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At least 307 records · Page 17Linked to original sources
PubMed1996

Radical differences in functions of closely related members of the human carcinoembryonic antigen gene family.

The immunoglobulin superfamily represents an ancient, highly diversified group of cell surface and extracellular molecules responsible for a wide range of molecular and cellular recognition functions. The human carcinoembryonic antigen (CEA) subfamily of the immunoglobulin superfamily presents evidence of continuing diversification of the immunoglobulin family, in that some of its members, including CEA itself and nonspecific cross-reacting antigen (NCA), are expressed only in primates and not in rodents. These "new" members are glycophosphatidylinositol linked to the external cell membrane and are up-regulated in cancer, unlike members present in both rodents and primates, i.e., biliary glycoprotein (BGP), which are transmembrane linked and down-regulated in cancer. CEA, NCA, and BGP have all been shown to function in vitro as intercellular adhesion molecules. We show here that the properties of adhesion are radically different, in that BGP-mediated adhesion is reversibly Ca2+ and Mg2+ dependent, temperature dependent, and ATP inhibitable, whereas CEA- and NCA-mediated adhesion is the opposite in all aspects. Also, the novel double-reciprocal, antiparallel binding observed for CEA-CEA interactions is not seen for BGP. Finally, the myogenic differentiation block demonstrated for the ectopic expression of CEA in myoblasts was also observed for NCA but not for BGP, which is consistent with the changes in expression seen in cancer. The appearance of new CEA family members with such different properties is discussed in the context of evolution and cancer.

Adenosine Triphosphate↗
PubMed1999

Evidence for breaking domain-domain functional communication in a synthetase-tRNA complex.

We report here evidence for mutations that break domain-domain functional communication in a synthetase-tRNA complex. Each synthetase is roughly divided into two major domains that are paralleled by the two arms of the L-shaped tRNA structure. The active-site-containing domain interacts with the acceptor arm of the tRNA. The second domain frequently interacts with the anticodon-containing arm. By an induced-fit mechanism, contacts with the anticodon can activate formation of a robust transition state at a site over 70 A away. This induced-fit-based activation is thought to occur through domain-domain signaling and is seen by the enhancement of aminoacylation of the anticodon-containing full tRNA versus a substrate based on the acceptor arm alone. Here we describe a rationally designed mutant methionyl-tRNA synthetase containing two point substitutions at sites that potentially link an anticodon-binding motif to the catalytic domain. The double mutation had no effect on interactions with either the isolated acceptor arm or the anticodon stem-loop. In contrast to interactions with the separate pieces, the mutant enzyme was severely impaired for binding the native tRNA and lost much of its ability to enhance the rate of charging of the full tRNA over that of a substrate based on the acceptor arm alone. We propose that these residues are part of a network for facilitating domain-domain communication for formation of an active synthetase-tRNA complex by induced fit.

Acylation↗
PubMed1992

Testing and characterizing enzymes and membrane-bound carrier proteins acting on amphipathic ligands in the presence of bilayer membrane material and soluble binding protein. Application to the uptake of oleate into isolated cells.

