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The Biochemical Journal Nov 1992The poly(A)(+)-binding protein (PABP) is a highly conserved protein that binds to the poly(A)+ tail of mRNAs. PABP has been shown to regulate message stability and...
The poly(A)(+)-binding protein (PABP) is a highly conserved protein that binds to the poly(A)+ tail of mRNAs. PABP has been shown to regulate message stability and translational efficiency, yet the mechanisms remain unknown. To facilitate further dissection of the functions of this protein, we have expressed and purified Xenopus PABP using a baculovirus expression system. At 48 h after infection of insect Spodoptera frugiperda (Sf9) cells with recombinant virus, approx. 3% of cell protein was PABP. Purification of PABP was achieved by affinity chromatography on poly(A)(+)-Sepharose. The purified protein was indistinguishable from Xenopus PABP with respect to its immunoreactivity and electrophoretic mobility. Furthermore, the recombinant PABP was expressed and purified as a functional protein as indicated by its ability to bind to poly(A)(+)-Sepharose and its ability to enhance the translation of adenylated messages in vitro. By comparing protein extracts from various developmental stages of Xenopus embryos with known amounts of purified PABP, we determined the amount of PABP per embryo. This analysis suggested that there is less than one PABP molecule available per PABP-binding site at early stages of development, and only a slight excess of PABP at later stages.
Topics: Animals; Baculoviridae; Blotting, Western; Carrier Proteins; Cell Line; Cloning, Molecular; Genetic Vectors; Moths; Poly A; Poly(A)-Binding Proteins; Protein Biosynthesis; RNA; Recombinant Proteins; Xenopus
PubMed: 1280105
DOI: 10.1042/bj2870761 -
Journal of Bacteriology Jul 1995We previously reported that group D streptococci exhibited immunoglobulin G (IgG)-binding activity and that a 52-kDa IgG-binding protein was present in all Streptococcus...
We previously reported that group D streptococci exhibited immunoglobulin G (IgG)-binding activity and that a 52-kDa IgG-binding protein was present in all Streptococcus suis strains examined (B. Serhir, R. Higgins, B. Foiry, and M. Jacques, J. Gen. Microbiol. 139:2953-2958, 1993). The objective of the present study was to purify and characterize this protein. Pig IgG were immobilized through their Fab fragments to ECH-Sepharose 4B, and the protein was purified by affinity chromatography. Electron microscopy observations of the purified material showed filamentous structures with a diameter of approximately 4 nm; these structures were not observed when the material was treated with either urea or ethanolamine. Electrophoretic and Western immunoblot analyses showed that the 52-kDa protein constituted the bulk of the recovered material. This protein was stained with either Coomassie brilliant blue or silver nitrate; it reacted with a large variety of mammalian IgG, human IgG (Fc) fragments, human IgA, and other human plasma proteins. The 52-kDa protein exhibited lower IgG-binding affinities than protein A and protein G. However, it was able to compete with protein A and protein G for binding to human IgG. In addition, it bound chicken IgG with high affinity. This last property differentiated the 52-kDa protein of S. suis from the six IgG-binding proteins described to date. The 52-kDa protein displayed similar affinities for untreated and deglycosylated pig IgG. The N-terminal amino acid sequence (SIITDVYAXEVLDSXGNPTLEV) revealed no homology with any bacterial proteins in the Swiss-Prot database. Its isoelectric point of approximately 4.6 and its amino acid composition, rich in aspartic and glutamic acids, showed that it had some similarities with other IgG-binding proteins. In this report, we have purified and characterized a 52-kDa IgG-binding protein from S. suis capsular type 2. Although this protein shares some similarities with other IgG- and/or IgA-binding proteins, it is unique in reacting with chicken IgG.
Topics: Amino Acid Sequence; Amino Acids; Animals; Bacterial Capsules; Bacterial Proteins; Binding, Competitive; Blotting, Western; Carrier Proteins; Chickens; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Glycoproteins; Immunoglobulin Isotypes; Isoelectric Focusing; Molecular Sequence Data; Protein Binding; Sequence Analysis; Species Specificity; Streptococcus suis; Swine
PubMed: 7601850
DOI: 10.1128/jb.177.13.3830-3836.1995 -
Journal of Biochemistry Nov 1994Midkine (MK) is a heparin-binding growth/differentiation factor with a molecular weight of 13 kDa, and is structurally unrelated to fibroblast growth factors (FGF). We...
