richard A; Alliston, Tamara; Amirtharajah, Mohana

    2013-09-01

    TGFβ plays a critical role in tendon formation and healing. While its downstream effector Smad3 has been implicated in the healing process, little is known about the role of Smad3 in normal tendon development or tenocyte gene expression. Using mice deficient in Smad3 (Smad3(-/-) ), we show that Smad3 ablation disrupts tendon architecture and has a dramatic impact on normal gene and protein expression during development as well as in mature tendon. In developing and adult tendon, loss of Smad3 results in reduced protein expression of the matrix components Collagen 1 and Tenascin-C. Additionally, when compared to wild type, tendon from adult Smad3(-/-) mice shows a down regulation of key tendon marker genes. Finally, we have established that Smad3 has the ability to physically interact with the critical transcriptional regulators Scleraxis and Mohawk. Together these results indicate a central role for Smad3 in normal tendon formation and in the maintenance of mature tendon. Copyright © 2013 Orthopaedic Research Society.

  8. Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides

    Directory of Open Access Journals (Sweden)

    Weonu Choe

    2016-12-01

    Full Text Available The rapidly increasing application of antibodies has inspired the development of several novel methods to isolate and target antibodies using smart biomaterials that mimic the binding of Fc-receptors to antibodies. The Fc-binding domain of antibodies is the primary binding site for e.g., effector proteins and secondary antibodies, whereas antigens bind to the Fab region. Protein A, G, and L, surface proteins expressed by pathogenic bacteria, are well known to bind immunoglobulin and have been widely exploited in antibody purification strategies. Several difficulties are encountered when bacterial proteins are used in antibody research and application. One of the major obstacles hampering the use of bacterial proteins is sample contamination with trace amounts of these proteins, which can invoke an immune response in the host. Many research groups actively develop synthetic ligands that are able to selectively and strongly bind to antibodies. Among the reported ligands, peptides that bind to the Fc-domain of antibodies are attractive tools in antibody research. Besides their use as high affinity ligands in antibody purification chromatography, Fc-binding peptides are applied e.g., to localize antibodies on nanomaterials and to increase the half-life of proteins in serum. In this review, recent developments of Fc-binding peptides are presented and their binding characteristics and diverse applications are discussed.

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

    Science.gov (United States)

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

    1988-11-25

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

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

    Directory of Open Access Journals (Sweden)

    Wan Raihana Wan Aasim

    2017-01-01

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

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

    Directory of Open Access Journals (Sweden)

    TIJANA SAVIC

    2006-02-01

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

  12. Cost Function Network-based Design of Protein-Protein Interactions: predicting changes in binding affinity.

    Science.gov (United States)

    Viricel, Clément; de Givry, Simon; Schiex, Thomas; Barbe, Sophie

    2018-02-20

    Accurate and economic methods to predict change in protein binding free energy upon mutation are imperative to accelerate the design of proteins for a wide range of applications. Free energy is defined by enthalpic and entropic contributions. Following the recent progresses of Artificial Intelligence-based algorithms for guaranteed NP-hard energy optimization and partition function computation, it becomes possible to quickly compute minimum energy conformations and to reliably estimate the entropic contribution of side-chains in the change of free energy of large protein interfaces. Using guaranteed Cost Function Network algorithms, Rosetta energy functions and Dunbrack's rotamer library, we developed and assessed EasyE and JayZ, two methods for binding affinity estimation that ignore or include conformational entropic contributions on a large benchmark of binding affinity experimental measures. If both approaches outperform most established tools, we observe that side-chain conformational entropy brings little or no improvement on most systems but becomes crucial in some rare cases. as open-source Python/C ++ code at sourcesup.renater.fr/projects/easy-jayz. thomas.schiex@inra.fr and sophie.barbe@insa-toulouse.fr. Supplementary data are available at Bioinformatics online.

  13. AHM1, a Novel Type of Nuclear Matrix–Localized, MAR Binding Protein with a Single AT Hook and a J Domain–Homologous Region

    Science.gov (United States)

    Morisawa, Gaku; Han-yama, Atsushi; Moda, Ichiro; Tamai, Atsushi; Iwabuchi, Masaki; Meshi, Tetsuo

    2000-01-01

    Interactions between the nuclear matrix and special regions of chromosomal DNA called matrix attachment regions (MARs) have been implicated in various nuclear functions. We have identified a novel protein from wheat, AT hook–containing MAR binding protein1 (AHM1), that binds preferentially to MARs. A multidomain protein, AHM1 has the special combination of a J domain–homologous region and a Zn finger–like motif (a J-Z array) and an AT hook. For MAR binding, the AT hook at the C terminus was essential, and an internal portion containing the Zn finger–like motif was additionally required in vivo. AHM1 was found in the nuclear matrix fraction and was localized in the nucleoplasm. AHM1 fused to green fluorescent protein had a speckled distribution pattern inside the nucleus. AHM1 is most likely a nuclear matrix component that functions between intranuclear framework and MARs. J-Z arrays can be found in a group of (hypothetical) proteins in plants, which may share some functions, presumably to recruit specific Hsp70 partners as co-chaperones. PMID:11041885

  14. Prediction of MHC class II binding affinity using SMM-align, a novel stabilization matrix alignment method

    DEFF Research Database (Denmark)

    Nielsen, Morten; Lundegaard, Claus; Lund, Ole

    2007-01-01

    the correct alignment of a peptide in the binding groove a crucial part of identifying the core of an MHC class II binding motif. Here, we present a novel stabilization matrix alignment method, SMM-align, that allows for direct prediction of peptide:MHC binding affinities. The predictive performance...... of the method is validated on a large MHC class II benchmark data set covering 14 HLA-DR (human MHC) and three mouse H2-IA alleles. RESULTS: The predictive performance of the SMM-align method was demonstrated to be superior to that of the Gibbs sampler, TEPITOPE, SVRMHC, and MHCpred methods. Cross validation...... between peptide data set obtained from different sources demonstrated that direct incorporation of peptide length potentially results in over-fitting of the binding prediction method. Focusing on amino terminal peptide flanking residues (PFR), we demonstrate a consistent gain in predictive performance...

  15. Interactions of rat repetitive sequence MspI8 with nuclear matrix proteins during spermatogenesis

    International Nuclear Information System (INIS)

    Rogolinski, J.; Widlak, P.; Rzeszowska-Wolny, J.

    1996-01-01

    Using the Southwestern blot analysis we have studied the interactions between rat repetitive sequence MspI8 and the nuclear matrix proteins of rats testis cells. Starting from 2 weeks the young to adult animal showed differences in type of testis nuclear matrix proteins recognizing the MspI8 sequence. The same sets of nuclear matrix proteins were detected in some enriched in spermatocytes and spermatids and obtained after fractionation of cells of adult animal by the velocity sedimentation technique. (author). 21 refs, 5 figs

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

    International Nuclear Information System (INIS)

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

    2009-01-01

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

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

    International Nuclear Information System (INIS)

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

    1989-01-01

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

  18. Decorin core protein (decoron) shape complements collagen fibril surface structure and mediates its binding.

    Science.gov (United States)

    Orgel, Joseph P R O; Eid, Aya; Antipova, Olga; Bella, Jordi; Scott, John E

    2009-09-15

    Decorin is the archetypal small leucine rich repeat proteoglycan of the vertebrate extracellular matrix (ECM). With its glycosaminoglycuronan chain, it is responsible for stabilizing inter-fibrillar organization. Type I collagen is the predominant member of the fibrillar collagen family, fulfilling both organizational and structural roles in animal ECMs. In this study, interactions between decoron (the decorin core protein) and binding sites in the d and e(1) bands of the type I collagen fibril were investigated through molecular modeling of their respective X-ray diffraction structures. Previously, it was proposed that a model-based, highly curved concave decoron interacts with a single collagen molecule, which would form extensive van der Waals contacts and give rise to strong non-specific binding. However, the large well-ordered aggregate that is the collagen fibril places significant restraints on modes of ligand binding and necessitates multi-collagen molecular contacts. We present here a relatively high-resolution model of the decoron-fibril collagen complex. We find that the respective crystal structures complement each other well, although it is the monomeric form of decoron that shows the most appropriate shape complementarity with the fibril surface and favorable calculated energies of interaction. One molecule of decoron interacts with four to six collagen molecules, and the binding specificity relies on a large number of hydrogen bonds and electrostatic interactions, primarily with the collagen motifs KXGDRGE and AKGDRGE (d and e(1) bands). This work helps us to understand collagen-decorin interactions and the molecular architecture of the fibrillar ECM in health and disease.

  19. Decorin core protein (decoron shape complements collagen fibril surface structure and mediates its binding.

    Directory of Open Access Journals (Sweden)

    Joseph P R O Orgel

    2009-09-01

    Full Text Available Decorin is the archetypal small leucine rich repeat proteoglycan of the vertebrate extracellular matrix (ECM. With its glycosaminoglycuronan chain, it is responsible for stabilizing inter-fibrillar organization. Type I collagen is the predominant member of the fibrillar collagen family, fulfilling both organizational and structural roles in animal ECMs. In this study, interactions between decoron (the decorin core protein and binding sites in the d and e(1 bands of the type I collagen fibril were investigated through molecular modeling of their respective X-ray diffraction structures. Previously, it was proposed that a model-based, highly curved concave decoron interacts with a single collagen molecule, which would form extensive van der Waals contacts and give rise to strong non-specific binding. However, the large well-ordered aggregate that is the collagen fibril places significant restraints on modes of ligand binding and necessitates multi-collagen molecular contacts. We present here a relatively high-resolution model of the decoron-fibril collagen complex. We find that the respective crystal structures complement each other well, although it is the monomeric form of decoron that shows the most appropriate shape complementarity with the fibril surface and favorable calculated energies of interaction. One molecule of decoron interacts with four to six collagen molecules, and the binding specificity relies on a large number of hydrogen bonds and electrostatic interactions, primarily with the collagen motifs KXGDRGE and AKGDRGE (d and e(1 bands. This work helps us to understand collagen-decorin interactions and the molecular architecture of the fibrillar ECM in health and disease.

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

    Directory of Open Access Journals (Sweden)

    Sarai Akinori

    2011-02-01

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

  1. Standardization for cortisol determination in human blood by competitive protein-binding

    International Nuclear Information System (INIS)

    Okada, H.

