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Biochemistry Mar 2010We identified a homologue of the molluscan acetylcholine-binding protein (AChBP) in the marine polychaete Capitella teleta, from the annelid phylum. The amino acid...
We identified a homologue of the molluscan acetylcholine-binding protein (AChBP) in the marine polychaete Capitella teleta, from the annelid phylum. The amino acid sequence of C. teleta AChBP (ct-AChBP) is 21-30% identical with those of known molluscan AChBPs. Sequence alignments indicate that ct-AChBP has a shortened Cys loop compared to other Cys loop receptors, and a variation on a conserved Cys loop triad, which is associated with ligand binding in other AChBPs and nicotinic ACh receptor (nAChR) alpha subunits. Within the D loop of ct-AChBP, a conserved aromatic residue (Tyr or Trp) in nAChRs and molluscan AChBPs, which has been implicated directly in ligand binding, is substituted with an isoleucine. Mass spectrometry results indicate that Asn122 and Asn216 of ct-AChBP are glycosylated when expressed using HEK293 cells. Small-angle X-ray scattering data suggest that the overall shape of ct-AChBP in the apo or unliganded state is similar to that of homologues with known pentameric crystal structures. NMR experiments show that acetylcholine, nicotine, and alpha-bungarotoxin bind to ct-AChBP with high affinity, with K(D) values of 28.7 microM, 209 nM, and 110 nM, respectively. Choline bound with a lower affinity (K(D) = 163 microM). Our finding of a functional AChBP in a marine annelid demonstrates that AChBPs may exhibit variations in hallmark motifs such as ligand-binding residues and Cys loop length and shows conclusively that this neurotransmitter binding protein is not limited to the phylum Mollusca.
Topics: Acetylcholine; Amino Acid Sequence; Animals; Carrier Proteins; Cell Line; Computational Biology; Humans; Magnetic Resonance Spectroscopy; Mass Spectrometry; Models, Molecular; Molecular Sequence Data; Polychaeta; Protein Conformation; Scattering, Small Angle; X-Ray Diffraction
PubMed: 20136097
DOI: 10.1021/bi902023y -
European Journal of Medical Research 2010Prevotella nigrescens, lacking siderophores was found to bind to the hemoproteins. The binding was observed also in the envelope which was prepared by sonication of the...
Prevotella nigrescens, lacking siderophores was found to bind to the hemoproteins. The binding was observed also in the envelope which was prepared by sonication of the cell. The binding occurred in the pH-dependent manner; the binding was observed below neutral pHs of the incubation mixtures but only slightly observed in the neutral and alkaline pHs. Furthermore, hemoglobin bound to the envelope was dissociated at high pHs buffers. Maximum amounts of hemoglobin bound to 1 mg envelope was 51.2 mug. Kd for the reaction at pH 5.0 was 2.1 x 10¹⁰ M (210 pM). From the dot blot assay, hemoglobin could bind to a protein solubilized from the envelope by a detergent, referred to as hemoglobin-binding protein (HbBP), then it was purified by the sequential procedures of ion exchange chromatography, affinity chromatography and isoelectric focusing. Molecular weight and isoelectric point of the HbBP were 46 kDa and 6.1, respectively.
Topics: Bacterial Proteins; Carrier Proteins; Hemoglobins; Prevotella nigrescens
PubMed: 20696644
DOI: 10.1186/2047-783x-15-7-314 -
The Journal of Biological Chemistry Aug 1988The Rd gene is expressed in the livers and oviducts of laying hens and codes for the riboflavin-binding protein (RfBP) of egg yolk and egg white. A lambda gt11 cDNA... (Comparative Study)
Comparative Study
The Rd gene is expressed in the livers and oviducts of laying hens and codes for the riboflavin-binding protein (RfBP) of egg yolk and egg white. A lambda gt11 cDNA library derived from chicken oviduct poly(A)+ RNA was screened with polyclonal rabbit antiserum to chicken RfBP. Positive clones were isolated and rescreened with a mixed oligonucleotide probe corresponding to residues 20-25 of the mature protein. The largest cDNA clone (969 base pairs) was subcloned into plasmid pIBI21, and the nucleotide sequence was determined by the dideoxynucleotide method. This clone contained the entire coding region for RfBP. The published amino acid sequence of the mature protein was confirmed. In addition, the following 17-residue signal peptide was deduced: Met-Leu-Arg-Phe-Ala-Ile-Thr-Leu-Phe-Ala-Val-Ile-Thr-Ser-Ser-Thr-Cys. Unexpectedly, the nucleotide sequence codes for 2 adjacent arginine residues at the carboxyl terminus that are not observed in the mature protein. The amino acid sequence of RfBP is homologous with bovine milk folate-binding protein. Eight of the nine pairs of cysteines involved in disulfide bonds in RfBP are conserved in folate-binding protein, as are all of the tryptophan residues. Sequence identity between homologous regions of these two vitamin-binding proteins is more than 30%.