1. A multiphasic modelling approach [Heirwegh, Meuwissen, Vermeir & De Smedt (1988) Biochem. J. 254, 101-108] is applied to systems containing poorly water-soluble amphipathic reactants, membrane material, soluble binding protein and acceptor protein (enzyme or membrane-bound carrier protein). 2. The field of application is constrained by the assumptions (i) that the amount of acceptor-bound substrate is small compared with the total amount and (ii) that all preceding chemical reactions and steps of mass transport are rapid compared with the chemical change monitored. 3. Initial-rate formulae for systems in which an acceptor interacts with unbound or protein-bound ligand are given. The saturation curves are near-hyperbolic or sigmoidal, depending both (i) on the form of ligand (unbound or protein-bound) acted upon by the acceptor and (ii) on whether the assays are performed at constant concentration of soluble binding protein Cp or at constant substrate/binding-site molar ratio RS. 4. Several diagnostic features permit unequivocal distinction between acceptor action on unbound or protein-bound substrate. In the former case, saturation curves, run at the same constant concentration of one of several binding proteins of increasing binding affinity, will show progressively increasing inhibition, the shape changing from near-hyperbolic at Km' less than K1' to sigmoidal at Km' greater than K1'.Km' is the effective Michaelis constant of the acceptor and K1' the effective dissociation constant of the binding sites of the soluble protein (for the sites with the higher binding affinity, if several classes of binding site are present on the protein). Alternatively, the maximum velocity obtained at constant RS less than or equal to 1 should increase hyperbolically with RS/(1-RS) for a binding protein with a single class of binding site. The formula that applies when the binding protein contains two classes of independent binding site is also available. When the acceptor acts on protein-bound ligand, the maximum velocity obtained at constant binding-protein concentration, Cp, increases hyperbolically with Cp. 5. Application of these and additional criteria to initial-rate data on the uptake of oleate into isolated cells supports a mechanism of carrier-mediated uptake of the unbound ligand and allows one to clarify some observations that hitherto had been poorly explained. 6. The influence of soluble binding protein on the reaction and substrate specificities of ligand/acceptor interaction is also discussed. 7. In its present state, data treatment for 'double binding-protein systems' generally requires separate determination of the binding parameters of the soluble binding protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Carrier Proteins↗
PubMed2003

RNA binding and intramolecular interactions modulate the regulation of gene expression by nuclear factor 110.

Nuclear factor 110 (NF110) belongs to the nuclear factor 90 (NF90) family of double-stranded RNA (dsRNA) binding proteins that regulate gene expression at the transcriptional level in vertebrates. The proteins are identical at their N terminus, which functions as a negative regulatory region, but have distinct C termini as a result of alternate splicing. Maximal transcriptional activity of NF110 requires its C-terminal domain and a central domain that contains a nuclear localization signal and two dsRNA-binding motifs (dsRBMs). We find that dsRNA binding is reduced by RGG and GQSY motifs present in the C-terminal region. To directly evaluate the role of RNA binding in transactivation, we conducted site-directed mutagenesis to substitute conserved residues in one or both of the dsRBMs. The mutations reduced the ability of NF110 to stimulate gene expression to an extent that paralleled the mutants' reduced ability to bind dsRNA. Full activity was restored when the dsRBM-containing region of NF110 was replaced with the RNA-binding region of the protein kinase PKR. Finally, NF110-mediated transactivation was inhibited by cotransfection of a plasmid encoding an artificial highly structured RNA. These data suggest that NF110 and its homologs are regulated by cis-acting domains present in some of the protein isoforms, and via interactions with RNAs that bind to their dsRBMs. We propose a model in which structured RNAs regulate gene expression by modulating transcription through interactions with members of the NF90 protein family.

Base Sequence↗
PubMed1976

Tissue-specific chromosomal non-histone protein interactions with DNA.

A group of chromosomal non-histone proteins with affinity for DNA (NP) was isolated from rat liver and Novikoff hepatoma. This fraction, which represents less than 5% of the total chromatin protein content, binds preferentially to unique, double-stranded sequences of fractionated homologous DNA. The interactions are strong at low ionic strength (Km = 6.7 X 10(-9) M) and decrease with rising salt concentration. Complexes of the NP protein fraction with homologous DNA are immunologically tissue-specific. As determined by microcomplement fixation, the NP proteins in Novikoff hepatoma are associated with the transcriptionally active, diffuse fraction of chromatin.

Animals↗
PubMed2004

Adeno-associated virus Rep78/Rep68 promotes localized melting of the rep binding element in the absence of adenosine triphosphate.