Midkine (MK) is a heparin-binding growth/differentiation factor with a molecular weight of 13 kDa, and is structurally unrelated to fibroblast growth factors (FGF). We studied MK-binding proteins in order to clarify the action mechanism of MK. A 100-kDa protein was identified in PYS-2, 3T3, and L cells as an MK-binding protein by a ligand blot experiment. This MK-binding protein was purified by affinity chromatography on an MK-agarose column followed by SDS polyacrylamide gel electrophoresis. Sequence determination of N-terminal 23 amino acid residues revealed that the MK-binding protein was nucleolin, a major nucleolar protein, which functions as a shuttle protein between the nucleus and cytoplasm and is located also on the cell surface. Heparin-binding growth associated molecule (HB-GAM), which has 50% sequence identity with MK, fused to maltose-binding protein also bound to nucleolin. On the other hand, basic FGF (bFGF) scarcely bound to nucleolin in the absence of heparin, while both MK and bFGF bound weakly to nucleolin in the presence of heparin. Nuclear localization of MK was shown in hemangioma cells by immunohistochemical staining. These findings supported the hypothesis that parts of the MK and HB-GAM are translocated to the nucleus after binding with nucleolin.
Topics: Amino Acid Sequence; Animals; Base Sequence; Carrier Proteins; Cells, Cultured; Cytokines; Fibroblast Growth Factor 2; Mice; Microscopy, Fluorescence; Microscopy, Phase-Contrast; Midkine; Molecular Sequence Data; Nuclear Proteins; Phosphoproteins; RNA-Binding Proteins; Sequence Homology; Nucleolin
PubMed: 7896734
DOI: 10.1093/oxfordjournals.jbchem.a124628 -
Nature Communications 2013Acetylcholine-binding protein is a water-soluble homologue of the extracellular ligand-binding domain of cys-loop receptors. It is used as a structurally accessible...
Acetylcholine-binding protein is a water-soluble homologue of the extracellular ligand-binding domain of cys-loop receptors. It is used as a structurally accessible prototype for studying ligand binding to these pharmaceutically important pentameric ion channels, in particular to nicotinic acetylcholine receptors, due to conserved binding site residues present at the interface between two subunits. Here we report that an aromatic conjugated small molecule binds acetylcholine-binding protein in an ordered π-π stack of three identical molecules per binding site, two parallel and one antiparallel. Acetylcholine-binding protein stabilizes the assembly of the stack by aromatic contacts. Thanks to the plasticity of its ligand-binding site, acetylcholine-binding protein can accommodate the formation of aromatic stacks of different size by simple loop repositioning and minimal adjustment of the interactions. This type of supramolecular binding provides a novel paradigm in drug design.
Topics: Acetylcholine; Acridine Orange; Animals; Binding Sites; Carrier Proteins; Crystallography, X-Ray; Electrons; Fluorescence; Ligands; Models, Molecular; Protein Binding
PubMed: 23695669
DOI: 10.1038/ncomms2900 -
FEBS Letters Dec 1989The human regulatory complement component C4b-binding protein (C4BP) circulates in plasma either as a free protein or in a bimolecular complex with the vitamin...
The human regulatory complement component C4b-binding protein (C4BP) circulates in plasma either as a free protein or in a bimolecular complex with the vitamin K-dependent protein S. The major form of C4BP is composed of 7 identical, disulfide-linked 70 kDa subunits (alpha-chains), the arrangement of which gives the C4BP molecule a spider-like appearance. Recently, we identified a unique 45 kDa subunit (beta-chain) in C4BP. We have now isolated a subpopulation of C4BP, which does not bind protein S. This C4BP species, which had a molecular weight slightly lower than that of the predominant form, was found to lack the beta-chain. Another lower molecular weight form of C4BP was also purified. It contained the beta-chain and was efficient in binding protein S. Its subunit composition was judged to comprise six alpha-chains and one beta-chain. These results indicate C4BP in plasma to be heterogeneous at a molecular level vis-a-vis subunit composition and/or protein S binding ability and provide support for the concept that the beta-chain of C4BP contains the single protein S binding site.
Topics: Blotting, Western; Carrier Proteins; Complement Inactivator Proteins; Complement System Proteins; Glycoproteins; Humans; Macromolecular Substances; Molecular Weight; Protein Binding; Protein S
PubMed: 2532155
DOI: 10.1016/0014-5793(89)81492-9 -
PloS One 2014A previously uncharacterized protein with a carotenoid-binding function has been isolated and characterized from the gonad of the New Zealand sea urchin Evechinus...