Topics: Amino Acid Sequence; Animals; Base Sequence; Carrier Proteins; Cattle; Chickens; DNA; Folate Receptors, GPI-Anchored; Membrane Transport Proteins; Milk; Molecular Sequence Data; Receptors, Cell Surface
PubMed: 3403518
DOI: No ID Found -
Proceedings of the National Academy of... Jul 1986An odorant-binding protein (OBP) was isolated from bovine olfactory and respiratory mucosa. We have produced polyclonal antisera to this protein and report its...
An odorant-binding protein (OBP) was isolated from bovine olfactory and respiratory mucosa. We have produced polyclonal antisera to this protein and report its immunohistochemical localization to mucus-secreting glands of the olfactory and respiratory mucosa. Although OBP was originally isolated as a pyrazine binding protein, both rat and bovine OBP also bind the odorants [3H]methyldihydrojasmonate and 3,7-dimethyl-octan-1-ol as well as 2-isobutyl-3-[3H]methoxypyrazine. We detect substantial odorant-binding activity attributable to OBP in secreted rat nasal mucus and tears but not in saliva, suggesting a role for OBP in transporting or concentrating odorants.
Topics: Animals; Binding Sites; Carrier Proteins; Cattle; Chemoreceptor Cells; Fatty Alcohols; Immunosorbent Techniques; Molecular Weight; Nasal Mucosa; Pyrazines; Rats; Receptors, Odorant; Smell; Tears
PubMed: 3523479
DOI: 10.1073/pnas.83.13.4942 -
Infection and Immunity Sep 1992Many streptococcal strains are known to bind the two most abundant plasma proteins, namely, immunoglobulin G and albumin. Protein G isolated from group C and G...
Many streptococcal strains are known to bind the two most abundant plasma proteins, namely, immunoglobulin G and albumin. Protein G isolated from group C and G streptococci has been demonstrated to have separate binding regions for each of these proteins. However, some group G streptococcal strains bind only serum albumin. This report describes the isolation of a 48-kDa albumin-binding protein from such a strain (DG12). The affinity constant of this protein for human serum albumin was determined to be 5 x 10(9) M-1, and the protein interacted strongly also with serum albumin from several other mammalian species. The gene encoding the albumin-binding protein was cloned and expressed in Escherichia coli. DNA sequence analysis of this gene revealed a unique NH2-terminal sequence and three types of repeats in the encoded protein. One of these repeated sequences has significant homology with the albumin-binding domains of protein G, and it was demonstrated that the albumin binding of the DG12 protein was localized within these domains. Another type of repeat is localized in the putative wall-spanning region of the molecule. This repeat sequence, which has the length of only 4 amino acids (LysProGluVal), is repeated 14 times. The relationship of the albumin-binding protein to other cell-wall-associated proteins of pathogenic streptococci is discussed.
Topics: Amino Acid Sequence; Base Sequence; Carrier Proteins; Cloning, Molecular; Molecular Sequence Data; Serum Albumin; Streptococcus
PubMed: 1500168
DOI: 10.1128/iai.60.9.3601-3608.1992 -
The Biochemical Journal May 1988The complete amino acid sequence of a fatty acid-binding protein from human heart was determined by automated Edman degradation of CNBr, BNPS-skatole...