We have applied fluorescence anisotropy and molecular beacon fluorescence methods to study the interactions between the Adeno-associated virus Rep78/Rep68 protein and the 23-bp Rep binding element (RBE). Rep78/Rep68 stably interacted with both the single- and double-stranded conformations of the RBE, but the interaction mechanisms of single- and double-stranded DNA appeared to be fundamentally different. The stoichiometry of Rep78 association with both the separate top and bottom strands of the RBE was 1:1, and the relative dissociation constant (K(D)) values of these associations were calculated to be 2.3x10(-8) and 3.2x10(-8) M, respectively. In contrast, the stoichiometry of Rep78 association with the double-stranded RBE was 2:1, and the dissociation constant was determined to be 4.2x10(-15) M(2). Moreover, Rep78/Rep68 interaction with the 23-bp duplex RBE appeared to cause localized melting of the double-stranded DNA substrate in the absence of adenosine triphosphate (ATP). This melting activity showed slower kinetics than binding and may contribute to the initiation of ATP-dependent Rep78 helicase activity.

Adenosine Triphosphate↗
PubMed1997

Discrimination of amino acids mediating Ras binding from noninteracting residues affecting raf activation by double mutant analysis.

The contribution of residues outside the Ras binding domain of Raf (RafRBD) to Ras-Raf interaction and Ras-dependent Raf activation has remained unresolved. Here, we utilize a double mutant approach to identify complementary interacting amino acids that are involved in Ras-Raf interaction and activation. Biochemical analysis demonstrates that Raf-Arg59 and Raf-Arg67 from RafRBD are interacting residues complementary to Ras-Glu37 located in the Ras effector region. Raf-Arg59 and Raf-Arg67 also mediate interaction with Ras-Glu37 in Ras-dependent Raf activation. The characteristics observed here can be used as criteria for a role of residues from other regions of Raf in Ras-Raf interaction and activation. We developed a quantitative two-hybrid system as a tool to investigate the effect of point mutations on protein-protein interactions that elude biochemical analysis of bacterially expressed proteins. This assay shows that Raf-Ser257 in the RafCR2 domain does not contribute to Ras-Raf interaction and that the Raf-S257L mutation does not restore Raf binding to Ras-E37G. Yet, Raf-S257L displays high constitutive kinase activity and further activation by Ras-G12V/E37G is still impaired as compared with activation by Ras-G12V. This strongly suggests that the RafCR2 domain is an independent domain involved in the control of Raf activity and a common mechanism for constitutively activating mutants may be the interference with the inactive ground state of the kinase.

Amino Acid Substitution↗
PubMed2000

A surface plasmon resonance method for detecting multiple modes of DNA-ligand interactions.

A simple and general surface plasmon resonance (SPR) based method has been developed to detect and quantitate binding of low molecular weight compounds (200-1,200 Da) to double stranded DNA. Several compounds were chosen to probe three different modes of binding interactions, intercalation, minor groove binding and electrostatic interactions. Ethidium bromide (MW 390 Da), a probe of intercalative binding, was tested by plotting the steady state SPR responses measured on a DNA modified surface versus ethidium bromide concentration. The best fit of the binding isotherm gave a Keq of 1.8 x 105 M-1. Co-solvents such as DMSO are often used in activity assays to increase the solubility of poorly water-soluble drugs. The effect of DMSO on the ethidium bromide/DNA interaction was also tested by measuring binding in the presence of 0, 1 and 5%, DMSO. No effect on the measured Keq was observed at these DMSO concentrations. The binding of actinomycin (MW 1,255 Da), an antibiotic known to bind DNA through intercalation and minor groove binding, was also tested. The Keq estimated from the steady state responses on a DNA surface was 1.9 x 106 M-1. DAPI (MW 350 Da) (4',6-diamidino-2-phenylindole) a fluorescent probe which binds the minor groove of DNA was also tested and gave a Keq of 1.8 x 106 M-1 measured by SPR. Finally, spermine (MW 202) a compound known to bind DNA through ionic interactions gave the weakest Keq of 1.7 x 104 M-1. All the Keq values measured by SPR and reported for these compounds were in good agreement with literature values measured by other techniques.