    1978-01-01

    Standardization for determination of cortisol from human plasma (17-hydroxycorticosteroids) using competitive protein-binding method is presented. Activated carbon coated with dextrans is used for separation of the hormone-protein complexe and hormone labelled free [pt

  2. Proteome scale identification, classification and structural analysis of iron-binding proteins in bread wheat.

    Science.gov (United States)

    Verma, Shailender Kumar; Sharma, Ankita; Sandhu, Padmani; Choudhary, Neha; Sharma, Shailaja; Acharya, Vishal; Akhter, Yusuf

    2017-05-01

    Bread wheat is one of the major staple foods of worldwide population and iron plays a significant role in growth and development of the plant. In this report, we are presenting the genome wide identification of iron-binding proteins in bread wheat. The wheat genome derived putative proteome was screened for identification of iron-binding sequence motifs. Out of 602 putative iron-binding proteins, 130 were able to produce reliable structural models by homology techniques and further analyzed for the presence of iron-binding structural motifs. The computationally identified proteins appear to bind to ferrous and ferric ions and showed diverse coordination geometries. Glu, His, Asp and Cys amino acid residues were found to be mostly involved in iron binding. We have classified these proteins on the basis of their localization in the different cellular compartments. The identified proteins were further classified into their protein folds, families and functional classes ranging from structure maintenance of cellular components, regulation of gene expression, post translational modification, membrane proteins, enzymes, signaling and storage proteins. This comprehensive report regarding structural iron binding proteome provides useful insights into the diversity of iron binding proteins of wheat plants and further utilized to study their roles in plant growth, development and physiology. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Coating magnesium hydroxide on surface of carbon microspheres and interface binding with poly (ethylene terephthalate) matrix

    Energy Technology Data Exchange (ETDEWEB)

    Xue, Baoxia [Key Laboratory of Interface Science and Engineering in Advanced Materials, Taiyuan University of Technology, Ministry of Education, Taiyuan 030024 (China); Research Center on Advanced Materials Science and Technology, Taiyuan University of Technology, Taiyuan 030024 (China); College of Textile Engineering, Taiyuan University of Technology, Yuci 030600 (China); Niu, Mei, E-mail: niumei@tyut.edu.cn [Key Laboratory of Interface Science and Engineering in Advanced Materials, Taiyuan University of Technology, Ministry of Education, Taiyuan 030024 (China); College of Textile Engineering, Taiyuan University of Technology, Yuci 030600 (China); Yang, Yongzhen, E-mail: yyztyut@126.com [Key Laboratory of Interface Science and Engineering in Advanced Materials, Taiyuan University of Technology, Ministry of Education, Taiyuan 030024 (China); Research Center on Advanced Materials Science and Technology, Taiyuan University of Technology, Taiyuan 030024 (China); Bai, Jie; Song, Yinghao; Peng, Yun [College of Textile Engineering, Taiyuan University of Technology, Yuci 030600 (China); Liu, Xuguang [Key Laboratory of Interface Science and Engineering in Advanced Materials, Taiyuan University of Technology, Ministry of Education, Taiyuan 030024 (China); College of Chemistry and Chemical Engineering, Taiyuan University of Technology, Taiyuan 030024 (China)

    2017-08-01

    Highlights: • Magnesium hydroxide (MH) as a capsule wall was firstly coated on the surface of carbon microspheres (CMSs) to obtain MH@CMSs by liquid phase deposition method. • An organic layer of 3-Aminopropyltriethoxysilane (APTS) was then introduced on the surface of MH@CMSs. • The formed two layers provided the FMH@CMSs/PET with good mechanical and flame-retardant properties. - Abstract: In this account, magnesium hydroxide (MH) employed as a capsule wall was firstly coated on the surface of carbon microspheres (CMSs) to obtain MH@CMSs using liquid phase deposition, then was modified by 3-Aminopropyltriethoxysilane (APTS) to form FMH@CMSs. To investigate the interface binding forces, a series of PET composites was prepared by melt compounding with MH@CMSs or FMH@CMSs. Field-emission scanning electron microscopy (FESEM), Transmission electron microscopy (TEM), Fourier-transform Infrared spectrometry (FTIR) and X-ray photoelectron spectroscopy (XPS) were used to characterize the morphology, chemical structure, and effect of functionalization of CMSs. The coating degree and thermal stability were investigated by thermogravimetry analysis. The results showed that CMSs were coated by an inorganic shell layer of MH as a capsule wall. On the other hand, MH@CMSs were coated with an organic layer of APTS. When compared to MH@CMSs, the interface binding forces between FMH@CMSs and PET matrix were significantly improved, and the tensile strength of FMH@CMSs/PET was higher than that of MH@CMSs/PET. At 1 wt% mass fraction of FMH@CMSs, the limiting oxygen index (LOI) value of PET composites increased from 21% to 27.6% following a V-0 rating. The tensile strength of FMH@CMSs/PET increased by 66.2% to reach 47.20 MPa, a value nearly similar to that of PET. Overall, the formed two layers provided the FMH@CMSs/PET with good mechanical and flame-retardant properties, which would broaden their scope of application.

  4. Extreme sequence divergence but conserved ligand-binding specificity in Streptococcus pyogenes M protein.

    Directory of Open Access Journals (Sweden)

    2006-05-01

    Full Text Available Many pathogenic microorganisms evade host immunity through extensive sequence variability in a protein region targeted by protective antibodies. In spite of the sequence variability, a variable region commonly retains an important ligand-binding function, reflected in the presence of a highly conserved sequence motif. Here, we analyze the limits of sequence divergence in a ligand-binding region by characterizing the hypervariable region (HVR of Streptococcus pyogenes M protein. Our studies were focused on HVRs that bind the human complement regulator C4b-binding protein (C4BP, a ligand that confers phagocytosis resistance. A previous comparison of C4BP-binding HVRs identified residue identities that could be part of a binding motif, but the extended analysis reported here shows that no residue identities remain when additional C4BP-binding HVRs are included. Characterization of the HVR in the M22 protein indicated that two relatively conserved Leu residues are essential for C4BP binding, but these residues are probably core residues in a coiled-coil, implying that they do not directly contribute to binding. In contrast, substitution of either of two relatively conserved Glu residues, predicted to be solvent-exposed, had no effect on C4BP binding, although each of these changes had a major effect on the antigenic properties of the HVR. Together, these findings show that HVRs of M proteins have an extraordinary capacity for sequence divergence and antigenic variability while retaining a specific ligand-binding function.

  5. Predicting Flavin and Nicotinamide Adenine Dinucleotide-Binding Sites in Proteins Using the Fragment Transformation Method

    Directory of Open Access Journals (Sweden)

    Chih-Hao Lu

    2015-01-01

    Full Text Available We developed a computational method to identify NAD- and FAD-binding sites in proteins. First, we extracted from the Protein Data Bank structures of proteins that bind to at least one of these ligands. NAD-/FAD-binding residue templates were then constructed by identifying binding residues through the ligand-binding database BioLiP. The fragment transformation method was used to identify structures within query proteins that resembled the ligand-binding templates. By comparing residue types and their relative spatial positions, potential binding sites were identified and a ligand-binding potential for each residue was calculated. Setting the false positive rate at 5%, our method predicted NAD- and FAD-binding sites at true positive rates of 67.1% and 68.4%, respectively. Our method provides excellent results for identifying FAD- and NAD-binding sites in proteins, and the most important is that the requirement of conservation of residue types and local structures in the FAD- and NAD-binding sites can be verified.

  6. Combined role of type IX collagen and cartilage oligomeric matrix protein in cartilage matrix assembly: Cartilage oligomeric matrix protein counteracts type IX collagen-induced limitation of cartilage collagen fibril growth in mouse chondrocyte cultures

    NARCIS (Netherlands)

    Blumbach, K.; Bastiaansen-Jenniskens, Y.M.; Groot, J. de; Paulsson, M.; Osch, G.J.V.M. van; Zaucke, F.

    2009-01-01

    Objective. Defects in the assembly and composition of cartilage extracellular matrix are likely to result in impaired matrix integrity and increased susceptibility to cartilage degeneration. The aim of this study was to determine the functional interaction of the collagen fibril-associated proteins

  7. Photorhabdus adhesion modification protein (Pam) binds extracellular polysaccharide and alters bacterial attachment.

    Science.gov (United States)

    Jones, Robert T; Sanchez-Contreras, Maria; Vlisidou, Isabella; Amos, Matthew R; Yang, Guowei; Muñoz-Berbel, Xavier; Upadhyay, Abhishek; Potter, Ursula J; Joyce, Susan A; Ciche, Todd A; Jenkins, A Toby A; Bagby, Stefan; Ffrench-Constant, Richard H; Waterfield, Nicholas R

    2010-05-12

    Photorhabdus are Gram-negative nematode-symbiotic and insect-pathogenic bacteria. The species Photorhabdus asymbiotica is able to infect humans as well as insects. We investigated the secreted proteome of a clinical isolate of P. asymbiotica at different temperatures in order to identify proteins relevant to the infection of the two different hosts. A comparison of the proteins secreted by a clinical isolate of P. asymbiotica at simulated insect (28 degrees C) and human (37 degrees C) temperatures led to the identification of a small and highly abundant protein, designated Pam, that is only secreted at the lower temperature. The pam gene is present in all Photorhabdus strains tested and shows a high level of conservation across the whole genus, suggesting it is both ancestral to the genus and probably important to the biology of the bacterium. The Pam protein shows limited sequence similarity to the 13.6 kDa component of a binary toxin of Bacillus thuringiensis. Nevertheless, injection or feeding of heterologously produced Pam showed no insecticidal activity to either Galleria mellonella or Manduca sexta larvae. In bacterial colonies, Pam is associated with an extracellular polysaccharide (EPS)-like matrix, and modifies the ability of wild-type cells to attach to an artificial surface. Interestingly, Surface Plasmon Resonance (SPR) binding studies revealed that the Pam protein itself has adhesive properties. Although Pam is produced throughout insect infection, genetic knockout does not affect either insect virulence or the ability of P. luminescens to form a symbiotic association with its host nematode, Heterorhabditis bacteriophora. We studied a highly abundant protein, Pam, which is secreted in a temperature-dependent manner in P. asymbiotica. Our findings indicate that Pam plays an important role in enhancing surface attachment in insect blood. Its association with exopolysaccharide suggests it may exert its effect through mediation of EPS properties. Despite

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

    OpenAIRE

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

    2015-01-01

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

  9. Recombinant fusion protein of albumin-retinol binding protein inactivates stellate cells

    International Nuclear Information System (INIS)

    Choi, Soyoung; Park, Sangeun; Kim, Suhyun; Lim, Chaeseung; Kim, Jungho; Cha, Dae Ryong; Oh, Junseo

    2012-01-01

    Highlights: ► We designed novel recombinant albumin-RBP fusion proteins. ► Expression of fusion proteins inactivates pancreatic stellate cells (PSCs). ► Fusion proteins are successfully internalized into and inactivate PSCs. ► RBP moiety mediates cell specific uptake of fusion protein. -- Abstract: Quiescent pancreatic- (PSCs) and hepatic- (HSCs) stellate cells store vitamin A (retinol) in lipid droplets via retinol binding protein (RBP) receptor and, when activated by profibrogenic stimuli, they transform into myofibroblast-like cells which play a key role in the fibrogenesis. Despite extensive investigations, there is, however, currently no appropriate therapy available for tissue fibrosis. We previously showed that the expression of albumin, composed of three homologous domains (I–III), inhibits stellate cell activation, which requires its high-affinity fatty acid-binding sites asymmetrically distributed in domain I and III. To attain stellate cell-specific uptake, albumin (domain I/III) was coupled to RBP; RBP-albumin domain III (R-III) and albumin domain I -RBP-albumin III (I-R-III). To assess the biological activity of fusion proteins, cultured PSCs were used. Like wild type albumin, expression of R-III or I-R-III in PSCs after passage 2 (activated PSCs) induced phenotypic reversal from activated to fat-storing cells. On the other hand, R-III and I-R-III, but not albumin, secreted from transfected 293 cells were successfully internalized into and inactivated PSCs. FPLC-purified R-III was found to be internalized into PSCs via caveolae-mediated endocytosis, and its efficient cellular uptake was also observed in HSCs and podocytes among several cell lines tested. Moreover, tissue distribution of intravenously injected R-III was closely similar to that of RBP. Therefore, our data suggest that albumin-RBP fusion protein comprises of stellate cell inactivation-inducing moiety and targeting moiety, which may lead to the development of effective anti

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

    Science.gov (United States)

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

    2015-01-01

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

  11. Spectroscopic analysis on the binding interaction between tetracycline hydrochloride and bovine proteins β-casein, α-lactalbumin

    International Nuclear Information System (INIS)

    Bi, Hongna; Tang, Lin; Gao, Xin; Jia, Jingjing; Lv, Henghui

    2016-01-01

    We investigated the binding interaction between tetracycline hydrochloride (TCH) and bovine proteins β-casein (β-CN), α-lactalbumin (α-LA) in aqueous solution by multi-spectroscopic methods and molecular modeling techniques. Fluorescence and time-resolved fluorescence showed that TCH effectively quenched the intrinsic fluorescence of bovine proteins via static quenching, while there was a single class of binding site on protein. Thermodynamic parameters revealed that electrostatic forces played major roles in the interaction between β-CN and TCH, whereas α-LA-TCH complex were stabilized by hydrogen bonds and van der Waals forces. Moreover, circular dichroism spectra (CD spectra), ultraviolet visible absorption spectra (UV–vis absorption spectra), and fluorescence Excitation-Emission Matrix (EEM) spectra results indicated the secondary structure of bovine proteins was changed in the presence of TCH with the α-helix percentage of protein-TCH complexes decreased. Molecular modeling analysis supported the experimental results well. In addition, the research of surface hydrophobicity further verified tertiary structure of proteins was changed in the presence of TCH and the possible changes of protein function. These results achieved from experiments may be valuable in the milk industry and food safety.

