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Results for “Double Bind Interaction”

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At least 217 records · Page 12Linked to original sources

PubMed2004

A double-deletion method to quantifying incremental binding energies in proteins from experiment: example of a destabilizing hydrogen bonding pair.

The contribution of a specific hydrogen bond in apoflavodoxin to protein stability is investigated by combining theory, experiment and simulation. Although hydrogen bonds are major determinants of protein structure and function, their contribution to protein stability is still unclear and widely debated. The best method so far devised to estimate the contribution of side-chain interactions to protein stability is double mutant cycle analysis, but the interaction energies so derived are not identical to incremental binding energies (the energies quantifying net contributions of two interacting groups to protein stability). Here we introduce double-deletion analysis of 'isolated' residue pairs as a means to precisely quantify incremental binding. The method is exemplified by studying a surface-exposed hydrogen bond in a model protein (Asp96/Asn128 in apoflavodoxin). Combined substitution of these residues by alanines slightly destabilizes the protein due to a decrease in hydrophobic surface burial. Subtraction of this effect, however, clearly indicates that the hydrogen-bonded groups in fact destabilize the native conformation. In addition, molecular dynamics simulations and classic double mutant cycle analysis explain quantitatively that, due to frustration, the hydrogen bond must form in the native structure because when the two groups get approximated upon folding their binding becomes favorable. We would like to remark that 1), this is the first time the contribution of a specific hydrogen bond to protein stability has been measured by experiment; and 2), more hydrogen bonds need to be analyzed to draw general conclusions on protein hydrogen bond energetics. To that end, the double-deletion method should be of help.

Amino Acid Substitution↗

PubMed2006

The dicationic derivatives of DBTAA: Interactions with DNA/RNA and antiproliferative effects on human cell lines.

Three dibenzotetraaza[14]annulenes non-covalently interacted with double-stranded DNA and RNA by mixed minor groove and/or intercalative binding mode. Observed interactions were strongly dependent on the steric exposure of positive charges and the length of the linkers of studied compounds as well as on the secondary structure and basepair composition of DNA/RNA. Compound 2 showed pronounced selectivity toward dA-dT-rich sequences and binding mode switch from dominant minor groove binding to ds-DNA to dominant intercalation into ds-RNA. Antiproliferative effect of studied compounds on human tumor and normal cell lines was in good agreement with the strength of observed interactions with DNA/RNA.

Antineoplastic Agents↗

PubMed1996

Characterization of a polypyrimidine/polypurine tract in the promoter of the gene for chicken malic enzyme.

Starvation inhibits and refeeding stimulates transcription of the malic enzyme gene in chick liver. DNA between -320 and +72 base pairs (bp) is DNase I-hypersensitive in hepatic nuclei from fed but not starved chicks (Ma, X. J., and Goodridge, A. G. (1992) Nucleic Acids Res. 20, 4997-5002). A polypyrimidine/polypurine (PPY/PPU) tract lies within the DNase I-hypersensitive region. In hepatocytes transiently transfected with plasmids containing triiodothyronine response elements and a minimal promoter from the malic enzyme gene linked to the chloramphenicol acetyltransferase gene, deletion of the PPY/PPU tract inhibited chloramphenicol acetyltransferase activity by about 90% with or without triiodothyronine. Fine mapping of S1 nuclease-sensitive sites suggests that the PPY/PPU tract can assume different isoforms of non-B-DNA, some of which may be triplex structures. The PPY/PPU tract contains specific binding sites for single- and double-stranded DNA binding proteins and, with 8 bp 3' of the tract, can function as a promoter. A (CT)7 repeat binds single-stranded DNA-binding protein and is essential for promoter activity. Two C-rich elements bind single-stranded DNA-binding proteins and may mediate inhibition of promoter function. The single- and double-stranded DNA-binding proteins that interact with the PPY/PPU tract may regulate transcription of the malic enzyme gene.

Animals↗

PubMed2004

Entropy-enthalpy compensation in ionic interactions probed in a zinc finger peptide.