Biotin↗
PubMed1997

Charged residues in transmembrane domains II and XI of a vesicular monoamine transporter form a charge pair that promotes high affinity substrate recognition.

Vesicular monoamine transporters package monoamine neurotransmitters into secretory vesicles for regulated exocytotic release. Both vesicular monoamine transporter 1 and 2 contain several charged residues predicted to reside within transmembrane domains (TMDs), and conservation of these residues in multiple species and in other members of the gene family suggest important roles in transporter structure and function. To determine the role of these residues, we have used site-directed mutagenesis. Replacement of Asp-263 in TMD6 with Asn (D263N) had no effect on transport activity. However, replacement of Lys-139 in TMD2 with Ala (K139A), Asp-400 in TMD10 with Asn (D400N), or Asp-427 in TMD11 with Asn (D427N) eliminated transport activity despite normal levels of protein expression. Remarkably, the double mutant K139A/D427N showed substantial transport activity, suggesting that Lys-139 and Asp-427 interact to form an ion pair in the native protein and hence that TMD2 occurs next to TMD11. Nonetheless, the double mutant showed reduced apparent affinity for serotonin and reduced ability of serotonin to inhibit reserpine binding, suggesting that although not required for activity, the ion pair promotes high affinity interaction with the substrate. In addition, a double mutant in which the polarity of the charged residues was reversed (K139D/D427K) showed no active transport. Remarkably, however, this mutant displayed normal reserpine binding that remained coupled to DeltaH+, but serotonin failed to inhibit reserpine binding, suggesting that the charge reversal specifically disrupts substrate recognition.

Animals↗
PubMed1996

The P58 cellular inhibitor complexes with the interferon-induced, double-stranded RNA-dependent protein kinase, PKR, to regulate its autophosphorylation and activity.

The 58-kDa protein, referred to as P58, is a cellular inhibitor of the interferon-induced, double-stranded RNA-activated protein kinase, PKR. The P58 protein inhibits both the autophosphorylation of PKR and the phosphorylation of the PKR natural substrate, the alpha subunit of eukaryotic initiation factor eIF-2. Sequence analysis revealed that P58 is a member of the tetratricopeptide family of proteins. Utilizing experimental approaches, which included coprecipitation or coselection of native and recombinant wild-type and mutant proteins, we found that P58 can efficiently complex with the PKR protein kinase. Attempts to map the P58 interactive sites revealed a correlation between the ability of P58 to inhibit PKR in vitro and bind to PKR. The DnaJ sequences, present at the carboxyl terminus of P58, were dispensable for binding in vitro, while sequences containing the eIF-2 alpha similarity region were essential for efficient complex formation. Furthermore, not all tetratricopeptide motifs were necessary for PKR-P58 interactions. Initial experiments to map the binding domains present in PKR showed that P58 complexed with PKR molecules that lacked the first RNA binding domain but did not bind to a PKR mutant containing only the amino terminus. These data, taken together, demonstrate that P58 inhibits PKR through a direct interaction, which is likely independent of the binding of double-stranded RNA to the protein kinase.

3T3 Cells↗
PubMed2001

Double-stranded DNA binding characteristics and subcellular distribution of a minor groove binding diphenyl ether bisbenzimidazole.