  12. Spectroscopic analysis on the binding interaction between tetracycline hydrochloride and bovine proteins β-casein, α-lactalbumin

    Energy Technology Data Exchange (ETDEWEB)

    Bi, Hongna; Tang, Lin, E-mail: tanglin@sdnu.edu.cn; Gao, Xin; Jia, Jingjing; Lv, Henghui

    2016-10-15

    We investigated the binding interaction between tetracycline hydrochloride (TCH) and bovine proteins β-casein (β-CN), α-lactalbumin (α-LA) in aqueous solution by multi-spectroscopic methods and molecular modeling techniques. Fluorescence and time-resolved fluorescence showed that TCH effectively quenched the intrinsic fluorescence of bovine proteins via static quenching, while there was a single class of binding site on protein. Thermodynamic parameters revealed that electrostatic forces played major roles in the interaction between β-CN and TCH, whereas α-LA-TCH complex were stabilized by hydrogen bonds and van der Waals forces. Moreover, circular dichroism spectra (CD spectra), ultraviolet visible absorption spectra (UV–vis absorption spectra), and fluorescence Excitation-Emission Matrix (EEM) spectra results indicated the secondary structure of bovine proteins was changed in the presence of TCH with the α-helix percentage of protein-TCH complexes decreased. Molecular modeling analysis supported the experimental results well. In addition, the research of surface hydrophobicity further verified tertiary structure of proteins was changed in the presence of TCH and the possible changes of protein function. These results achieved from experiments may be valuable in the milk industry and food safety.

  13. Competitive protein binding analysis for thyroxine using Sephadex column (Tetralute)

    International Nuclear Information System (INIS)

    Miyai, Kiyoshi; Katayama, Yoshiaki; Sawazaki, Norio; Ishibashi, Kaichiro; Kawashima, Minoru.

    1975-01-01

    The method of competitive protein binding analysis of thyroxine (T 4 ) using Tetralute kit was evaluated. The net retention was decreased when the procedure of competition and separation was performed at a higher temperature but the final T 4 -I values were constant when the standard and test sera were treated identically. Coefficient of variation (C.V.) was 4% (within-assay) and 6% (between-assay) respectively. However, the T 4 -I values of pooled serum for quality control were slightly lower in earlier experiments in which correction factors (1.03--1.62 in 18 out of 21 assays) were necessary. T 4 -I values were determined by the Tetralute in 155 cases. They were as follows: 4.9+-0.8 μg/dl (euthyroid subjects), 6.4+-1.2 μg/dl (cord serum), 7.1+-1.1 μg/dl (pregnant women). 9.0+-3.6 μg/dl (trophoblastic disease), 13.3+-4.8 μg/dl (Graves' disease), 6.3+-1.6 μg/dl (Plummer's disease), 4 -I values determined by Tetralute and Res-O-Mat T 4 (r=0.96). Following oral administration of Telepaque the serum protein-bound iodine was markedly elevated, while the T 4 -I determined by Tetralute did not change. In vitro addition of diphenylhydantoin (500 μg/ml), salicylate (4 mg/ml) and phenobarbital (1 mg/ml) had no or little effect on T 4 determination by Tetralute. A high concentration of benzbromarone (0.1 mg/ml) caused a higher value of T 4 -I determined by Tetralute when added to a TBG solution but there was only a slight increase when it was added to serum. (auth.)

  14. RNAcontext: a new method for learning the sequence and structure binding preferences of RNA-binding proteins.

    Directory of Open Access Journals (Sweden)

    Hilal Kazan

    2010-07-01

    Full Text Available Metazoan genomes encode hundreds of RNA-binding proteins (RBPs. These proteins regulate post-transcriptional gene expression and have critical roles in numerous cellular processes including mRNA splicing, export, stability and translation. Despite their ubiquity and importance, the binding preferences for most RBPs are not well characterized. In vitro and in vivo studies, using affinity selection-based approaches, have successfully identified RNA sequence associated with specific RBPs; however, it is difficult to infer RBP sequence and structural preferences without specifically designed motif finding methods. In this study, we introduce a new motif-finding method, RNAcontext, designed to elucidate RBP-specific sequence and structural preferences with greater accuracy than existing approaches. We evaluated RNAcontext on recently published in vitro and in vivo RNA affinity selected data and demonstrate that RNAcontext identifies known binding preferences for several control proteins including HuR, PTB, and Vts1p and predicts new RNA structure preferences for SF2/ASF, RBM4, FUSIP1 and SLM2. The predicted preferences for SF2/ASF are consistent with its recently reported in vivo binding sites. RNAcontext is an accurate and efficient motif finding method ideally suited for using large-scale RNA-binding affinity datasets to determine the relative binding preferences of RBPs for a wide range of RNA sequences and structures.

  15. Tetrodotoxin- and tributyltin-binding abilities of recombinant pufferfish saxitoxin and tetrodotoxin binding proteins of Takifugu rubripes.

    Science.gov (United States)

    Satone, Hina; Nonaka, Shohei; Lee, Jae Man; Shimasaki, Yohei; Kusakabe, Takahiro; Kawabata, Shun-Ichiro; Oshima, Yuji

    2017-01-01

    We investigated the ability of recombinant pufferfish saxitoxin and tetrodotoxin binding protein types 1 and 2 of Takifugu rubripes (rTrub.PSTBP1 and rTrub.PSTBP2) to bind to tetrodotoxin (TTX) and tributyltin. Both rTrub.PSTBPs bound to tributyltin in an ultrafiltration binding assay but lost this ability on heat denaturation. In contrast, only rTrub.PSTBP2 bound to TTX even heat denaturation. This result suggests that the amino acid sequence of PSTBP2 may be contributed for its affinity for TTX. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Crystal Structures and Binding Dynamics of Odorant-Binding Protein 3 from two aphid species Megoura viciae and Nasonovia ribisnigri.

    Science.gov (United States)

    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

    2016-04-22

    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.

  17. Matrix proteins as centralized organizers of negative-sense RNA virions.

    Science.gov (United States)

    Liljeroos, Lassi; Butcher, Sarah J

    2013-01-01

    Matrix proteins are essential components of most negative-sense RNA, enveloped viruses. They serve a wide range of duties ranging from self-driven membrane budding and coordination of other viral components to modulation of viral transcription. The functional similarity between these proteins is striking, despite major differences in their structures. Whereas biochemical and structural studies have partly been hindered by the inherent aggregation properties of these proteins, their cellular functions are beginning to be understood. In this review we summarize the current knowledge on negative-sense RNA virus matrix proteins and their interactions with other viral and cellular proteins. We also discuss the similarities and differences in matrix protein functions between the different families within the negative-sense RNA viruses.

  18. Cholesterol Promotes Protein Binding by Affecting Membrane Electrostatics and Solvation Properties.

    Science.gov (United States)

    Doktorova, Milka; Heberle, Frederick A; Kingston, Richard L; Khelashvili, George; Cuendet, Michel A; Wen, Yi; Katsaras, John; Feigenson, Gerald W; Vogt, Volker M; Dick, Robert A

    2017-11-07

    Binding of the retroviral structural protein Gag to the cellular plasma membrane is mediated by the protein's matrix (MA) domain. Prominent among MA-PM interactions is electrostatic attraction between the positively charged MA domain and the negatively charged plasma membrane inner leaflet. Previously, we reported that membrane association of HIV-1 Gag, as well as purified Rous sarcoma virus (RSV) MA and Gag, depends strongly on the presence of acidic lipids and is enhanced by cholesterol (Chol). The mechanism underlying this enhancement was unclear. Here, using a broad set of in vitro and in silico techniques we addressed molecular mechanisms of association between RSV MA and model membranes, and investigated how Chol enhances this association. In neutron scattering experiments with liposomes in the presence or absence of Chol, MA preferentially interacted with preexisting POPS-rich clusters formed by nonideal lipid mixing, binding peripherally to the lipid headgroups with minimal perturbation to the bilayer structure. Molecular dynamics simulations showed a stronger MA-bilayer interaction in the presence of Chol, and a large Chol-driven increase in lipid packing and membrane surface charge density. Although in vitro MA-liposome association is influenced by disparate variables, including ionic strength and concentrations of Chol and charged lipids, continuum electrostatic theory revealed an underlying dependence on membrane surface potential. Together, these results conclusively show that Chol affects RSV MA-membrane association by making the electrostatic potential at the membrane surface more negative, while decreasing the penalty for lipid headgroup desolvation. The presented approach can be applied to other viral and nonviral proteins. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  19. Ultra-fast optical manipulation of single proteins binding to the actin cytoskeleton

    Science.gov (United States)

    Capitanio, Marco; Gardini, Lucia; Pavone, Francesco Saverio

    2014-02-01

    In the last decade, forces and mechanical stresses acting on biological systems are emerging as regulatory factors essential for cell life. Emerging evidences indicate that factors such as applied forces or the rigidity of the extracellular matrix (ECM) determine the shape and function of cells and organisms1. Classically, the regulation of biological systems is described through a series of biochemical signals and enzymatic reactions, which direct the processes and cell fate. However, mechanotransduction, i.e. the conversion of mechanical forces into biochemical and biomolecular signals, is at the basis of many biological processes fundamental for the development and differentiation of cells, for their correct function and for the development of pathologies. We recently developed an in vitro system that allows the investigation of force-dependence of the interaction of proteins binding the actin cytoskeleton, at the single molecule level. Our system displays a delay of only ~10 μs between formation of the molecular bond and application of the force and is capable of detecting interactions as short as 100 μs. Our assay allows direct measurements of load-dependence of lifetimes of single molecular bonds and conformational changes of single proteins and molecular motors. We demonstrate our technique on molecular motors, using myosin II from fast skeletal muscle and on protein-DNA interaction, specifically on Lactose repressor (LacI). The apparatus is stabilized to less than 1 nm with both passive and active stabilization, allowing resolving specific binding regions along the actin filament and DNA molecule. Our technique extends single-molecule force-clamp spectroscopy to molecular complexes that have been inaccessible up to now, opening new perspectives for the investigation of the effects of forces on biological processes.

  20. A Single Rainbow Trout Cobalamin-binding Protein Stands in for Three Human Binders

    DEFF Research Database (Denmark)

    Greibe, Eva Holm; Fedosov, Sergey; Sorensen, Boe S

    2012-01-01

    affinity for the cobalamin analog cobinamide. Like haptocorrin and transcobalamin, the trout cobalamin-binding protein was present in plasma and recognized ligands with altered nucleotide moiety. Like intrinsic factors, the trout cobalamin-binding protein was present in the stomach and resisted degradation...... by trypsin and chymotrypsin. It also resembled intrinsic factor in the composition of conserved residues in the primary cobalamin-binding site in the C terminus. The trout cobalamin-binding protein was glycosylated and displayed spectral properties comparable with those of haptocorrin and intrinsic factor...

  1. Novel serological neo-epitope markers of extracellular matrix proteins for the detection of portal hypertension

    DEFF Research Database (Denmark)

    Leeming, Diana Julie; Karsdal, M A; Byrjalsen, I

    2013-01-01

    The hepatic venous pressure gradient (HVPG) is an invasive, but important diagnostic and prognostic marker in cirrhosis with portal hypertension (PHT). During cirrhosis, remodelling of fibrotic tissue by matrix metalloproteinases (MMPs) is a permanent process generating small fragments of degrade...... extracellular matrix (ECM) proteins known as neoepitopes, which are then released into the circulation....