The complete amino acid sequence of a fatty acid-binding protein from human heart was determined by automated Edman degradation of CNBr, BNPS-skatole [3'-bromo-3-methyl-2-(2-nitrobenzenesulphenyl)indolenine], hydroxylamine, Staphylococcus aureus V8 proteinase, tryptic and chymotryptic peptides, and by digestion of the protein with carboxypeptidase A. The sequence of the blocked N-terminal tryptic peptide from citraconylated protein was determined by collisionally induced decomposition mass spectrometry. The protein contains 132 amino acid residues, is enriched with respect to threonine and lysine, lacks cysteine, has an acetylated valine residue at the N-terminus, and has an Mr of 14768 and an isoelectric point of 5.25. This protein contains two short internal repeated sequences from residues 48-54 and from residues 114-119 located within regions of predicted beta-structure and decreasing hydrophobicity. These short repeats are contained within two longer repeated regions from residues 48-60 and residues 114-125, which display 62% sequence similarity. These regions could accommodate the charged and uncharged moieties of long-chain fatty acids and may represent fatty acid-binding domains consistent with the finding that human heart fatty acid-binding protein binds 2 mol of oleate or palmitate/mol of protein. Detailed evidence for the amino acid sequences of the peptides has been deposited as Supplementary Publication SUP 50143 (23 pages) at the British Library Lending Division, Boston Spa, Yorkshire LS23 7BQ, U.K., from whom copies may be obtained as indicated in Biochem. J. (1988) 249, 5.
Topics: Amino Acid Sequence; Carrier Proteins; Chromatography, Gel; Circular Dichroism; Fatty Acid-Binding Protein 7; Fatty Acid-Binding Proteins; Fatty Acids; Humans; Mass Spectrometry; Molecular Sequence Data; Myocardium; Neoplasm Proteins; Tumor Suppressor Proteins
PubMed: 3421901
DOI: 10.1042/bj2520191 -
Protein Science : a Publication of the... May 2018Computational protein design is still a challenge for advancing structure-function relationships. While recent advances in this field are promising, more information for...
Computational protein design is still a challenge for advancing structure-function relationships. While recent advances in this field are promising, more information for genuine predictions is needed. Here, we discuss different approaches applied to install novel glutamine (Gln) binding into the Lysine/Arginine/Ornithine binding protein (LAOBP) from Salmonella typhimurium. We studied the ligand binding behavior of two mutants: a binding pocket grafting design based on a structural superposition of LAOBP to the Gln binding protein QBP from Escherichia coli and a design based on statistical coupled positions. The latter showed the ability to bind Gln even though the protein was not very stable. Comparison of both approaches highlighted a nonconservative shared point mutation between LAOBP_graft and LAOBP_sca. This context dependent L117K mutation in LAOBP turned out to be sufficient for introducing Gln binding, as confirmed by different experimental techniques. Moreover, the crystal structure of LAOBP_L117K in complex with its ligand is reported.
Topics: Amino Acids; Bacterial Proteins; Binding Sites; Carrier Proteins; Ligands; Models, Molecular; Mutation; Protein Conformation; Salmonella typhimurium; Thermodynamics
PubMed: 29524280
DOI: 10.1002/pro.3403 -
The Journal of Biological Chemistry Feb 1993The 49-kDa poly(A)-binding protein II (PAB II) was purified to homogeneity from calf thymus. The 70-kDa poly(A)-binding protein I (PAB I) was obtained in different...
The 49-kDa poly(A)-binding protein II (PAB II) was purified to homogeneity from calf thymus. The 70-kDa poly(A)-binding protein I (PAB I) was obtained in different fractions of the same preparation. Whereas PAB II stimulated poly(A) polymerase, PAB I was an inhibitor. In analytical ultracentrifugation, the predominant form of PAB II was a monomer of 50.3 kDa. A sedimentation constant of only 2.2 S indicated a distinctly non-spherical shape. Binding was specific for single-stranded purine polyribonucleotides. The dependence of the dissociation constant on the length of oligoriboadenylate indicated a binding site size of 12 nucleotides. A single site was bound with a KD of 2 x 10(-9) M, as determined by nitrocellulose filter binding assays. From fluorescence quenching and gel retardation experiments, the packing ratio on poly(A) was estimated as 23 nucleotides/protein monomer.