The interactions of Hoechst 33377 (H1) with 20 different oligomeric duplexes have been investigated via spectrofluorometric titrations and/or thermal denaturation experiments. H1 is shown to form 2:1 complexes with dsDNA binding sites of at least four contiguous A/T base pairs. H1 is also shown to possess the rare ability to meaningfully distinguish between different A.T rich sequences. For example, the combined equilibrium constants for complexation of the oligomeric duplex 5'-GCAATTGC-3' (15) by H1 are found to be 110-fold greater than for the duplex 5'-GCTTAAGC-3' (16). It is believed that the 5'-TpA-3' dinucleotide step in 16 disrupts the rigid "A-tract" conformation of 15 and discourages minor groove binding by agents capable of recognizing longer dsDNA sequences. Molecular models are presented which elucidate the structure of the (H1)(2)-dsDNA minor groove complex. The two H1 molecules bind to an A/T rich sequence of 6 bp in a slightly staggered, side-by-side, and antiparallel arrangement. Evidence suggests that the piperazine rings of the H1 side-by-side complex are capable of resting in the minor groove of G/C base pairs. Fluorescence microscopy studies using NIH3T3 cells indicate that H1 is capable of traversing the cytoplasmic membrane and selectively localizing to nuclear DNA. H1 also demonstrated the ability to inhibit endogenous transcription of the c-fos gene in NIH3T3 cells at micromolar concentrations. Cytotoxicity studies employing the same cell type show H1 to possess an LD(50) of 3.5 microM.

3T3 Cells↗
PubMed1978

The interaction of ethidium with synthetic double-stranded polynucleotides at low ionic strength.

The interaction of ethidium with synthetic DNA and RNA double-stranded polymers at 0.01 M ionic strength, pH 7.0, has been studied by fluorimetry at low drug to nucleotide ratios. Binding constants have been calculated assuming an excluded-neighbouring site model for the interaction of ethidium with double-stranded polymers. The values obtained are poly d(AT).poly d(AT), 9.5 X 10(6) M-1; poly dA.poly dT, 6.5 X 10(5) M-1; poly d(GC).poly d(GC), 9.9 X 10(6) M-1; poly dG,poly dC, 4.5 X 1-(6) M-1; poly d(AC); poly d(GT), 9.8 X 10(6) M-1; poly d(AG).poly d(CT), 1.3 X 10(6) M-1; poly rA.poly rU, 4.1 X 10(7) M-1. The displacement of ethidium from poly d(AT).poly d(AT) by 9-aminoacridine and an acridine-containing antitumor agent (NSC 156303; 4'-(9-acridinylamino)methanesulphon-m-anisidide) has also been examined.

Acridines↗
PubMed1998

DNA-dependent protein kinase: DNA binding and activation in the absence of Ku.

In mammalian cells, double-strand break repair and V(D)J recombination require DNA-dependent protein kinase (DNA-PK), a serine/threonine kinase that is activated by DNA. DNA-PK consists of a 460-kDa subunit (p460) that contains a putative kinase domain and a heterodimeric subunit (Ku) that binds to double-stranded DNA ends. Previous reports suggested that the activation of DNA-PK requires the binding of Ku to DNA. To investigate this further, p460 and Ku were purified separately to homogeneity. Surprisingly, p460 was capable of binding to DNA in the absence of Ku. The binding of p460 to double-stranded DNA ends was salt-labile and could be disrupted by single-stranded or supercoiled DNA, properties distinct from the binding of Ku to DNA. Under low salt conditions, which permitted the binding of p460 to DNA ends, the kinase was activated. Under higher salt conditions, which inhibited the binding of p460, activation of the kinase required the addition of Ku. Significantly, when the length of DNA decreased to 22 bp, Ku competed with p460 for DNA binding and inhibited kinase activity. These data demonstrate that p460 is a self-contained kinase that is activated by direct interaction with double-stranded DNA and that the role of Ku is to stabilize the binding of p460 to DNA ends.

Animals↗
PubMed1996

Mutation of the cysteine residues in human initiation factor 4E: effects on mRNA cap binding ability.

To investigate the role of four Cys residues of eukaryotic initiation factor (eIF)-4E in the recognition of the mRNA cap structure, single, double and quadruple mutant genes which encoded the Ala residues in the place of the respective Cys residues were prepared using a synthetic human eIF-4E gene by the site-directed mutagenesis, and were expressed in E. coli with the same way as the wild type. The cap binding abilities of respective mutated eIF-4Es were compared with that of the wild-type by m7GTP affinity chromatography. The results suggest that, although all four of Cys residues participate in the recognition of the mRNA cap structure, they contribute indirectly to the stabilization of overall tertiary structure, especially of the cap binding pocket, rather than by direct interaction. A comparison among the cap binding abilities of single, double and quadruple mutants indicated no existence of internal disulfide bonds in eIF-4E.

