An ABA-binding protein with nucleic acid-binding property

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A cold-regulated nucleic acid-binding protein of winter wheat shares a domain with bacterial cold shock proteins

The molecular mechanisms of cold acclimation are still largely unknown; however, it has been established that overwintering plants such as winter wheat increases freeze tolerance during cold treatments. In prokaryotes, cold shock proteins are induced by temperature downshifts and have been proposed to function as RNA chaperones. A wheat cDNA encoding a putative nucleic acid-binding protein, WCSP1, was isolated and found to be homologous to the predominant CspA of Escherichia coli. The putative WCSP1 protein contains a three-domain structure consisting of an N-terminal cold shock domain with two internal conserved consensus RNA binding domains and an internal glycine-rich region, which is interspersed with three C-terminal CX(2)CX(4)HX(4)C (CCHC) zinc fingers. Each domain has been described independently within several nucleotide-binding proteins. Northern and Western blot analyses showed that WCSP1 mRNA and protein levels steadily increased during cold acclimation, respectively. WCSP1 induction was cold-specific because neither abscisic acid treatment, drought, salinity, nor heat stress induced WCSP1 expression. Nucleotide binding assays determined that WCSP1 binds ssDNA, dsDNA, and RNA homopolymers. The capacity to bind dsDNA was nearly eliminated in a mutant protein lacking C-terminal zinc fingers. Structural and expression similarities to E. coli CspA suggest that WCSP1 may be involved in gene regulation during cold acclimation.     

Annotating nucleic acid-binding function based on protein structure

Many of the targets of structural genomics will be proteins with little or no structural similarity to those currently in the database. Therefore, novel function prediction methods that do not rely on sequence or fold similarity to other known proteins are needed. We present an automated approach to predict nucleic-acid-binding (NA-binding) proteins, specifically DNA-binding proteins. The method is based on characterizing the structural and sequence properties of large, positively charged electrostatic patches on DNA-binding protein surfaces, which typically coincide with the DNA-binding-sites. Using an ensemble of features extracted from these electrostatic patches, we predict DNA-binding proteins with high accuracy. We show that our method does not rely on sequence or structure homology and is capable of predicting proteins of novel-binding motifs and protein structures solved in an unbound state. Our method can also distinguish NA-binding proteins from other proteins that have similar, large positive electrostatic patches on their surfaces, but that do not bind nucleic acids.     

Cloning and tissue expression of chicken heart fatty acid-binding protein and intestine fatty acid-binding protein genes

Fatty acid-binding proteins (FABPs) are members of a superfamily of lipid-binding proteins, occurring intracellularly in invertebrates and vertebrates. This study was designed to clone and characterize the genes of heart fatty acid-binding protein and intestine fatty acid-binding protein in the chicken. PCR primers were designed according to the chicken EST sequences to amplify cDNA of H-FABP and I-FABP genes from chicken heart and intestinal tissues. Analysis of sequence showed that the cDNA of the chicken H-FABP gene is 75 to 77% homologues to human, mouse, and pig H-FABP genes, and the chicken I-FABP gene is 71 to 72% homologues to human, mouse, and pig I-FABP genes. In addition, Northern blot analysis indicated that of the two genes, similar to the copartner of the mammal, H-FABP gene was expressed in a wide variety of tissues, and I-FABP gene was expressed only in intestinal tissues. The expression levels of the chicken H-FABP mRNA in heart and I-FABP mRNA in intestine had significant differences between the broilers from fat line and Bai'er layers at six weeks of age. The results of this study provided basic molecular information for studying the role of two FABPs in the regulation of fatty acid metabolism in avian species.     

A human gene coding for a membrane-associated nucleic acid-binding protein

Studies to clone a cell-surface DNA-binding protein involved in the binding and internalization of extracellular DNA have led to the isolation of a gene for a membrane-associated nucleic acid-binding protein (MNAB). The full-length cDNA is 4.3 kilobases with an open reading frame of 3576 base pairs encoding a protein of approximately 130 kDa (GenBank accession numbers and ). The MNAB gene is on human chromosome 9 with wide expression in normal tissues and tumor cells. A C3HC4 RING finger and a CCCH zinc finger have been identified in the amino-terminal half of the protein. MNAB bound DNA (K(D) approximately 4 nm) and mutagenesis of a single conserved amino acid in the zinc finger reduced DNA binding by 50%. A potential transmembrane domain exists near the carboxyl terminus. Antibodies against the amino-terminal half of the protein immunoprecipitated a protein of molecular mass approximately 150 kDa and reacted with cell surfaces. The MNAB protein is membrane-associated and primarily localized to the perinuclear space, probably to the endoplasmic reticulum or trans-Golgi network. Characterization of the MNAB protein as a cell-surface DNA-binding protein, critical in binding and internalization of extracellular DNA, awaits confirmation of its localization to cell surfaces.     