A previously uncharacterized protein with a carotenoid-binding function has been isolated and characterized from the gonad of the New Zealand sea urchin Evechinus chloroticus. The main carotenoid bound to the protein was determined by reversed phase-high performance liquid chromatography to be 9'-cis-echinenone and hence this 15 kDa protein has been called an echinenone-binding protein (EBP). Purification of the EBP in quantity from the natural source proved to be challenging. However, analysis of EBP by mass spectrometry combined with information from the Strongylocentrotus purpuratus genome sequence and the recently published E. chloroticus transcriptome database, enabled recombinant expression of wild type EBP and also of a cysteine61 to serine mutant that had improved solubility characteristics. Circular dichroism data and ab initio structure prediction suggests that the EBP adopts a 10-stranded β-barrel fold consistent with that of fatty acid-binding proteins. Therefore, EBP may represent the first report of a fatty acid-binding protein in complex with a carotenoid.
Topics: Amino Acid Sequence; Animals; Carotenoids; Carrier Proteins; Fatty Acid-Binding Proteins; Gene Expression; Gonads; Mass Spectrometry; Molecular Sequence Data; Protein Binding; Protein Structure, Secondary; Recombinant Proteins; Sea Urchins; Sequence Alignment
PubMed: 25192378
DOI: 10.1371/journal.pone.0106465 -
Bioscience, Biotechnology, and... Sep 2000The biliverdin-binding protein from the larval hemolymph of the swallowtail butterfly, Papilio xuthus L., was purified and characterized. The crude biliverdin-binding...
The biliverdin-binding protein from the larval hemolymph of the swallowtail butterfly, Papilio xuthus L., was purified and characterized. The crude biliverdin-binding protein, obtained by ammonium sulfate fractionation, was purified in two steps, the first one by gel filtration chromatography and the second one by ion-exchange chromatography. The molecular mass of the purified protein was analyzed by SDS-polyacrylamide gel electrophoresis and estimated to be 21 kDa. The Namino terminal sequence of P. xuthus biliverdin-binding protein analyzed up to the 19th residue showed that 42% of the amino acid sequence are sequence similarity to the bilin-binding protein from Pieris brassicae. These results suggest that the P. xuthus biliverdin-binding protein belongs to the insecticyanin-type.
Topics: Amino Acid Sequence; Amino Acids; Animals; Biliverdine; Butterflies; Carrier Proteins; Electrophoresis, Polyacrylamide Gel; Hemolymph; Insect Proteins; Invertebrate Hormones; Larva; Molecular Sequence Data; Molecular Weight; Sequence Alignment; Sequence Homology, Amino Acid
PubMed: 11055407
DOI: 10.1271/bbb.64.1978 -
The Journal of Biological Chemistry Apr 2003Retinoid interactions determine the function of the cellular retinaldehyde binding protein (CRALBP) in the rod visual cycle where it serves as an 11-cis-retinol acceptor...
Retinoid interactions determine the function of the cellular retinaldehyde binding protein (CRALBP) in the rod visual cycle where it serves as an 11-cis-retinol acceptor for the enzymatic isomerization of all-trans- to 11-cis-retinol and as a substrate carrier for 11-cis-retinol dehydrogenase (RDH5). Based on preliminary NMR studies suggesting retinoid interactions with Met and Trp residues, human recombinant CRALBP (rCRALBP) with altered Met or Trp were produced and analyzed for ligand interactions. The primary structures of the purified proteins were verified for mutants M208A, M222A, M225A, W165F, and W244F, then retinoid binding properties and substrate carrier functions were evaluated. All the mutant proteins bound 11-cis- and 9-cis-retinal and therefore were not grossly misfolded. Altered UV-visible spectra and lower retinoid binding affinities were observed for the mutants, supporting modified ligand interactions. Altered kinetic parameters were observed for RDH5 oxidation of 11-cis-retinol bound to rCRALBP mutants M222A, M225A, and W244F, supporting impaired substrate carrier function. Heteronuclear single quantum correlation NMR analyses confirmed localized structural changes upon photoisomerization of rCRALBP-bound 11-cis-retinal and demonstrated ligand-dependent conformational changes for residues Met-208, Met-222, Trp-165, and Trp-244. Furthermore, residues Met-208, Met-222, Met-225, and Trp-244 are within a region exhibiting high homology to the ligand binding cavity of phosphatidylinositol transfer protein. Overall the data implicate Trp-165, Met-208, Met-222, Met-225, and Trp-244 as components of the CRALBP ligand binding cavity.