Zinc(II) and cobalt(II) binding to a series of zinc finger peptides with different charged residue pairs across from one another in a beta-sheet were examined. Previous studies revealed a narrow range of interaction free energies (<0.5 kcal/mol) between these residues. Here, isothermal titration calorimetry studies were performed, revealing a range of over 3 kcal/mol in relative binding enthalpies. Double mutant cycle analysis revealed a range of interaction enthalpies ranging from -3.1 to -3.4 kcal/mol for the Arg-Asp pair to -0.8 kcal/mol for the Lys-Glu pair. The large range of interaction enthalpies coupled with the small range of interaction free energies reveals substantial entropy-enthalpy compensation. The magnitudes of the effects are consistent with the formation of a structurally rigid Arg-Asp contact ion pair but less direct and more mobile interactions involving the other combinations.

Calorimetry↗

PubMed2000

Potent inhibition of werner and bloom helicases by DNA minor groove binding drugs.

Maintenance of genomic integrity is vital to all organisms. A number of human genetic disorders, including Werner Syndrome, Bloom Syndrome and Rothmund-Thomson Syndrome, exhibit genomic instability with some phenotypic characteristics of premature aging and cancer predisposition. Presumably the aberrant cellular and clinical phenotypes in these disorders arise from defects in important DNA metabolic pathways such as replication, recombination or repair. These syndromes are all characterized by defects in a member of the RecQ family of DNA helicases. To obtain a better understanding of how these enzymes function in DNA metabolic pathways that directly influence chromosomal integrity, we have examined the effects of non-covalent DNA modifications on the catalytic activities of purified Werner (WRN) and Bloom (BLM) DNA helicases. A panel of DNA-binding ligands displaying unique properties for interacting with double helical DNA was tested for their effects on the unwinding activity of WRN and BLM helicases on a partial duplex DNA substrate. The levels of inhibition by a number of these compounds were distinct from previously reported values for viral, prokaryotic and eukaryotic helicases. The results demonstrate that BLM and WRN proteins exhibit similar sensitivity profiles to these DNA-binding ligands and are most potently inhibited by the structurally related minor groove binders distamycin A and netropsin (K(i) </=1 microM). The distinct inhibition of WRN and BLM helicases by the minor groove binders suggest that these helicases unwind double-stranded DNA by a related mechanism.

Adenosine Triphosphatases↗

PubMed1993

Inactivation of the endogenous argininosuccinate lyase activity of duck delta-crystallin by modification of an essential histidine residue with diethyl pyrocarbonate.

The argininosuccinate lyase activity of duck delta-crystallin was inactivated by diethyl pyrocarbonate at 0 degrees C and pH 7.5. The inactivation followed pseudo-first-order kinetics after appropriate correction for the decomposition of the reagent during the modification period. The plot of the observed pseudo-first-order rate constant versus diethyl pyrocarbonate concentration in the range of 0.17-1.7 mM was linear and went through the origin with a second-order rate constant of 1.45 +/- 0.1 M-1.s-1. The double-logarithmic plot was also linear, with slope of 1.13, which suggested a 1:1 stoichiometry for the reaction between diethyl pyrocarbonate and delta-crystallin. L-Arginine, L-norvaline or L-citrulline protected the argininosuccinate lyase activity of delta-crystallin from diethyl pyrocarbonate inactivation. The dissociation constants for the delta-crystallin-L-arginine and delta-crystallin-L-citrulline binary complexes, determined by the protection experiments, were 4.2 +/- 0.2 and 0.12 +/- 0.04 mM respectively. Fumarate alone had no protective effect. However, fumarate plus L-arginine gave synergistic protection with a ligand binding interacting factor of 0.12 +/- 0.02. The double-protection data conformed to a random Uni Bi kinetic mechanism. Fluorescence-quenching studies indicated that the modified delta-crystallin had minimum, if any, conformational changes as compared with the native delta-crystallin. Inactivation of the enzyme activity was accompanied by an increasing absorbance at 240 nm of the protein. The absorption near 280 nm did not change. Treatment of the modified protein with hydroxylamine regenerated the enzyme activity to the original level. These results strongly indicated the modification of an essential histidine residue. Calculation from the 240 nm absorption changes indicated that only one histidine residue per subunit was modified by the reagent. This super-active histidine residue has a pKa value of approximately 6.8 and acts as a general acid-base catalyst in the enzyme reaction mechanism. Our experimental data are compatible with an E1cB mechanism [Raushel (1984) Arch. Biochem. Biophys. 232, 520-525] for the argininosuccinate lyase with the essential histidine residue close to the arginine-binding domain of delta-crystallin. L-Citrulline, after binding to this domain, might form an extra hydrogen bond with the essential histidine residue.