A single-stranded nucleic acid-binding protein from Artemia salina. II. Interaction with nucleic acids

A helix-destabilizing protein, HD40 (Mr 40,000), isolated from the cytoplasm of Artemia salina (Marvil, D.K., Nowak, L., and Szer, W. (1980) J. Biol. Chem. 255, 6466-6472) stoichiometrically disrupts the secondary structures of synthetic single-stranded and helical polynucleotides (e.g. poly(rA), poly(dA), poly(rC), poly(dC), and poly(rU)) as well as those of natural polynucleotides (e.g. MS2 RNA and phi X174 viral DNA). The conformations of double-stranded DNA and double- or triple-stranded synthetic polynucleotides are not affected by the protein. Formation of duplexes, e.g. poly(rA . rU), is prevented by HD40 at 25 to 50 mM but not at 100 to 140 mM NaCl. The unwinding of the residual secondary structure of RNA and DNA by HD40 is not highly cooperative and has a stoichiometry of one HD40 per 12 to 15 nucleotides. The addition of HD40 in excess of 1 molecule per 12 to 15 nucleotides results in the cooperative formation of distinct bead-like structures along the nucleic acid strand. The beads are about 20 nm in diameter with a center to center distance of about 40 nm. The appearance of the beads is not accompanied by any spectral changes (CD and UV) beyond those obtained at a stoichiometry of one HD40 molecule per 12 to 15 nucleotides.     

Studies of the fatty acid-binding site of rat liver fatty acid-binding protein using fluorescent fatty acids

Rat liver fatty acid-binding protein (FABP) is a 14.3-kDa cytosolic protein which binds long chain free fatty acids (ffa) and is believed to participate in intracellular movement and/or distribution of ffa. In the studies described here fluorescently labeled ffa were used to examine the physical nature of the ffa-binding site on FABP. The fluorescent analogues were 16- and 18-carbon ffa with an anthracene moiety covalently attached at eight different points along the length of the hydrocarbon chain (AOffa). Emission maxima of all FABP-bound AOffa were found to be considerably blue-shifted with respect to emission of phospholipid membrane-bound AOffa, suggesting a high degree of motional constraint for protein-bound ffa. Large fluorescence quantum yields and long excited state life-times indicate that the FABP-binding site for ffa is highly hydrophobic. Analysis of rotational correlation times for the FABP-bound AOffa suggest that the ffa are tightly bound to the protein. Variation of the quantum yield with attachment site suggests that the carboxylic acid group of the fatty acyl chain is located near the aqueous surface of the FABP. The rest of the ffa hydrocarbon chain is buried within the protein in a hydrophobic pocket and is particularly constrained at the midportion of the acyl chain.     

Disulfide bonds in rat cutaneous fatty acid-binding protein

Unlike other fatty acid-binding proteins, cutaneous (epidermal) fatty acid-binding proteins contain a large number of cysteine residues. The status of the five cysteine residues in rat cutaneous fatty acid-binding protein was examined by chemical and mass-spectrometric analyses. Two disulfide bonds were identified, between Cys-67 and Cys-87, and between Cys-120 and Cys-127, though extent of formation of the first disulfide bond was rather low in another preparation. Cys-43 was free cysteine. Homology modeling study of the protein indicated the close proximity of the sulfur atoms of these cysteine pairs, supporting the presence of the disulfide bonds. These disulfide bonds appear not to be directly involved in fatty acid-binding activity, because a recombinant rat protein expressed in Escherichia coli in which all five cysteines are fully reduced showed fatty acid-binding activity as examined by displacement of a fluorescent fatty acid analog by long-chain fatty acids. However, the fact that the evolutionarily distant shark liver fatty acid-binding protein also has a disulfide bond corresponding to the one between Cys-120 and Cys-127, and that fatty acid-binding proteins play multiple roles suggests that some functions of cutaneous fatty acid-binding protein might be regulated by the cellular redox state through formation and reduction of disulfide bonds. Although we cannot completely exclude the possibility of oxidation during preparation and analysis, it is remarkable that a protein in cytosol under normally reducing conditions appears to contain disulfide bonds.     

SPKK, a new nucleic acid-binding unit of protein found in histone.

A new DNA-binding unit of a protein different from the alpha-helix, the beta-sheet and the Zn-finger is proposed based on the analysis of the structure of the N-terminus of sea urchin spermatogenous histone H1. DNA-binding arms of the sea urchin spermatogenous histones, H1 and H2B, are composed of repeats of Ser-Pro-Lys(Arg)-Lys(Arg) (SPKK) residues. A six-times repeat of SPKK (S6 peptide) was isolated from H1 and the competition of S6 for DNA binding with a DNA-binding dye, Hoechst 33258, was analysed. The S6 peptide is shown to be a competitive inhibitor of Hoechst 33258, and it is concluded that the SPKK repeat binds to DNA in its minor groove with a binding constant, KS6 = 1.67 X 10(10) M-1. The circular dichroism (CD) spectrum of a synthetic peptide, SPRKSPRK (S2 peptide), is quite different from those of both the alpha-helix and the beta-sheet and resembles that of a random coil. From statistical consideration of protein structures it is proposed that SPKK forms a compact beta-turn stabilized by an additional hydrogen bond. Since a repeated chain of such turn of SPKK offers a repeat of amides of Ser residues at a distance similar to that of DNA-binding amides of the drugs, Hoechst 33258 and netropsin, and since the amides of these drugs bind to DNA replacing the spine of hydration in a minor groove, it is proposed that a repeat of SPKK binds to DNA in the minor groove using similar hydrogen bonds.     