Topics: Amino Acid Sequence; Binding Sites; Carrier Proteins; Gene Expression; Humans; Kinetics; Ligands; Molecular Sequence Data; Mutagenesis, Site-Directed; Nuclear Magnetic Resonance, Biomolecular; Protein Interaction Mapping; Protein Structure, Tertiary; Retinoids; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet
PubMed: 12536149
DOI: 10.1074/jbc.M212775200 -
Crystal structures of the liganded and unliganded nickel-binding protein NikA from Escherichia coli.The Journal of Biological Chemistry Dec 2003Bacteria have evolved a number of tightly controlled import and export systems to maintain intracellular levels of the essential but potentially toxic metal nickel....
Bacteria have evolved a number of tightly controlled import and export systems to maintain intracellular levels of the essential but potentially toxic metal nickel. Nickel homeostasis systems include the dedicated nickel uptake system nik found in Escherichia coli, a member of the ABC family of transporters, that involves a periplasmic nickel-binding protein, NikA. This is the initial nickel receptor and mediator of the chemotactic response away from nickel. We have solved the crystal structure of NikA protein in the presence and absence of nickel, showing that it behaves as a "classical" periplasmic binding protein. In contrast to other binding proteins, however, the ligand remains accessible to the solvent and is not completely enclosed. No direct bonds are formed between the metal cation and the protein. The nickel binding site is apolar, quite unlike any previously characterized protein nickel binding site. Despite relatively weak binding, NikA is specific for nickel. Using isothermal titration calorimetry, the dissociation constant for nickel was found to be approximately 10 microm and that for cobalt was approximately 20 times higher.
Topics: ATP-Binding Cassette Transporters; Binding Sites; Calorimetry; Carrier Proteins; Cloning, Molecular; Crystallography, X-Ray; Dose-Response Relationship, Drug; Escherichia coli; Escherichia coli Proteins; Kinetics; Ligands; Models, Molecular; Nickel; Protein Binding; Protein Structure, Secondary; Ultracentrifugation
PubMed: 12960164
DOI: 10.1074/jbc.M307941200 -
PloS One 2013Polypyrimidine tract-binding (PTB) proteins are RNA-binding proteins that generally contain four RNA recognition motifs (RRMs). In potato, six cDNAs encoding full-length...
Polypyrimidine tract-binding (PTB) proteins are RNA-binding proteins that generally contain four RNA recognition motifs (RRMs). In potato, six cDNAs encoding full-length PTB proteins have been identified. In the present study Nova1-like protein, designated StNova1, was identified as a potential interacting partner of the StPTB proteins via yeast two-hybrid screening. Nova protein is a RNA-binding protein that contains three K-homology (KH) domains. In humans, these proteins are involved in regulation of neuronal RNA metabolism but the role of Nova-like proteins in plants is poorly understood. We have validated this interaction and mapped the protein binding region on StNova1 and StPTB1 and -6 using a novel domain interaction phage display (DIPP) technique. The interaction between the two RNA-binding proteins StPTB1/6 and StNova1 is mediated through linker regions that are distinctly separated from the RRMs. Furthermore, using a random 21-mer phage-peptide library, we have identified a number of peptides with the consensus sequence motif [S/G][V/I][L/V]G that recognize the StPTB proteins. One over-represented peptide that recognizes StPTB6 contains the GVLGPWP sequence that is similar to the GIGGRYP sequence in the glycine-rich linker region between the KH2 and KH3 domains of StNova1. We show, through site-specific mutations, the importance of glycine and proline residues in StNova1-StPTB interactions.
Topics: Amino Acid Motifs; Amino Acid Sequence; Antigens, Neoplasm; Binding Sites; Carrier Proteins; Cell Surface Display Techniques; Molecular Sequence Data; Mutation; Nerve Tissue Proteins; Neuro-Oncological Ventral Antigen; Peptide Library; Polypyrimidine Tract-Binding Protein; Protein Binding; Protein Interaction Domains and Motifs; Protein Interaction Mapping; RNA-Binding Proteins; Sequence Alignment; Solanum tuberosum
PubMed: 23717658
DOI: 10.1371/journal.pone.0064783