  2. Retinol binding protein 4, obesity, and insulin resistance in adolescents

    Directory of Open Access Journals (Sweden)

    Ronaldi Noor

    2017-02-01

    Full Text Available Background Obesity is a global problem. Even in poor and developing countries, obesity has reached alarming levels. In childhood, obesity may lead to insulin resistance. Retinol binding protein (RBP4, secreted primarily by liver and adipose tissues, was recently proposed as a link between obesity and insulin resistance. The role of RBP4 in pediatric obesity and its relationship with insulin resistance have not been well elucidated. Objective To compare RBP4 levels in obese and lean adolescents and to assess for a relationship between RBP4 levels and insulin resistance. Method This cross-sectional study was conducted in three senior high schools in Padang, West Sumatera, Indonesia. Subjects were adolescents aged 14-18 years, who were obese or normal weight (n=56. We measured subjects’ body mass index (BMI and serum RBP4 concentrations. Insulin resistance was assessed using the homeostasis model assessment of insulin resistance (HOMA-IR index. Results Similar RBP4 levels were found in the obese and normoweight groups (P>0.05. Higher RBP4 levels were found in the insulin resistant compared to the non-insulin resistant group, but the difference was not significant (P > 0.05. Conclusion There is no significant difference in mean RBP4 levels in obese adolescents compared to normoweight adolescents. Nor are mean RBP4 levels significantly different between obese adolescents with and without insulin resistance.

  3. Sertoli cell origin of testicular androgen-binding protein (ABP)

    Energy Technology Data Exchange (ETDEWEB)

    Hagenaes, L [Pediatric Endocrinology Unit, Stockholm; Ritzen, E M; Ploeen, L; Hansson, V; French, F S; Nayfeh, S N

    1975-05-01

    In this report it is suggested that the specific androgen-binding protein (ABP), previously shown to originate in the testes of rat and other species, is produced by the Sertoli cells. This suggestion is based upon the following experimental findings: (1) ABP was found in high concentrations in testicular efferent duct fluid but only in trace amounts in inter-tubular lymph. (2) ABP could be recovered from crude preparations of testes tubules, but not from Leydig cells from the same testes. (3) Testes whose germinal epithelium had been severely damaged by gamma irradiation showed no decrease in ABP content. The transport of ABP to epididymis was also preserved as judged from the levels of ABP in caput epididymis. (4) Testes that were completely devoid of germ cells following prenatal gamma irradiation showed high levels of ABP. These high levels approached zero following hypophysectomy, but could be restored by FSH administration to the hypophysectomized animals. ABP has been well characterized and now provides a valuable experimental tool as an indicator of Sertoli cell function.

  4. Matrix Gla Protein polymorphism, but not concentrations, is associated with radiographic hand osteoarthritis

    Science.gov (United States)

    Objective. Factors associated with mineralization and osteophyte formation in osteoarthritis (OA) are incompletely understood. Genetic polymorphisms of matrix Gla protein (MGP), a mineralization inhibitor, have been associated clinically with conditions of abnormal calcification. We therefore evalua...

  5. Effect of benzimidazol-derivatives on the DNA-protein binding formation after UV-radiation of chromatin

    International Nuclear Information System (INIS)

    Mil', E.M.; Binyukov, V.I.; Zhil'tsova, V.M.; Stolyarova, L.G.; Kuznetsov, Yu.V.

    1991-01-01

    Effect of benzimidazol-derivatives on the DNA-protein binding formation was studied after UV-radiation of chromatin. These derivatives were shown to protect chromatin from UV-induced DNA-protein binding formation. Structural analog contained two aminomethyl residuals sensibilized additional binding formation in chromatin. Results suggested, that benzimidazol interacted with DNA, while aminomethyl groups interacted with protein and sensibilized binding of DNA, whilt aminomethyl groups interacted with protein and sensibilized binding of DNA with histone H1

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

    International Nuclear Information System (INIS)

    Dodsworth, Jeremy A.; Leigh, John A.

    2007-01-01

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

  7. Species Differences in the Carbohydrate Binding Preferences of Surfactant Protein D

    DEFF Research Database (Denmark)

    Crouch, Erika C.; Smith, Kelly; McDonald, Barbara

    2006-01-01

    Interactions of surfactant protein D (SP-D) with micro-organisms and organic antigens involve binding to the trimeric neck plus carbohydrate recognition domain (neck+CRD). In these studies, we compared the ligand binding of homologous human, rat, and mouse trimeric neck+CRD fusion proteins, each ...

  8. Pumilio and nanos RNA-binding proteins counterbalance the transcriptional consequences of RB1 inactivation.

    Science.gov (United States)

    Miles, Wayne O; Dyson, Nicholas J

    2014-01-01

    The ability of the retinoblastoma protein (RB) tumor suppressor to repress transcription stimulated by the E2 promoter binding factors (E2F) is integral to its biological functions. Our recent report described a conserved feedback mechanism mediated by the RNA-binding proteins Pumilio and Nanos that increases in importance following RB loss and helps cells to tolerate deregulated E2F.

  9. Regulation of activity of the yeast TATA-binding protein through intra ...

    Indian Academy of Sciences (India)

    Unknown

    Abbreviations used: BMH, Bismaleimidohexane; TBP, TATA-binding protein; yTBP, yeast TBP. J. Biosci. | Vol. ... Therefore for full-length TBP, intra-molecular interactions can regulate its activity via a similar ..... simulations (Miaskeiwicz and Ornstein 1996). .... box binding protein (TBP): A molecular dynamics computa-.

  10. Distribution of PASTA domains in penicillin-binding proteins and serine/threonine kinases of Actinobacteria.

    Science.gov (United States)

    Ogawara, Hiroshi

    2016-09-01

    PASTA domains (penicillin-binding protein and serine/threonine kinase-associated domains) have been identified in penicillin-binding proteins and serine/threonine kinases of Gram-positive Firmicutes and Actinobacteria. They are believed to bind β-lactam antibiotics, and be involved in peptidoglycan metabolism, although their biological function is not definitively clarified. Actinobacteria, especially Streptomyces species, are distinct in that they undergo complex cellular differentiation and produce various antibiotics including β-lactams. This review focuses on the distribution of PASTA domains in penicillin-binding proteins and serine/threonine kinases in Actinobacteria. In Actinobacteria, PASTA domains are detectable exclusively in class A but not in class B penicillin-binding proteins, in sharp contrast to the cases in other bacteria. In penicillin-binding proteins, PASTA domains distribute independently from taxonomy with some distribution bias. Particularly interesting thing is that no Streptomyces species have penicillin-binding protein with PASTA domains. Protein kinases in Actinobacteria possess 0 to 5 PASTA domains in their molecules. Protein kinases in Streptomyces can be classified into three groups: no PASTA domain, 1 PASTA domain and 4 PASTA domain-containing groups. The 4 PASTA domain-containing groups can be further divided into two subgroups. The serine/threonine kinases in different groups may perform different functions. The pocket region in one of these subgroup is more dense and extended, thus it may be involved in binding of ligands like β-lactams more efficiently.

  11. Vitamin D, vitamin D binding protein, lung function and structure in COPD

    DEFF Research Database (Denmark)

    Berg, Isaac; Hanson, Corrine; Sayles, Harlan

    2013-01-01

    Vitamin D and vitamin D binding protein (DBP) have been associated with COPD and FEV1. There are limited data regarding emphysema and vitamin D and DBP.......Vitamin D and vitamin D binding protein (DBP) have been associated with COPD and FEV1. There are limited data regarding emphysema and vitamin D and DBP....

  12. Size versus polarizability in protein-ligand interactions: binding of noble gases within engineered cavities in phage T4 lysozyme.

    Science.gov (United States)

    Quillin, M L; Breyer, W A; Griswold, I J; Matthews, B W

    2000-09-29

    To investigate the relative importance of size and polarizability in ligand binding within proteins, we have determined the crystal structures of pseudo wild-type and cavity-containing mutant phage T4 lysozymes in the presence of argon, krypton, and xenon. These proteins provide a representative sample of predominantly apolar cavities of varying size and shape. Even though the volumes of these cavities range up to the equivalent of five xenon atoms, the noble gases bind preferentially at highly localized sites that appear to be defined by constrictions in the walls of the cavities, coupled with the relatively large radii of the noble gases. The cavities within pseudo wild-type and L121A lysozymes each bind only a single atom of noble gas, while the cavities within mutants L133A and F153A have two independent binding sites, and the L99A cavity has three interacting sites. The binding of noble gases within two double mutants was studied to characterize the additivity of binding at such sites. In general, when a cavity in a protein is created by a "large-to-small" substitution, the surrounding residues relax somewhat to reduce the volume of the cavity. The binding of xenon and, to a lesser degree, krypton and argon, tend to expand the volume of the cavity and to return it closer to what it would have been had no relaxation occurred. In nearly all cases, the extent of binding of the noble gases follows the trend xenon>krypton>argon. Pressure titrations of the L99A mutant have confirmed that the crystallographic occupancies accurately reflect fractional saturation of the binding sites. The trend in noble gas affinity can be understood in terms of the effects of size and polarizability on the intermolecular potential. The plasticity of the protein matrix permits repulsion due to increased ligand size to be more than compensated for by attraction due to increased ligand polarizability. These results have implications for the mechanism of general anesthesia, the migration

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

    Directory of Open Access Journals (Sweden)

    Quddus M Ruhul

    2010-12-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Lee Sael

    2010-12-01

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

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

    Science.gov (United States)

    Sael, Lee; Kihara, Daisuke

    2010-01-01

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

  17. Extracellular matrix proteins: a positive feedback loop in lung fibrosis?

    NARCIS (Netherlands)

    Blaauboer, M.E.; van Boeijen, F.R.; Emson, C.L.; Turner, S.M.; Zandieh-Doulabi, B.; Hanemaaijer, R.; Smit, T.H.; Stoop, R.; Everts, V.

    2014-01-01

    Lung fibrosis is characterized by excessive deposition of extracellular matrix. This not only affects tissue architecture and function, but it also influences fibroblast behavior and thus disease progression. Here we describe the expression of elastin, type V collagen and tenascin C during the

  18. Extracellular matrix proteins: A positive feedback loop in lung fibrosis?

    NARCIS (Netherlands)

    Blaauboer, M.E.; Boeijen, F.R.; Emson, C.L.; Turner, S.M.; Zandieh-Doulabi, B.; Hanemaaijer, R.; Smit, T.H.; Stoop, R.; Everts, V.

    2014-01-01

    Lung fibrosis is characterized by excessive deposition of extracellular matrix. This not only affects tissue architecture and function, but it also influences fibroblast behavior and thus disease progression. Here we describe the expression of elastin, type V collagen and tenascin C during the

  19. D-fructose-binding proteins in bull seminal plasma: Isolation and characterization

    Czech Academy of Sciences Publication Activity Database

    Liberda, J.; Kraus, Marek; Ryšlavá, H.; Vlasáková, M.; Jonáková, Věra; Tichá, M.

    2001-01-01

    Roč. 47, č. 4 (2001), s. 113-119 ISSN 0015-5500 R&D Projects: GA ČR GA303/99/0357; GA ČR GV524/96/K162 Institutional research plan: CEZ:AV0Z5052915 Keywords : bull seminal plasma * non-heparin-binding and heparin-binding proteins * D-fructose-binding proteins Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.519, year: 2001

  20. Growth of thin films of low molecular weight proteins by matrix assisted pulsed laser evaporation (MAPLE)

    DEFF Research Database (Denmark)

    Matei, Andreea; Schou, Jørgen; Constantinescu, C.

    2011-01-01

    Thin films of lysozyme and myoglobin grown by matrix assisted pulsed laser evaporation (MAPLE) from a water ice matrix have been investigated. The deposition rate of these two low molecular weight proteins (lysozyme: 14307 amu and myoglobin: 17083 amu) exhibits a maximum of about 1–2 ng/cm2 per....... The results for lysozyme demonstrate that the fragmentation rate of the proteins during the MAPLE process is not influenced by the pH of the water solution prior to freezing....