Animals↗

PubMed2006

hSnm1B is a novel telomere-associated protein.

Artemis, a member of the beta-CASP family, has been implicated in the regulation of both telomere stability and length. Prompted by this, we examined whether the other two putative DNA-binding members of this family, hSnm1A and hSnm1B, may associate with telomeres. hSnm1A was found to not interact with the telomere. Conversely, hSnm1B was found to associate with telomeres in vivo by both immunofluorescence and chromatin immunoprecipitation. Furthermore, the C terminus of hSnm1B was shown to interact with the TRF homology domain of TRF2 indicating that hSnm1B is likely recruited to the telomere via interaction with the double-stranded telomere-binding protein TRF2.

Binding Sites↗

PubMed1999

High precision solution structure of the C-terminal KH domain of heterogeneous nuclear ribonucleoprotein K, a c-myc transcription factor.

Among it's many reported functions, heterogeneous nuclear ribonucleoprotein (hnRNP) K is a transcription factor for the c- myc gene, a proto-oncogene critical for the regulation of cell growth and differentiation. We have determined the solution structure of the Gly26-->Arg mutant of the C-terminal K-homology (KH) domain of hnRNP K by NMR spectroscopy. This is the first structure investigation of hnRNP K. Backbone residual dipolar couplings, which provide information that is fundamentally different from the standard NOE-derived distance restraints, were employed to improve structure quality. An independent assessment of structure quality was achieved by comparing the backbone15N T1/T2ratios to the calculated structures. The C-terminal KH module of hnRNP K (KH3) is revealed to be a three-stranded beta-sheet stacked against three alpha-helices, two of which are nearly parallel to the strands of the beta-sheet. The Gly26-->Arg mutation abolishes single-stranded DNA binding without altering the overall fold of the protein. This provides a clue to possible nucleotide binding sites of KH3. It appears unlikely that the solvent-exposed side of the beta-sheet will be the site of protein-nucleic acid complex formation. This is in contrast to the earlier theme for protein-RNA complexes incorporating proteins structurally similar to KH3. We propose that the surface of KH3 that interacts with nucleic acid is comparable to the region of DNA interaction for the double-stranded DNA-binding domain of bovine papillomavirus-1 E2 that has a three-dimensional fold similar to that of KH3.

Amino Acid Sequence↗

PubMed1982

Stability decrease of RNA double helices by phenylalanine-, tyrosine- and tryptophane-amides. Analysis in terms of site binding and relation to melting proteins.

The amides of L-phenylalanine, L-tyrosine and L-tryptophane decrease the melting temperatures tm of poly(A)*poly(U) and poly(I)*poly(C) double helices at low concentrations (1 mM), whereas high concentrations finally lead to an increase of tm. This dependence of the tm-values upon the ligand concentration can be represented quantitatively by a simple site binding model, providing binding parameters for the interaction between the amides and the nucleic acids both in the double- and the single-stranded conformation. According to these data the affinity to the single strands is higher than that to the double strands and increases in the series Phe less than Tyr less than Trp. The binding constants decrease with increasing salt concentration as expected for an interaction driven by electrostatic attraction. However, part of the interaction is also due to stacking between the aromatic amides and the nucleic acid bases. The present results indicate a direct correlation between the presence of aromatic amino acids at the binding site of helix destabilising proteins and the properties of simple derivatives of these amino acids. Furthermore the results suggest that very simple peptides containing aromatic amino acids served as a starting point for the evolution of helix destabilising proteins.