The 12 kDa protein of potato virus M displays properties of a nucleic acid-binding regulatory protein

The 3' terminal 1.4 kb segment of potato virus M (PVM) genomic RNA was cloned and sequenced. This part of the viral genome encodes the capsid protein CP as well as a 12 kDa protein of as yet unknown function. Both proteins were expressed in bacteria and their nucleic acid-binding properties studied. The 12 kDa protein (pr12), but not the capsid protein bound single- and double-stranded nucleic acids. This property of pr12 in conjunction with a zinc finger motif located adjacent to a basic region of the 12 kDa protein suggests that it may act as a regulatory factor during virus replication.     

Characterization of a fatty acid-binding protein from rat heart

A fatty acid-binding protein has been isolated from rat heart and purified by gel filtration chromatography on Sephadex G-75 and anion-exchange chromatography on DE52. The circular dichroic spectrum of this protein was not affected by protein concentration, suggesting that it does not aggregate into multimers. Computer analyses of the circular dichroic spectrum predicted that rat heart fatty acid-binding protein contains approximately 22% alpha-helix, 45% beta-form and 33% unordered structure. Immunological studies showed that the fatty acid-binding proteins from rat heart and rat liver are immunochemically unrelated. The amino acid composition and partial amino acid sequence of the heart protein indicated that it is structurally related to, but distinct from, other fatty acid-binding proteins from liver, intestine, and 3T3 adipocytes. Using a binding assay which measures the transfer of fatty acids between donor liposomes and protein (Brecher, P., Saouaf, R., Sugarman, J. M., Eisenberg, D., and LaRosa, K. (1984) J. Biol. Chem. 259, 13395-13401), it was shown that both rat heart and liver fatty acid-binding proteins bind 2 mol of oleic acid or palmitic acid/mol of protein. The structural and functional relationship of rat heart fatty acid-binding protein to fatty acid-binding proteins from other tissues is discussed.     

A novel nucleic acid-binding protein that interacts with human rad51 recombinase.

Using the yeast two-hybrid system, we isolated a cDNA encoding a novel human protein, named Pir51, that strongly interacts with human Rad51 recombinase. Analysis in vitro confirmed the interaction between Rad51 and Pir51. Pir51 mRNA is expressed in a number of human organs, most notably in testis, thymus, colon and small intestine. The Pir51 gene locus was mapped to chromosome 12p13.1-13. 2 by fluorescence in situ hybridization. The Pir51 protein was expressed in Escherichia coli and purified to near homogeneity. Biochemical analysis shows that the Pir51 protein binds both single- and double-stranded DNA, and is capable of aggregating DNA. The protein also binds RNA. The Pir51 protein may represent a new member of the multiprotein complexes postulated to carry out homologous recombination and DNA repair in mammalian cells.     

Modern tools for identification of nucleic acid-binding proteins

Numerous biological mechanisms depend on nucleic acid--protein interactions. The first step to the understanding of these mechanisms is to identify interacting molecules. Knowing one partner, the identification of other associated molecular species can be carried out using affinity-based purification procedures. When the nucleic acid-binding protein is known, the nucleic acid can be isolated and identified by sensitive techniques such as polymerase chain reaction followed by DNA sequencing or hybridization on chips. The reverse identification procedure is less straightforward in part because interesting nucleic acid-binding proteins are generally of low abundance and there are no methods to amplify amino acid sequences. In this article, we will review the strategies that have been developed to identify nucleic acid-binding proteins. We will focus on methods permitting the identification of these proteins without a priori knowledge of protein candidates.     

A single-stranded nucleic acid-binding protein from Artemia salina. I. Purification and characterization

A protein that binds tightly to single-stranded but not to double-strained nucleic acids has been purified to homogeneity from a high salt wash of ribosomes from cryptobiotic Artemia saline gastrulae. The protein, designated HD40 to indicate a helix-destabilizing protein with a molecular weight of 40,000, is present in the high-salt ribosomal wash at a level of about 2 molecules per 80 S ribosome. The protein is monomeric at salt concentrations from 0.01 to 0.5 M and has an alpha-helix content of approximately 15%. The amino acid composition of HD40 is characterized by a high glycine content (19.5 mol%), the absence of cysteine, and the presence of the unusual amino acid dimethylarginine. The isolated protein binds preferentially to natural RNA over denatured DNA. HD40 inhibits protein synthesis directed by poly(rU) and by Artemia poly(A+) RNA in cell-free systems derived from Artemia and from wheat germ; inhibition is relieved by excess of mRNA. Single-stranded ribo- and deoxyribopolynucleotides are largely protected from degradation by nucleases when complexed with HD40.