  1. Ion Binding Energies Determining Functional Transport of ClC Proteins

    Science.gov (United States)

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

    2014-06-01

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

  2. Influenza A virus infection engenders a poor antibody response against the ectodomain of matrix protein 2

    Directory of Open Access Journals (Sweden)

    Wunner William

    2006-12-01

    Full Text Available Abstract Background Matrix protein 2 (M2 is an integral tetrameric membrane protein of influenza A virus (IAV. Its ectodomain (M2e shows remarkably little diversity amongst human IAV strains. As M2e-specific antibodies (Abs have been shown to reduce the severity of infection in animals, M2e is being studied for its capability of providing protection against a broad range of IAV strains. Presently, there is little information about the concentration of M2e-specific Abs in humans. Two previous studies made use of ELISA and Western blot against M2e peptides and recombinant M2 protein as immunosorbents, respectively, and reported Ab titers to be low or undetectable. An important caveat is that these assays may not have detected all Abs capable of binding to native tetrameric M2e. Therefore, we developed an assay likely to detect all M2e tetramer-specific Abs. Results We generated a HeLa cell line that expressed full length tetrameric M2 (HeLa-M2 or empty vector (HeLa-C10 under the control of the tetracycline response element. These cell lines were then used in parallel as immunosorbents in ELISA. The assay was standardized and M2e-specific Ab titers quantified by means of purified murine or chimeric (mouse variable regions, human constant regions M2e-specific Abs in the analysis of mouse and human sera, respectively. We found that the cell-based ELISA was substantially more effective than immobilized M2e peptide in detecting M2e-specific Abs in sera of mice that had recovered from repetitive IAV infections. Still, titers remained low ( Conclusion The results provide convincing evidence that M2e-specific Ab-mediated protection is currently lacking or suboptimal in humans.

  3. Sequence similarity between the erythrocyte binding domain of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals a functional heparin binding motif involved in binding to the Duffy antigen receptor for chemokines

    Directory of Open Access Journals (Sweden)

    Bolton Michael J

    2011-11-01

    Full Text Available Abstract Background The HIV surface glycoprotein gp120 (SU, gp120 and the Plasmodium vivax Duffy binding protein (PvDBP bind to chemokine receptors during infection and have a site of amino acid sequence similarity in their binding domains that often includes a heparin binding motif (HBM. Infection by either pathogen has been found to be inhibited by polyanions. Results Specific polyanions that inhibit HIV infection and bind to the V3 loop of X4 strains also inhibited DBP-mediated infection of erythrocytes and DBP binding to the Duffy Antigen Receptor for Chemokines (DARC. A peptide including the HBM of PvDBP had similar affinity for heparin as RANTES and V3 loop peptides, and could be specifically inhibited from heparin binding by the same polyanions that inhibit DBP binding to DARC. However, some V3 peptides can competitively inhibit RANTES binding to heparin, but not the PvDBP HBM peptide. Three other members of the DBP family have an HBM sequence that is necessary for erythrocyte binding, however only the protein which binds to DARC, the P. knowlesi alpha protein, is inhibited by heparin from binding to erythrocytes. Heparitinase digestion does not affect the binding of DBP to erythrocytes. Conclusion The HBMs of DBPs that bind to DARC have similar heparin binding affinities as some V3 loop peptides and chemokines, are responsible for specific sulfated polysaccharide inhibition of parasite binding and invasion of red blood cells, and are more likely to bind to negative charges on the receptor than cell surface glycosaminoglycans.

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

    Science.gov (United States)

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

    2015-12-01

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

  5. Role of Electrostatics in Protein-RNA Binding: The Global vs the Local Energy Landscape.

    Science.gov (United States)

    Ghaemi, Zhaleh; Guzman, Irisbel; Gnutt, David; Luthey-Schulten, Zaida; Gruebele, Martin

    2017-09-14

    U1A protein-stem loop 2 RNA association is a basic step in the assembly of the spliceosomal U1 small nuclear ribonucleoprotein. Long-range electrostatic interactions due to the positive charge of U1A are thought to provide high binding affinity for the negatively charged RNA. Short range interactions, such as hydrogen bonds and contacts between RNA bases and protein side chains, favor a specific binding site. Here, we propose that electrostatic interactions are as important as local contacts in biasing the protein-RNA energy landscape toward a specific binding site. We show by using molecular dynamics simulations that deletion of two long-range electrostatic interactions (K22Q and K50Q) leads to mutant-specific alternative RNA bound states. One of these states preserves short-range interactions with aromatic residues in the original binding site, while the other one does not. We test the computational prediction with experimental temperature-jump kinetics using a tryptophan probe in the U1A-RNA binding site. The two mutants show the distinct predicted kinetic behaviors. Thus, the stem loop 2 RNA has multiple binding sites on a rough RNA-protein binding landscape. We speculate that the rough protein-RNA binding landscape, when biased to different local minima by electrostatics, could be one way that protein-RNA interactions evolve toward new binding sites and novel function.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-01-15

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

  7. Change of conformation and internal dynamics of supercoiled DNA upon binding of Escherichia coli single-strand binding protein

    International Nuclear Information System (INIS)

    Langowski, J.; Benight, A.S.; Fujimoto, B.S.; Schurr, J.M.; Schomburg, U.

    1985-01-01

    The influence of Escherichia coli single-strand binding (SSB) protein on the conformation and internal dynamics of pBR322 and pUC8 supercoiled DNAs has been investigated by using dynamic light scattering at 632.8 and 351.1 nm and time-resolved fluorescence polarization anisotropy of intercalated ethidium. SSB protein binds to both DNAs up to a stoichiometry that is sufficient to almost completely relax the superhelical turns. Upon saturation binding, the translational diffusion coefficients (D 0 ) of both DNAs decrease by approximately 20%. Apparent diffusion coefficients (D/sub app/) obtained from dynamic light scattering display the well-known increase with K 2 (K = scattering vector), leveling off toward a plateau value (D/sub plat/) at high K 2 . For both DNAs, the difference D/sub plat/ - D 0 increases upon relaxation of supercoils by SSB protein, which indicates a corresponding enhancement of the subunit mobilities in internal motions. Fluorescence polarization anisotropy measurements on free and complexed pBR322 DNA indicate a (predominantly) uniform torsional rigidity for the saturated DNA/SSB protein complex that is significantly reduced compared to the free DNA. These observations are all consistent with the notion that binding of SSB protein is accompanied by a gradual loss of supercoils and saturates when the superhelical twist is largely removed

  8. Lactoferrin binding protein B - a bi-functional bacterial receptor protein.

    Directory of Open Access Journals (Sweden)

    Nicholas K H Ostan

    2017-03-01

    Full Text Available Lactoferrin binding protein B (LbpB is a bi-lobed outer membrane-bound lipoprotein that comprises part of the lactoferrin (Lf receptor complex in Neisseria meningitidis and other Gram-negative pathogens. Recent studies have demonstrated that LbpB plays a role in protecting the bacteria from cationic antimicrobial peptides due to large regions rich in anionic residues in the C-terminal lobe. Relative to its homolog, transferrin-binding protein B (TbpB, there currently is little evidence for its role in iron acquisition and relatively little structural and biophysical information on its interaction with Lf. In this study, a combination of crosslinking and deuterium exchange coupled to mass spectrometry, information-driven computational docking, bio-layer interferometry, and site-directed mutagenesis was used to probe LbpB:hLf complexes. The formation of a 1:1 complex of iron-loaded Lf and LbpB involves an interaction between the Lf C-lobe and LbpB N-lobe, comparable to TbpB, consistent with a potential role in iron acquisition. The Lf N-lobe is also capable of binding to negatively charged regions of the LbpB C-lobe and possibly other sites such that a variety of higher order complexes are formed. Our results are consistent with LbpB serving dual roles focused primarily on iron acquisition when exposed to limited levels of iron-loaded Lf on the mucosal surface and effectively binding apo Lf when exposed to high levels at sites of inflammation.

  9. Mannose-Binding Lectin Binds to Amyloid Protein and Modulates Inflammation

    Directory of Open Access Journals (Sweden)

    Mykol Larvie

    2012-01-01

    Full Text Available Mannose-binding lectin (MBL, a soluble factor of the innate immune system, is a pattern recognition molecule with a number of known ligands, including viruses, bacteria, and molecules from abnormal self tissues. In addition to its role in immunity, MBL also functions in the maintenance of tissue homeostasis. We present evidence here that MBL binds to amyloid β peptides. MBL binding to other known carbohydrate ligands is calcium-dependent and has been attributed to the carbohydrate-recognition domain, a common feature of other C-type lectins. In contrast, we find that the features of MBL binding to Aβ are more similar to the reported binding characteristics of the cysteine-rich domain of the unrelated mannose receptor and therefore may involve the MBL cysteine-rich domain. Differences in MBL ligand binding may contribute to modulation of inflammatory response and may correlate with the function of MBL in processes such as coagulation and tissue homeostasis.

  10. Dynamic SPR monitoring of yeast nuclear protein binding to a cis-regulatory element

    International Nuclear Information System (INIS)

    Mao, Grace; Brody, James P.

    2007-01-01

    Gene expression is controlled by protein complexes binding to short specific sequences of DNA, called cis-regulatory elements. Expression of most eukaryotic genes is controlled by dozens of these elements. Comprehensive identification and monitoring of these elements is a major goal of genomics. In pursuit of this goal, we are developing a surface plasmon resonance (SPR) based assay to identify and monitor cis-regulatory elements. To test whether we could reliably monitor protein binding to a regulatory element, we immobilized a 16 bp region of Saccharomyces cerevisiae chromosome 5 onto a gold surface. This 16 bp region of DNA is known to bind several proteins and thought to control expression of the gene RNR1, which varies through the cell cycle. We synchronized yeast cell cultures, and then sampled these cultures at a regular interval. These samples were processed to purify nuclear lysate, which was then exposed to the sensor. We found that nuclear protein binds this particular element of DNA at a significantly higher rate (as compared to unsynchronized cells) during G1 phase. Other time points show levels of DNA-nuclear protein binding similar to the unsynchronized control. We also measured the apparent association complex of the binding to be 0.014 s -1 . We conclude that (1) SPR-based assays can monitor DNA-nuclear protein binding and that (2) for this particular cis-regulatory element, maximum DNA-nuclear protein binding occurs during G1 phase

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

    International Nuclear Information System (INIS)

    Heemstra, V.L.

    1983-11-01

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

  12. Prediction of vitamin interacting residues in a vitamin binding protein using evolutionary information

    Directory of Open Access Journals (Sweden)

    Panwar Bharat

    2013-02-01

    Full Text Available Abstract Background The vitamins are important cofactors in various enzymatic-reactions. In past, many inhibitors have been designed against vitamin binding pockets in order to inhibit vitamin-protein interactions. Thus, it is important to identify vitamin interacting residues in a protein. It is possible to detect vitamin-binding pockets on a protein, if its tertiary structure is known. Unfortunately tertiary structures of limited proteins are available. Therefore, it is important to develop in-silico models for predicting vitamin interacting residues in protein from its primary structure. Results In this study, first we compared protein-interacting residues of vitamins with other ligands using Two Sample Logo (TSL. It was observed that ATP, GTP, NAD, FAD and mannose preferred {G,R,K,S,H}, {G,K,T,S,D,N}, {T,G,Y}, {G,Y,W} and {Y,D,W,N,E} residues respectively, whereas vitamins preferred {Y,F,S,W,T,G,H} residues for the interaction with proteins. Furthermore, compositional information of preferred and non-preferred residues along with patterns-specificity was also observed within different vitamin-classes. Vitamins A, B and B6 preferred {F,I,W,Y,L,V}, {S,Y,G,T,H,W,N,E} and {S,T,G,H,Y,N} interacting residues respectively. It suggested that protein-binding patterns of vitamins are different from other ligands, and motivated us to develop separate predictor for vitamins and their sub-classes. The four different prediction modules, (i vitamin interacting residues (VIRs, (ii vitamin-A interacting residues (VAIRs, (iii vitamin-B interacting residues (VBIRs and (iv pyridoxal-5-phosphate (vitamin B6 interacting residues (PLPIRs have been developed. We applied various classifiers of SVM, BayesNet, NaiveBayes, ComplementNaiveBayes, NaiveBayesMultinomial, RandomForest and IBk etc., as machine learning techniques, using binary and Position-Specific Scoring Matrix (PSSM features of protein sequences. Finally, we selected best performing SVM modules and

  13. Crystallographic structure and substrate-binding interactions of the molybdate-binding protein of the phytopathogen Xanthomonas axonopodis pv. citri.