Chemical Phenomena↗

PubMed1997

Binding of cationic (+4) alanine- and glycine-containing oligopeptides to double-stranded DNA: thermodynamic analysis of effects of coulombic interactions and alpha-helix induction.

Coulombic interactions and coupled conformational changes make important contributions to stability and specificity of many protein-nucleic acid complexes. As models of these phenomena in simpler systems, we have investigated the binding to mononucleosomal (160 base-pair) calf thymus DNA of a high charge density (compact) 5-residue (+4) oligopeptide (with 4 lysines and 1 tryptophan) and of four lower charge density (extended) 17-residue (+4) oligopeptides (each with 4 lysines, 10-12 alanines, 0-2 glycines, and 1 tryptophan). The fractional helicity (f(h)) of each oligopeptide before and after DNA binding was determined using circular dichroism. At low univalent cation concentration ([M+] = 6.4 mM), binding to DNA increases f(h) significantly for all but one of the extended oligopeptides. Oligopeptide-DNA binding constants (K(obs)) and apparent binding site sizes (n) were quantified using the noncooperative McGhee-von Hippel isotherm to fit tryptophan fluorescence quenching data. For each of the oligopeptides studied, n is found to be approximately equal to four, the number of lysine charges. In the range 6.4 mM < or = [M+] < or = 21.5 mM, power dependences of K(obs) on [M+] (SK(obs) = d log K(obs)/d log[M+]) for all 17-residue (+4) oligopeptides are similar with an average value of -3.7 +/- 0.4, which is indistinguishable (outside uncertainty) from the value obtained here for the compact (+4) oligopeptide and from values reported elsewhere for another compact tetralysine and for spermine (+4). Our results are consistent with the conclusion that the nonspecific binding to DNA of all these tetravalent ligands is driven primarily by coulombic interactions. At any [M+] investigated, values of K(obs) for the four extended (+4) oligopeptides differ by less than an order of magnitude, but all are 1-2 orders of magnitude less than values of K(obs) for two compact (+4) oligopeptides and for spermine. The differences in K(obs) for oligopeptide-DNA complexes, which all have similar n and similar SK(obs) indicate that when an extended oligopeptide binds to DNA it becomes more compact as a result of conformational changes, such as the additional alpha-helix formation detected by circular dichroism.

Alanine↗

PubMed1977

Inhibition of squalene synthetase by farnesyl pyrophosphate analogues.

The pyrophosphates of the following farnesol analogues have been synthesized: 2-methylfarnesol; 7,11-dimethyl-3-ethyl-2,6,10-dodecatrien-1-ol; 3-demethylfarnesol; 4-methylthiofarnesol; 7,11-dimethyl-3-iodo-2,6,10-dodecatrien-1-ol; 7,11-dimethyl02-iodo-2,6,10-dodecatrien-1-ol; 7,11-dimethyldodeca-6,10-dien-2-yn-1-ol; phytol; 3,7,11-trimethyl-2-dodecen-1-ol; 3,7,11-trimethyldodecan-1-ol; and geraniol. The double bonds in all the above compounds were in the E configuration, except phytol, which was a 7:3 mixture of 2E and 2Z isomers. Each of the pyrophosphates inhibits the incorporation of labeled farnesyl pyrophosphate into squalene by a yeast enzyme preparation. Free alcohols and monophosphates are inactive. The analogues, listed in order of decreasing inhibitory strength, are, by kinetic analysis, competitive or mixed inhibitors. Irreversible inhibition is not observed. The results suggest that binding to the enzyme is primarily mediated by the pyrophosphate moiety assisted by relatively nonspecific lipophilic interactions. Decreasing the chain length and saturating double bonds severely reduces binding, while substitution at the 2,3, and 4 positions, and lengthening of the chain, is well tolerated.