Topics: Animals; Binding, Competitive; Carrier Proteins; Cattle; Chromatography; Molecular Weight; Poly A; Poly(A)-Binding Proteins; Protein Binding; RNA-Binding Proteins; Spectrometry, Fluorescence; Ultracentrifugation
PubMed: 8428968
DOI: No ID Found -
The Journal of Biological Chemistry Jun 1990The plasma of the crayfish Pacifastacus leniusculus contains a protein which is able to bind to laminarin (a soluble beta-1,3-glucan) and which has been isolated by two... (Comparative Study)
Comparative Study
The plasma of the crayfish Pacifastacus leniusculus contains a protein which is able to bind to laminarin (a soluble beta-1,3-glucan) and which has been isolated by two independent methods, affinity precipitation with a beta-1,3-glucan or immunoaffinity chromatography. The purified beta-1,3-glucan binding protein was homogenous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is a monomeric glycoprotein with a molecular mass of approximately 100,000 Da and an isoelectric point of approximately 5.0. Amino acid analysis showed a very high similarity with the amino acid composition of beta-1,3-glucan binding proteins recently purified from two insects, the cockroach Blaberus craniifer and the silkworm Bombyx mori. The N-terminal amino acid sequence was determined to be: H2N-Asp-Ala-Gly-X-Ala-Ser-Leu-Val-Thr-Asn-Phe-Asn-Ser-Ala-Lys-Leu-X-X-Ly s--- Using monospecific rabbit polyclonal antibodies, the presence of this protein has also been shown within the blood cells. The purified beta-1,3-glucan binding protein did not show any peptidase or phenoloxidase activity but was able to enhance the activation of hemocyte-derived peptidase and prophenoloxidase only in the presence of the beta-1,3-glucan, laminarin, whereas mannan, dextran (alpha-glucan), or cellulose (beta-1,4-glucan) incubated with the beta-1,3-glucan binding protein had no effect on these enzyme activities. The beta-1,3-glucan binding protein could only be affinity-precipitated from crayfish plasma by the beta-1,3-glucans laminarin or curdlan (an insoluble beta-1,3-glucan), while mannan or dextran did not bind to the beta-1,3-glucan binding protein. No hemagglutinating activity of the purified beta-1,3-glucan binding protein could be detected.
Topics: Amino Acid Sequence; Amino Acids; Animals; Astacoidea; Carrier Proteins; Chemical Precipitation; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Glucans; Immunoassay; Isoelectric Point; Molecular Sequence Data; Molecular Weight; Monophenol Monooxygenase; Peptide Hydrolases; Polysaccharides; beta-Glucans
PubMed: 2111817
DOI: No ID Found -
The Plant Cell Dec 1992Sucrose transport from the apoplasm, across the plasma membrane, and into the symplast is critical for growth and development in most plant species. Phloem loading, the...
Sucrose transport from the apoplasm, across the plasma membrane, and into the symplast is critical for growth and development in most plant species. Phloem loading, the process of transporting sucrose against a concentration gradient into the phloem, is an essential first step in long-distance transport of sucrose and carbon partitioning. We report here that a soybean 62-kD sucrose binding protein is associated with the plasma membrane of several cell types engaged in sucrose transport, including the mesophyll cells of young sink leaves, the companion cells of mature phloem, and the cells of the developing cotyledons. Furthermore, the temporal expression of the gene and the accumulation pattern of the protein closely parallel the rate of sucrose uptake in the cotyledon. Molecular cloning and sequence analysis of a full-length cDNA for this 62-kD sucrose binding protein indicated that the protein is not an invertase, contains a 29-amino acid leader peptide that is absent from the mature protein, and is not an integral membrane protein. We conclude that the 62-kD sucrose binding protein is involved in sucrose transport, but is not performing this function independently.
Topics: Amino Acid Sequence; Base Sequence; Carrier Proteins; DNA; Membrane Transport Proteins; Microscopy, Fluorescence; Microscopy, Immunoelectron; Molecular Sequence Data; Plant Lectins; Plant Proteins; Soybean Proteins; Glycine max; Sucrose
PubMed: 1467654
DOI: 10.1105/tpc.4.12.1561