    Science.gov (United States)

    Balan, Andrea; Santacruz-Pérez, Carolina; Moutran, Alexandre; Ferreira, Luís Carlos Souza; Neshich, Goran; Gonçalves Barbosa, João Alexandre Ribeiro

    2008-02-01

    In Xanthomonas axonopodis pv. citri (Xac or X. citri), the modA gene codes for a periplasmic protein (ModA) that is capable of binding molybdate and tungstate as part of the ABC-type transporter required for the uptake of micronutrients. In this study, we report the crystallographic structure of the Xac ModA protein with bound molybdate. The Xac ModA structure is similar to orthologs with known three-dimensional structures and consists of two nearly symmetrical domains separated by a hinge region where the oxyanion-binding site lies. Phylogenetic analysis of different ModA orthologs based on sequence alignments revealed three groups of molybdate-binding proteins: bacterial phytopathogens, enterobacteria and soil bacteria. Even though the ModA orthologs are segregated into different groups, the ligand-binding hydrogen bonds are mostly conserved, except for Archaeglobus fulgidus ModA. A detailed discussion of hydrophobic interactions in the active site is presented and two new residues, Ala38 and Ser151, are shown to be part of the ligand-binding pocket.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-03-06

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

  15. Protein S binding to human endothelial cells is required for expression of cofactor activity for activated protein C

    NARCIS (Netherlands)

    Hackeng, T. M.; Hessing, M.; van 't Veer, C.; Meijer-Huizinga, F.; Meijers, J. C.; de Groot, P. G.; van Mourik, J. A.; Bouma, B. N.

    1993-01-01

    An important feedback mechanism in blood coagulation is supplied by the protein C/protein S anticoagulant pathway. In this study we demonstrate that the binding of human protein S to cultured human umbilical vein endothelial cells (HUVECs) is required for the expression of cofactor activity of

  16. Binding specificity and in vivo targets of the EH domain, a novel protein-protein interaction module

    DEFF Research Database (Denmark)

    Salcini, A E; Confalonieri, S; Doria, M

    1997-01-01

    EH is a recently identified protein-protein interaction domain found in the signal transducers Eps15 and Eps15R and several other proteins of yeast nematode. We show that EH domains from Eps15 and Eps15R bind in vitro to peptides containing an asparagine-proline-phenylalanine (NPF) motif. Direct...

  17. Specificity and sensitivity of binding proteins in the radioimmunoassay of cortisol

    International Nuclear Information System (INIS)

    Gijzen, A.H.J.

    1977-01-01

    A comparison concerning avidity towards cortisol and 10 other steroids was made between several binding proteins either in solution or bound to cellulose as so called ''solid phase'' reagent. Human blood cortisol binding protein (CBP, transcortin), and two distinctly different cortisol-binding rabbit antisera and the isolated immunoglobulins thereof were compared in their avidity to bind cortisol and several other steroids. The antisera were harvested from rabbits immunized with either cortisol-21-succinyl-albumin (CSA) or cortisol-3-oxim-albumin (COA). The latter antiserum, having the highest titre in cortisol titration, showed the greatest specificity and was most useful as a binding reagent in cortisol radioimmunoassay when used as a solid phase reagent. The determination of cortisol in micro samples of blood serum is possible without steroid extraction or serum protein denaturation and with only minor influence of steroid impurities in the sample to be analyzed. Affinity constants for all compared binding reagents and steroids are given

  18. Binding of tryptophan and iron by reptilion plasnna proteins

    African Journals Online (AJOL)

    transport functions. Albumin of the alligator (Alligator mississippiensis) and other reptiles binds, amongst other ions, tryptophan (McMenamy & Watson 1968) and transferrin binds iron (Barber & Sheeler 1963). Multiple transferrins are present in the plasma of many reptiles. (Dessauer et af 1962) and the albumin region of the.

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

    Indian Academy of Sciences (India)

    2012-06-25

    Jun 25, 2012 ... Support Vector Machine (SVM) is a state-of-the-art classifica- tion technique. Using canonical binding model, the C2H2 zinc finger protein–DNA interaction interface is modelled by the pairwise amino acid–base interactions. Using a classification framework, known examples of non-binding ZF–DNA pairs.

  20. Zinc ions bind to and inhibit activated protein C

    DEFF Research Database (Denmark)

    Zhu, Tianqing; Ubhayasekera, Wimal; Nickolaus, Noëlle

    2010-01-01

    fold enhanced, presumably due to the Ca2+-induced conformational change affecting the conformation of the Zn2+-binding site. The inhibition mechanism was non-competitive both in the absence and presence of Ca2+. Comparisons of sequences and structures suggested several possible sites for zinc binding...

  1. Surfactant protein D binds to human immunodeficiency virus (HIV) envelope protein gp120 and inhibits HIV replication

    DEFF Research Database (Denmark)

    Meschi, Joseph; Crouch, Erika C; Skolnik, Paul

    2005-01-01

    The envelope protein (gp120) of human immunodeficiency virus (HIV) contains highly conserved mannosylated oligosaccharides. These glycoconjugates contribute to resistance to antibody neutralization, and binding to cell surface lectins on macrophages and dendritic cells. Mannose-binding lectin (MBL......) binds to gp120 and plays a role in defence against the virus. In this study it is demonstrated that surfactant protein D (SP-D) binds to gp120 and inhibits HIV infectivity at significantly lower concentrations than MBL. The binding of SP-D was mediated by its calcium-dependent carbohydrate......-binding activity and was dependent on glycosylation of gp120. Native dodecameric SP-D bound to HIV gp120 more strongly than native trimeric SP-D. Since one common polymorphic form of SP-D is predominantly expressed as trimers and associated with lower blood levels, these individuals may have less effective innate...

  2. [Morphology of basement membrane and associated matrix proteins in normal and pathological tissues].

    Science.gov (United States)

    Nerlich, A

    1995-01-01

    Basement membranes (BM) are specialized structures of the extracellular matrix. Their composition is of particular importance for the maintenance of normal morphological and functional properties of a multitude of organs and tissue systems and it is thus required for regular homeostasis of body function. Generally, they possess three main functions, i.e. participation in the maintenance of tissue structure, control of fluid and substrate exchange, and regulation of cell growth and differentiation. BMs are made up by various components which are in part specifically localized within the BM zone, or which represent ubiquitous matrix constituents with specific quantitative and/or qualitative differences in their localization. On the basis of a thorough immunohistochemical analysis of normal and diseased tissues, we provide here a concept of "functional morphology/pathomorphology" of the different BM components analyzed: 1.) The ubiquitous BM-constituent collagen IV primarily stabilizes the BM-zone and thus represents the "backbone" of the BM providing mechanical strength. Its loss leads to cystic tissue transformation as it is evidenced from the analysis of polycystic nephropathies. Thus, in other cystic tissue transformations a similar formal pathogenesis may be present. 2.) The specific localization of collagen VII as the main structural component of anchoring fibrils underlines the mechanical anchoring function of this collagenous protein. Defects in this protein lead to hereditary epidermolysis. The rapid re-occurrence of epidermal collagen VII during normal human wound healing indicates a quick reconstitution of the mechanical tensile strength of healing wounds. 3.) The BM-specific heparan sulfate proteoglycan (HSPG, Perlecan) with its highly negative anionic charge can be assumed to exert filter control. This assumption is corroborated by the localizatory findings of a preferential deposition of HSPG in endothelial and particularly in glomerular BM. Similarly

  3. Mixed matrix membrane adsorbers for protein and blood purification

    NARCIS (Netherlands)

    Saiful, S.

    2007-01-01

    Biotechnology and bio-manufacturing markets are continuously growing, generating new sources of many valuable healthcare and life science products including therapeutic proteins and polysaccharides, monoclonals, vaccines, diagnostics, pharmaceutical chemicals and enzymes. These bioproducts have to

  4. Strong Ligand-Protein Interactions Derived from Diffuse Ligand Interactions with Loose Binding Sites.

    Science.gov (United States)

    Marsh, Lorraine

    2015-01-01

    Many systems in biology rely on binding of ligands to target proteins in a single high-affinity conformation with a favorable ΔG. Alternatively, interactions of ligands with protein regions that allow diffuse binding, distributed over multiple sites and conformations, can exhibit favorable ΔG because of their higher entropy. Diffuse binding may be biologically important for multidrug transporters and carrier proteins. A fine-grained computational method for numerical integration of total binding ΔG arising from diffuse regional interaction of a ligand in multiple conformations using a Markov Chain Monte Carlo (MCMC) approach is presented. This method yields a metric that quantifies the influence on overall ligand affinity of ligand binding to multiple, distinct sites within a protein binding region. This metric is essentially a measure of dispersion in equilibrium ligand binding and depends on both the number of potential sites of interaction and the distribution of their individual predicted affinities. Analysis of test cases indicates that, for some ligand/protein pairs involving transporters and carrier proteins, diffuse binding contributes greatly to total affinity, whereas in other cases the influence is modest. This approach may be useful for studying situations where "nonspecific" interactions contribute to biological function.

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  6. Guanine nucleotide binding proteins in zucchini seedlings: Characterization and interactions with the NPA receptor

    International Nuclear Information System (INIS)

    Lindeberg, M.; Jacobs, M.

    1989-01-01

    A microsomal membrane preparation from hypocotyls of dark-grown Cucurbita pepo L. seedlings contains specific high-affinity binding sites for the non-hydrolyzable GTP analog guanosine 5'-[γ-thio] triphosphate (GTP-γ-S). Both the binding affinity and the pattern of binding specificity for GTP and GTP analogs are similar to animal G-proteins, and two zucchini membrane proteins are recognized in western blots by antiserum specific for the σ subunit of platelet G s protein. GTP-γ-S can increase specific naphthylphthalamic acid (NPA) binding in zucchini microsomal membrane preparations, with its stimulation increasing with large tissue age. Al +3 and F - agents known to activate G-proteins - decreased NPA specific binding by ca. 15%. In tests of in vitro auxin transport employing zucchini plasma membrane vesicles, AlF - 4 strongly inhibited 3 H-indoleacetic acid nor accumulation; GTP-γ-S effects on this system will be discussed

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

    Directory of Open Access Journals (Sweden)

    Naresh Arora

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

  8. Biomimetic conformation-specific assembly of proteins at artificial binding sites nano-patterned on silicon

    Science.gov (United States)

    de la Rica, Roberto; Matsui, Hiroshi

    2009-01-01

    Biomolecules such as enzymes and antibodies possess binding sites where the molecular architecture and the physicochemical properties are optimum for their interaction with a particular target, in some cases even differentiating between stereoisomers. Here, we mimic this exquisite specificity via the creation of a suitable chemical environment by fabricating artificial binding sites for the protein calmodulin (CaM). By downscaling well-known surface chemical modification methodologies to the nanometer scale via silicon nanopatterning, the Ca2+-CaM conformer was found to selectively bind the biomimetic binding sites. The methodology could be adapted to mimic other protein-receptor interactions for sensing and catalysis. PMID:19757782

  9. Escherichia coli Protein Expression System for Acetylcholine Binding Proteins (AChBPs.

    Directory of Open Access Journals (Sweden)

    Nikita Abraham

    Full Text Available Nicotinic acetylcholine receptors (nAChR are ligand gated ion channels, identified as therapeutic targets for a range of human diseases. Drug design for nAChR related disorders is increasingly using structure-based approaches. Many of these structural insights for therapeutic lead development have been obtained from co-crystal structures of nAChR agonists and antagonists with the acetylcholine binding protein (AChBP. AChBP is a water soluble, structural and functional homolog of the extracellular, ligand-binding domain of nAChRs. Currently, AChBPs are recombinantly expressed in eukaryotic expression systems for structural and biophysical studies. Here, we report the establishment of an Escherichia coli (E. coli expression system that significantly reduces the cost and time of production compared to the existing expression systems. E. coli can efficiently express unglycosylated AChBP for crystallography and makes the expression of isotopically labelled forms feasible for NMR. We used a pHUE vector containing an N-terminal His-tagged ubiquitin fusion protein to facilitate AChBP expression in the soluble fractions, and thus avoid the need to recover protein from inclusion bodies. The purified protein yield obtained from the E. coli expression system is comparable to that obtained from existing AChBP expression systems. E. coli expressed AChBP bound nAChR agonists and antagonists with affinities matching those previously reported. Thus, the E. coli expression system significantly simplifies the expression and purification of functional AChBP for structural and biophysical studies.