Escherichia coli↗
PubMed1975

8-Anilinonaphthalene-1-sulphonate interaction with whole and disrupted mitochondria: a re-evaluation of the use of double-reciprocal plots in the derivation of binding parameters for fluorescent probes binding to mitochondrial membranes.

It is shown that 8-anilinonaphthalene-1-sulphonate is a permeant anion of whole mitochondrial membranes. It is also shown experimentally and algebraically that plots of reciprocal fluorescence against reciprocal membrane concentration, at a fixed 8-anilino-naphthalene-1-sulphonate concentration, are straight lines even when more than one binding site is involved.

Anilino Naphthalenesulfonates↗
PubMed2000

The cysteine- and glycine-rich LIM domain protein CRP2 specifically interacts with a novel human protein (CRP2BP).

We used the interaction trap to isolate a novel human protein that specifically interacts with the double LIM domain protein CRP2. This protein, designated CRP2BP (for CRP2 binding partner), was previously postulated by sequencing contigs of human chromosome 20. The observed interaction is mediated via the LIM1 domain of CRP2 and is of functional relevance in cellular environment. This novel single copy gene spans approximately 45-bp and is organized into at least ten exons. CRP2BP is expressed in all human tissues tested, with a major mRNA of 4-kb in size and an additional 3.2-kb transcript in placenta.

Adaptor Proteins, Signal Transducing↗
PubMed2003

RP4 repressor protein KorB binds to the major groove of the operator DNA: a Raman study.

KorB is a member of the ParB family of bacterial partitioning proteins. The protein encoded by the conjugative plasmid RP4 is part of the global control circuit and regulates the expression of plasmid genes, the products of which are involved in replication, transfer, and stable inheritance. KorB is a homodimeric protein which binds to palindromic 13 bp DNA sequences [5'-TTTAGC((G)/(C))GCTAAA-3'] present 12 times in the 60 kb plasmid. Each KorB subunit is composed of two domains; the C-domain is responsible for the dimerization of the protein, whereas the N-terminal domain recognizes and binds to the operator sequence (O(B)). Here we describe results of a Raman spectroscopic study of the interaction of the N-domain with a double-stranded model oligonucleotide composed of the palindromic binding sequence and terminal 5'-A(Br)U and AG-3' bases. Comparison of the Raman spectra of the free KorB N-domain and O(B) DNA with the spectrum of the complex reveals large differences. KorB-N binds in the major groove of the O(B) DNA, and the interactions induce changes in the DNA backbone and in the secondary structure of the protein.

DNA Primase↗
PubMed2004

Atomic force microscopy study of cellulose surface interaction controlled by cellulose binding domains.

Colloidal probe microscopy has been used to study the interaction between model cellulose surfaces and the role of cellulose binding domain (CBD), peptides specifically binding to cellulose, in interfacial interaction of cellulose surfaces modified with CBDs. The interaction between pure cellulose surfaces in aqueous electrolyte solution is dominated by double layer repulsive forces with the range and magnitude of the net force dependent on electrolyte concentration. AFM imaging reveals agglomeration of CBD adsorbed on cellulose surface. Despite an increase in surface charge owing to CBD binding to cellulose surface, force profiles are less repulsive for interactions involving, at least, one modified surface. Such changes are attributed to irregularity of the topography of protein surface and non-uniform distribution of surface charges on the surface of modified cellulose. Binding double CBD hybrid protein to cellulose surfaces causes adhesive forces at retraction, whereas separation curves obtained with cellulose modified with single CBD show small adhesion only at high ionic strength. This is possibly caused by the formation of the cross-links between cellulose surfaces in the case of double CBD.

Cellulose↗