Diphosphates↗

PubMed1991

Use of ion exchange chromatography for the study of RecA-DNA interaction.

In vitro binding of RecA protein to double-stranded DNA (dsDNA) was studied using ion-exchange liquid chromatography. The method allowed quantification of both free DNA and free protein. The results unambiguously showed a binding stoichiometry of 3 base pairs per RecA monomer. The binding exhibited cooperativity, and the stoichiometry suggested that RecA does not form complexes with two molecules of dsDNA. More than 90% of RecA molecules in the sample were active for DNA binding.

Adenosine Triphosphate↗

PubMed1999

Mapping of protein-protein interactions within the DNA-dependent protein kinase complex.

In mammalian cells, the Ku and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) proteins are required for the correct and efficient repair of DNA double-strand breaks. Ku comprises two tightly-associated subunits of approximately 69 and approximately 83 kDa, which are termed Ku70 and Ku80 (or Ku86), respectively. Previously, a number of regions of both Ku subunits have been demonstrated to be involved in their interaction, but the molecular mechanism of this interaction remains unknown. We have identified a region in Ku70 (amino acid residues 449-578) and a region in Ku80 (residues 439-592) that participate in Ku subunit interaction. Sequence analysis reveals that these interaction regions share sequence homology and suggests that the Ku subunits are structurally related. On binding to a DNA double-strand break, Ku is able to interact with DNA-PKcs, but how this interaction is mediated has not been defined. We show that the extreme C-terminus of Ku80, specifically the final 12 amino acid residues, mediates a highly specific interaction with DNA-PKcs. Strikingly, these residues appear to be conserved only in Ku80 sequences from vertebrate organisms. These data suggest that Ku has evolved to become part of the DNA-PK holo-enzyme by acquisition of a protein-protein interaction motif at the C-terminus of Ku80.

Amino Acid Sequence↗

PubMed2005

RNA silencing suppressor p21 of Beet yellows virus forms an RNA binding octameric ring structure.

Many plant viruses encode proteins that suppress the antiviral RNA silencing response mounted by the host. The suppressors p19 from tombusvirus and p21 from Beet yellows virus appear to block silencing by directly binding siRNA, a critical mediator in the process. Here, we report the crystal structure of p21, which reveals an octameric ring architecture with a large central cavity of approximately 90 A diameter. The all alpha-helical p21 monomer consists of N- and C-terminal domains that associate with their neighboring counterparts through symmetric head-to-head and tail-to-tail interactions. A putative RNA binding surface is identified in the conserved, positive-charged inner surface of the ring. In contrast to the specific p19-siRNA duplex interaction, p21 is a general nucleic acid binding protein, interacting with 21 nt or longer single- and double-stranded RNAs in vitro. This study reveals an RNA binding structure adopted by the p21 silencing suppressor.

Amino Acid Sequence↗

PubMed2001

Nuclear factor 90 is a substrate and regulator of the eukaryotic initiation factor 2 kinase double-stranded RNA-activated protein kinase.

Nuclear factor 90 (NF90) is a member of an expanding family of double-stranded (ds) RNA-binding proteins thought to be involved in gene expression. Originally identified in complex with nuclear factor 45 (NF45) as a sequence-specific DNA-binding protein, NF90 contains two double stranded RNA-binding motifs (dsRBMs) and interacts with highly structured RNAs as well as the dsRNA-activated protein kinase, PKR. In this report, we characterize the biochemical interactions between these two dsRBM containing proteins. NF90 binds to PKR through two independent mechanisms: an RNA-independent interaction occurs between the N terminus of NF90 and the C-terminal region of PKR, and an RNA-dependent interaction is mediated by the dsRBMs of the two proteins. Co-immunoprecipitation analysis demonstrates that NF90, NF45, and PKR form a complex in both nuclear and cytosolic extracts, and both proteins serve as substrates for PKR in vitro. NF90 is phosphorylated by PKR in its RNA-binding domain, and this reaction is partially blocked by the NF90 N-terminal region. The C-terminal region also inhibits PKR function, probably through competitive binding to dsRNA. A model for NF90-PKR interactions is proposed.