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

    Directory of Open Access Journals (Sweden)

    Nickolay A Khazanov

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

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

    Directory of Open Access Journals (Sweden)

    Tao Zhang

    2015-11-01

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

  12. Site-directed antibody immobilization using a protein A-gold binding domain fusion protein for enhanced SPR immunosensing.

    Science.gov (United States)

    de Juan-Franco, Elena; Caruz, Antonio; Pedrajas, J R; Lechuga, Laura M

    2013-04-07

    We have implemented a novel strategy for the oriented immobilization of antibodies onto a gold surface based on the use of a fusion protein, the protein A-gold binding domain (PAG). PAG consists of a gold binding peptide (GBP) coupled to the immunoglobulin-binding domains of staphylococcal protein A. This fusion protein provides an easy and fast oriented immobilization of antibodies preserving its native structure, while leaving the antigen binding sites (Fab) freely exposed. Using this immobilization strategy, we have demonstrated the performance of the immunosensing of the human Growth Hormone by SPR. A limit of detection of 90 ng mL(-1) was obtained with an inter-chip variability lower than 7%. The comparison of this method with other strategies for the direct immobilization of antibodies over gold surfaces has showed the enhanced sensitivity provided by the PAG approach.

  13. Structural and functional studies of Escherichia coli aggregative adherence fimbriae (AAF/V) reveal a deficiency in extracellular matrix binding

    DEFF Research Database (Denmark)

    Jønsson, Rie; Liu, Bing; Struve, Carsten

    2017-01-01

    A possesses an inserted helix at the beginning of the donor strand, which together with altered surface electrostatics, renders the protein unable to interact with fibronectin. Hence, here we characterize the first AAF variant with a binding mode that varies from previously described AAFs...

  14. Recombinant fusion protein of albumin-retinol binding protein inactivates stellate cells

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Soyoung; Park, Sangeun; Kim, Suhyun [Laboratory of Cellular Oncology, Korea University Graduate School of Medicine, Ansan, Gyeonggi do 425-707 (Korea, Republic of); Lim, Chaeseung [Department of Laboratory Medicine, Korea University Guro Hospital, Seoul 152-703 (Korea, Republic of); Kim, Jungho [Department of Life Science, Sogang University, Seoul 121-742 (Korea, Republic of); Cha, Dae Ryong [Department of Internal Medicine, Korea University Ansan Hospital, Ansan, Gyeonggi do 425-020 (Korea, Republic of); Oh, Junseo, E-mail: ohjs@korea.ac.kr [Laboratory of Cellular Oncology, Korea University Graduate School of Medicine, Ansan, Gyeonggi do 425-707 (Korea, Republic of)

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer We designed novel recombinant albumin-RBP fusion proteins. Black-Right-Pointing-Pointer Expression of fusion proteins inactivates pancreatic stellate cells (PSCs). Black-Right-Pointing-Pointer Fusion proteins are successfully internalized into and inactivate PSCs. Black-Right-Pointing-Pointer RBP moiety mediates cell specific uptake of fusion protein. -- Abstract: Quiescent pancreatic- (PSCs) and hepatic- (HSCs) stellate cells store vitamin A (retinol) in lipid droplets via retinol binding protein (RBP) receptor and, when activated by profibrogenic stimuli, they transform into myofibroblast-like cells which play a key role in the fibrogenesis. Despite extensive investigations, there is, however, currently no appropriate therapy available for tissue fibrosis. We previously showed that the expression of albumin, composed of three homologous domains (I-III), inhibits stellate cell activation, which requires its high-affinity fatty acid-binding sites asymmetrically distributed in domain I and III. To attain stellate cell-specific uptake, albumin (domain I/III) was coupled to RBP; RBP-albumin{sup domain} {sup III} (R-III) and albumin{sup domain} {sup I}-RBP-albumin{sup III} (I-R-III). To assess the biological activity of fusion proteins, cultured PSCs were used. Like wild type albumin, expression of R-III or I-R-III in PSCs after passage 2 (activated PSCs) induced phenotypic reversal from activated to fat-storing cells. On the other hand, R-III and I-R-III, but not albumin, secreted from transfected 293 cells were successfully internalized into and inactivated PSCs. FPLC-purified R-III was found to be internalized into PSCs via caveolae-mediated endocytosis, and its efficient cellular uptake was also observed in HSCs and podocytes among several cell lines tested. Moreover, tissue distribution of intravenously injected R-III was closely similar to that of RBP. Therefore, our data suggest that albumin-RBP fusion protein comprises

  15. Limited proteolysis combined with isotope labeling and quantitative LC-MALDI MS for monitoring protein conformational changes: a study on calcium-binding sites of cardiac Troponin C

    International Nuclear Information System (INIS)

    McDonald, Chris; Li Liang

    2005-01-01

    Studies of protein-protein and protein-ligand interactions are important for understanding biological functions of proteins. A new technique based on the partial proteolysis of proteins combined with quantitative mass spectrometry is developed as a means of tracking structural changes after the formation of a protein-ligand complex. In this technique, a protein of interest with and without the binding of a ligand is digested with an enzyme to generate a set of peptides, followed by separation of the peptides by liquid chromatography. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) is used to identify chromatographically separated peptides, and locate their sequence alignments in the parent protein. Using an isotopically labeled protein as a sample against an unlabeled protein standard, quantitative information can be gathered. This overcomes the inherent lack of quantitative capability of MALDI MS. The utility of the technique to investigate protein-ligand interactions is demonstrated in a model system involving calcium binding to cardiac Troponin C (cTnC). Using this technique, the general location of the three calcium-binding sites of cTnC can be determined by using several different enzymes to generate overlapping peptide maps of cTnC

  16. Sequence similarity between the erythrocyte binding domain of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals a functional heparin binding motif involved in binding to the Duffy antigen receptor for chemokines

    OpenAIRE

    Bolton, Michael J; Garry, Robert F

    2011-01-01

    Abstract Background The HIV surface glycoprotein gp120 (SU, gp120) and the Plasmodium vivax Duffy binding protein (PvDBP) bind to chemokine receptors during infection and have a site of amino acid sequence similarity in their binding domains that often includes a heparin binding motif (HBM). Infection by either pathogen has been found to be inhibited by polyanions. Results Specific polyanions that inhibit HIV infection and bind to the V3 loop of X4 strains also inhibited DBP-mediated infectio...

  17. Distribution in rat tissues of modulator-binding protein of particulate nature

    International Nuclear Information System (INIS)

    Sobue, K.; Muramoto, Y.; Kakiuchi, S.; Yamazaki, R.

    1979-01-01

    Studies on Ca 2+ -activatable cyclic nucleotide phosphodiesterase led to the discovery of a protein modulator that is required for the activation of this enzyme by Ca 2+ . Later, this protein has been shown to cause the Ca 2+ -dependent activation of several enzymes that include phosphodiesterase, adenylate cyclase, a protein kinase from muscles, phosphorylase b kinase, actomyosin ATPase, membranous ATPase from erythrocytes and nerve synapses. Thus, modulator protein appears to be an intracellular mediator of actions of Ca 2+ . The present work shows the distribution of this particulate modulator-binding component in rat tissues. This paper also describes the labeling of modulator protein with tritium without deteriorating its biological activities and application of this 3 H-modulator protein to the determination of the Ca ++ dependent binding of modulator protein with membranous protein. This technique proves to be useful in studying enzymes or proteins whose functions are regulated by Ca ++ /modulator protein system. (Auth.)

  18. Transcriptional factor DLX3 promotes the gene expression of enamel matrix proteins during amelogenesis.

    Directory of Open Access Journals (Sweden)

    Zhichun Zhang

    Full Text Available Mutation of distal-less homeobox 3 (DLX3 is responsible for human tricho-dento-osseous syndrome (TDO with amelogenesis imperfecta, indicating a crucial role of DLX3 in amelogenesis. However, the expression pattern of DLX3 and its specific function in amelogenesis remain largely unknown. The aim of this study was to investigate the effects of DLX3 on enamel matrix protein (EMP genes. By immunohistochemistry assays of mouse tooth germs, stronger immunostaining of DLX3 protein was identified in ameloblasts in the secretory stage than in the pre-secretory and maturation stages, and the same pattern was found for Dlx3 mRNA using Realtime PCR. In a mouse ameloblast cell lineage, forced expression of DLX3 up-regulated the expression of the EMP genes Amelx, Enam, Klk4, and Odam, whereas knockdown of DLX3 down-regulated these four EMP genes. Further, bioinformatics, chromatin immunoprecipitation, and luciferase assays revealed that DLX3 transactivated Enam, Amelx, and Odam through direct binding to their enhancer regions. Particularly, over-expression of mutant-DLX3 (c.571_574delGGGG, responsible for TDO inhibited the activation function of DLX3 on expression levels and promoter activities of the Enam, Amelx, and Odam genes. Together, our data show that DLX3 promotes the expression of the EMP genes Amelx, Enam, Klk4, and Odam in amelogenesis, while mutant-DLX3 disrupts this regulatory function, thus providing insights into the molecular mechanisms underlying the enamel defects of TDO disease.

  19. Binding of acyl CoA by fatty acid binding protein and the effect on fatty acid activation

    International Nuclear Information System (INIS)

    Burrier, R.E.; Manson, C.R.; Brecher, P.

    1987-01-01

    The ability of purified rat liver and heart fatty acid binding proteins (FABPs) to bind oleoyl CoA and modulate acyl CoA synthesis by microsomal membranes was investigated. Using binding assays employing either Lipidex 1000 or multilamellar liposomes to sequester unbound ligand, rat liver but not rat heart FABP was shown to bind radiolabeled acyl CoA. Binding studies suggest that liver FABP has a single binding site for acyl CoA which is separate from the two binding sites for fatty acids. Experiments were then performed to determine how binding may influence acyl CoA metabolism by liver microsomes or heart sarcoplasmic reticulum. Using liposomes as fatty acid donors, liver FABP stimulated acyl CoA production whereas heart FABP did not stimulate production over control values. 14 C-Fatty acid-FABP complexes were prepared, incubated with membranes and acyl CoA synthetase activity was determined. Up to 70% of the fatty acid could be converted to acyl CoA in the presence of liver FABP but in the presence of heart FABP, only 45% of the fatty acid was converted. The amount of product formed was not changed by additional membrane, enzyme cofactor, or incubation time. Liver but not heart FABP bound the acyl CoA formed and removed it from the membranes. These studies suggest that liver FABP can increase the amount of acyl CoA by binding this ligand thereby removing it from the membrane and possibly aiding transport within the cell

  20. Binding of acyl CoA by fatty acid binding protein and the effect on fatty acid activation

    Energy Technology Data Exchange (ETDEWEB)

    Burrier, R.E.; Manson, C.R.; Brecher, P.

    1987-05-01

    The ability of purified rat liver and heart fatty acid binding proteins (FABPs) to bind oleoyl CoA and modulate acyl CoA synthesis by microsomal membranes was investigated. Using binding assays employing either Lipidex 1000 or multilamellar liposomes to sequester unbound ligand, rat liver but not rat heart FABP was shown to bind radiolabeled acyl CoA. Binding studies suggest that liver FABP has a single binding site for acyl CoA which is separate from the two binding sites for fatty acids. Experiments were then performed to determine how binding may influence acyl CoA metabolism by liver microsomes or heart sarcoplasmic reticulum. Using liposomes as fatty acid donors, liver FABP stimulated acyl CoA production whereas heart FABP did not stimulate production over control values. /sup 14/C-Fatty acid-FABP complexes were prepared, incubated with membranes and acyl CoA synthetase activity was determined. Up to 70% of the fatty acid could be converted to acyl CoA in the presence of liver FABP but in the presence of heart FABP, only 45% of the fatty acid was converted. The amount of product formed was not changed by additional membrane, enzyme cofactor, or incubation time. Liver but not heart FABP bound the acyl CoA formed and removed it from the membranes. These studies suggest that liver FABP can increase the amount of acyl CoA by binding this ligand thereby removing it from the membrane and possibly aiding transport within the cell.

  1. Phytochrome regulates GTP-binding protein activity in the envelope of pea nuclei

    Science.gov (United States)

    Clark, G. B.; Memon, A. R.; Thompson, G. A. Jr; Roux, S. J.

    1993-01-01

    Three GTP-binding proteins with apparent molecular masses of 27, 28 and 30 kDa have been detected in isolated nuclei of etiolated pea plumules. After LDS-PAGE and transfer to nitrocellulose these proteins bind [32P]GTP in the presence of excess ATP, suggesting that they are monomeric G proteins. When nuclei are disrupted, three proteins co-purify with the nuclear envelope fraction and are highly enriched in this fraction. The level of [32P]GTP-binding for all three protein bands is significantly increased when harvested pea plumules are irradiated by red light, and this effect is reversed by far-red light. The results indicate that GTP-binding activity associated with the nuclear envelope of plant cells is photoreversibly regulated by the pigment phytochrome.

  2. Zinc(II) and the single-stranded DNA binding protein of bacteriophage T4

    International Nuclear Information System (INIS)

    Gauss, P.; Krassa, K.B.; McPheeters, D.S.; Nelson, M.A.; Gold, L.

    1987-01-01

    The DNA binding domain of the gene 32 protein of the bacteriophage T4 contains a single zinc-finger sequence. The gene 32 protein is an extensively studied member of a class of proteins that bind relatively nonspecifically to single-stranded DNA. The authors have sequenced and characterized mutations in gene 32 whose defective proteins are activated by increasing the Zn(II) concentration in the growth medium. The results identify a role for the gene 32 protein in activation of T4 late transcription. Several eukaryotic proteins with zinc fingers participate in activation of transcription, and the gene 32 protein of T4 should provide a simple, well-characterized system in which genetics can be utilized to study the role of a zinc finger in nucleic acid binding and gene expression

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

    DEFF Research Database (Denmark)

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

    1999-01-01

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

  4. Importin α5 negatively regulates importin β1-mediated nuclear import of Newcastle disease virus matrix protein and viral replication and pathogenicity in chicken fibroblasts.

    Science.gov (United States)

    Duan, Zhiqiang; Xu, Haixu; Ji, Xinqin; Zhao, Jiafu; Xu, Houqiang; Hu, Yan; Deng, Shanshan; Hu, Shunlin; Liu, Xiufan

    2018-12-31

    The matrix (M) protein of Newcastle disease virus (NDV) is demonstrated to localize in the nucleus via intrinsic nuclear localization signal (NLS), but cellular proteins involved in the nuclear import of NDV M protein and the role of M's nuclear localization in the replication and pathogenicity of NDV remain unclear. In this study, importin β1 was screened to interact with NDV M protein by yeast two-hybrid screening. This interaction was subsequently confirmed by co-immunoprecipitation and pull-down assays. In vitro binding studies indicated that the NLS region of M protein and the amino acids 336-433 of importin β1 that belonged to the RanGTP binding region were important for binding. Importantly, a recombinant virus with M/NLS mutation resulted in a pathotype change of NDV and attenuated viral replication and pathogenicity in chicken fibroblasts and SPF chickens. In agreement with the binding data, nuclear import of NDV M protein in digitonin-permeabilized HeLa cells required both importin β1 and RanGTP. Interestingly, importin α5 was verified to interact with M protein through binding importin β1. However, importin β1 or importin α5 depletion by siRNA resulted in different results, which showed the obviously cytoplasmic or nuclear accumulation of M protein and the remarkably decreased or increased replication ability and pathogenicity of NDV in chicken fibroblasts, respectively. Our findings therefore demonstrate for the first time the nuclear import mechanism of NDV M protein and the negative regulation role of importin α5 in importin β1-mediated nuclear import of M protein and the replication and pathogenicity of a paramyxovirus.

  5. Absence of serum growth hormone binding protein in patients with growth hormone receptor deficiency (Laron dwarfism)

    Energy Technology Data Exchange (ETDEWEB)

    Daughaday, W.H.; Trivedi, B.

    1987-07-01

    It has recently been recognized that human serum contains a protein that specifically binds human growth hormone (hGH). This protein has the same restricted specificity for hGH as the membrane-bound GH receptor. To determine whether the GH-binding protein is a derivative of, or otherwise related to, the GH receptor, the authors have examined the serum of three patients with Laron-type dwarfism, a condition in which GH refractoriness has been attributed to a defect in the GH receptor. The binding of /sup 125/I-labeled hGH incubated with serum has been measured after gel filtration of the serum through an Ultrogel AcA 44 minicolumn. Results are expressed as percent of specifically bound /sup 125/I-hGH and as specific binding relative to that of a reference serum after correction is made for endogenous GH. The mean +/- SEM of specific binding of sera from eight normal adults (26-46 years of age) was 21.6 +/- 0.45%, and the relative specific binding was 101.1 +/- 8.6%. Sera from 11 normal children had lower specific binding of 12.5 +/- 1.95% and relative specific binding of 56.6 +/- 9.1%. Sera from three children with Laron-type dwarfism lacked any demonstrable GH binding, whereas sera from 10 other children with other types of nonpituitary short stature had normal relative specific binding. They suggest that the serum GH-binding protein is a soluble derivative of the GH receptor. Measurement of the serum GH-binding protein may permit recognition of other abnormalities of the GH receptor.

  6. Absence of serum growth hormone binding protein in patients with growth hormone receptor deficiency (Laron dwarfism)

    International Nuclear Information System (INIS)

    Daughaday, W.H.; Trivedi, B.

    1987-01-01

    It has recently been recognized that human serum contains a protein that specifically binds human growth hormone (hGH). This protein has the same restricted specificity for hGH as the membrane-bound GH receptor. To determine whether the GH-binding protein is a derivative of, or otherwise related to, the GH receptor, the authors have examined the serum of three patients with Laron-type dwarfism, a condition in which GH refractoriness has been attributed to a defect in the GH receptor. The binding of 125 I-labeled hGH incubated with serum has been measured after gel filtration of the serum through an Ultrogel AcA 44 minicolumn. Results are expressed as percent of specifically bound 125 I-hGH and as specific binding relative to that of a reference serum after correction is made for endogenous GH. The mean +/- SEM of specific binding of sera from eight normal adults (26-46 years of age) was 21.6 +/- 0.45%, and the relative specific binding was 101.1 +/- 8.6%. Sera from 11 normal children had lower specific binding of 12.5 +/- 1.95% and relative specific binding of 56.6 +/- 9.1%. Sera from three children with Laron-type dwarfism lacked any demonstrable GH binding, whereas sera from 10 other children with other types of nonpituitary short stature had normal relative specific binding. They suggest that the serum GH-binding protein is a soluble derivative of the GH receptor. Measurement of the serum GH-binding protein may permit recognition of other abnormalities of the GH receptor

  7. The Leptospiral Antigen Lp49 is a Two-Domain Protein with Putative Protein Binding Function

    Energy Technology Data Exchange (ETDEWEB)

    Oliveira Giuseppe,P.; Oliveira Neves, F.; Nascimento, A.; Gomes Guimaraes, B.

    2008-01-01

    Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Currently available vaccines have limited effectiveness and therapeutic interventions are complicated by the difficulty in making an early diagnosis of leptospirosis. The genome of Leptospira interrogans was recently sequenced and comparative genomic analysis contributed to the identification of surface antigens, potential candidates for development of new vaccines and serodiagnosis. Lp49 is a membrane-associated protein recognized by antibodies present in sera from early and convalescent phases of leptospirosis patients. Its crystal structure was determined by single-wavelength anomalous diffraction using selenomethionine-labelled crystals and refined at 2.0 Angstroms resolution. Lp49 is composed of two domains and belongs to the all-beta-proteins class. The N-terminal domain folds in an immunoglobulin-like beta-sandwich structure, whereas the C-terminal domain presents a seven-bladed beta-propeller fold. Structural analysis of Lp49 indicates putative protein-protein binding sites, suggesting a role in Leptospira-host interaction. This is the first crystal structure of a leptospiral antigen described to date.

  8. Variations in riboflavin binding by human plasma: identification of immunoglobulins as the major proteins responsible

    International Nuclear Information System (INIS)

    Innis, W.S.; McCormick, D.B.; Merrill, A.H. Jr.

    1985-01-01

    Riboflavin binding by plasma proteins from healthy human subjects was examined by equilibrium dialysis using a physiological concentration of [2-14C]riboflavin (0.04 microM). Binding ranged from 0.080 to 0.917 pmole of riboflavin/mg of protein (with a mean +/- SD of 0.274 +/- 0.206), which corresponded to 4.14 to 49.4 pmole/ml of plasma (15.5 +/- 11.0) (N = 34). Males and females yielded similar results. Upon fractionation of plasma by gel filtration, the major riboflavin-binding components eluted with albumin and gamma-globulins. Albumin was purified and found to bind riboflavin only very weakly (Kd = 3.8 to 10.4 mM), although FMN and photochemical degradation products (e.g., lumiflavine and lumichrome) were more tightly bound. Binding in the gamma-globulin fraction was attributed to IgG and IGA because the binding protein(s) and immunoglobulins copurified using various methods were removed by treatment of plasma with protein A-agarose, and were coincident upon immunoelectrophoresis followed by autoradiography to detect [2-14C]riboflavin. Differences among the plasma samples correlated with the binding recovered with the immunoglobulins. Binding was not directly related to the total IgG or IgA levels of subjects. Hence, it appears that the binding is due to a subfraction of these proteins. These findings suggest that riboflavin-binding immunoglobulins are a major cause of variations in riboflavin binding in human circulation, and may therefore affect the utilization of this micronutrient

  9. A tetrodotoxin-binding protein in the hemolymph of shore crab Hemigrapsus sanguineus: purification and properties.

    Science.gov (United States)

    Nagashima, Yuji; Yamamoto, Kazuhiko; Shimakura, Kuniyoshi; Shiomi, Kazuo

    2002-06-01

    The shore crab Hemigrapsus sanguineus hemolymph contains soluble proteins that bind tetrodotoxin (TTX) and are responsible for high resistance of the crab to TTX. The TTX-binding protein was purified from the hemolymph by ultrafiltration, lectin affinity chromatography and gel filtration HPLC. The purified protein gave only one band in native-polyacrylamide gel electrophoresis (PAGE), confirming its homogeneity. Its molecular weight was estimated to be about 400k by gel filtration HPLC, while it was estimated to be about 82k under non-reducing conditions and about 72 and 82k under reducing conditions by SDS-PAGE, indicating that the TTX-binding protein was composed of at least two distinct subunits. The TTX-binding protein was an acidic glycoprotein with pI 3.5, abundant in Asp and Glu but absent in Trp, and contained 6% reducing sugar and 12% amino sugar. The protein selectively bound to TTX, with a neutralizing ability of 6.7 mouse unit TTX/mg protein, but not to paralytic shellfish poisoning toxins. However, its neutralizing activity was almost lost by treatments with enzymes (protease XIV, thermolysin, trypsin, amyloglucosidase and alpha-amylase) and denaturing agents (1% SDS, 1% dithiothreitol, 8 M urea and 6 M guanidine hydrochloride), suggesting the involvement of both proteinaceous and sugar moieties in the binding to TTX and the importance of the steric conformation of the TTX-binding protein. Copright 2002 Elsevier Science Ltd.

  10. New human erythrocyte protein with binding sites for both spectrin and calmodulin

    International Nuclear Information System (INIS)

    Gardner, K.; Bennett, V.

    1986-01-01

    A new cytoskeletal protein that binds calmodulin has been purified to greater than 95% homogeneity from human erythrocyte cytoskeletons. The protein is a heterodimer with subunits of 103,000 and 97,000 and M/sub r/ = 197,000 calculated from its Stokes radius of 6.9 nm and sedimentation coefficient of 6.8. A binding affinity of this protein for calmodulin has been estimated