Animals↗

PubMed2005

Label free analysis of transcription factors using microcantilever arrays.

We report the measurement of protein interaction with double-stranded DNA oligonucleotides using cantilever microarray technology. We investigated two different DNA-binding proteins, the transcription factors SP1 and NF-kappaB, using cantilever arrays as they allow label-free measurement of different biomolecular interactions in parallel. Double-stranded DNA oligonucleotides containing a specific binding site for a transcription factor were sensitized on gold-coated cantilevers. The binding of the transcription factor creates a surface stress, resulting in a bending of the cantilevers. Both transcription factors could be detected independently at concentrations of 80-100 nM. A concentration dependence of the bending signal was measured using concentrations from 100 to 400 nM of NF-kappaB. The experiments show that the recognition sequence of one transcription factor can serve as a reference for the other, highlighting the sequence specificity of transcription factor binding.

DNA↗

PubMed1993

RNA structural requirements for RNA binding, replication, and packaging in the yeast double-stranded RNA virus.

Several sites of interaction with viral proteins have been mapped in the plus strand of the Saccharomyces cerevisiae double-stranded RNA virus, ScV. These include a site necessary and sufficient for viral particle binding to plus strands (VBS) and a site necessary and sufficient for interference with replication of viral plus strands (INS). We show that the INS and VBS are identical and that they are necessary and sufficient for packaging. One of the viral RNAs has two adjacent VBS, which have additive INS activity. The second VBS has similar affinity for viral particles as the first but its complex with particles exhibits faster dissociation. Binding to the two sites in the viral RNA is independent but equivalent. This may mean two recognition sites per particle. This is what would be expected for two molecules of the protein thought to be responsible for sequence specific recognition, the cap-pol fusion polypeptide, per particle. Comparison of the secondary structures of several binding sites, as determined by site-directed mutagenesis and by ribonuclease mapping of single- and double-stranded regions, reveals a requirement for a specific loop sequence and two stem regions, in which there is no sequence specificity. Contrary to what was described in previous work, a bulge loop, but not a "bulged" A residue is necessary for binding of the second VBS. A minimal region of 30 bases is enough to cause both packaging and replication.

Base Sequence↗

PubMed1995

Structure and function of transcription-repair coupling factor. I. Structural domains and binding properties.

The 130-kDa mfd gene product is required for coupling transcription to repair in Escherichia coli. Mfd displaces E. coli RNA polymerase (Pol) stalled at a lesion, binds to the damage recognition protein UvrA, and increases the template strand repair rate during transcription. Here, the interactions of Mfd (transcription-repair coupling factor, TRCF) with DNA, RNA Pol, and UvrA were investigated. TRCF bound nonspecifically to double stranded DNA; binding to DNA produced alternating DNase I-protected and -hypersensitive regions, suggesting possible wrapping of the DNA around the enzyme. Weaker binding to single stranded DNA and no binding to single stranded RNA were observed. DNA binding required ATP, and hydrolysis of ATP promoted dissociation. Removal of a stalled RNA Pol also requires ATP hydrolysis. Apparently, TRCF recognizes a stalled elongation complex by directly interacting with RNA Pol, since binding to a synthetic transcription bubble was no stronger than binding to double stranded DNA, and binding to free RNA Pol holoenzyme and to initiation and elongation complexes in the absence of adenosine 5'-O-(thiotriphosphate) were observed. Structure-function analysis showed that residues 379-571 are involved in binding to a stalled RNAP. The helicase motifs region, residues 571-931, binds to ATP and duplex polynucleotide (DNA:DNA or DNA:RNA). Dissociation of the ternary complex upon hydrolysis of ATP also requires the carboxyl terminus of TRCF. Finally, residues 1-378 bind to UvrA and deliver the damage recognition component of the excision nuclease to the lesion.

Adenosine Triphosphatases↗