Showing papers on "Cooperative binding published in 1991"

Noncovalent DNA binding of bis(1,10-phenanthroline)copper(I) and related compounds.

Abstract: The noncovalent DNA binding of the bis(1,10-phenanthroline)copper(I) complex [(Phen)2CuI] was examined under anaerobic conditions by absorption and circular dichroism spectroscopy, and viscometry, as a function of phenanthroline concentration. Analyses according to the McGhee-von Hippel method indicated that binding exhibited both neighbor-exclusion and positive cooperativity effects, with a neighbor-exclusion parameter n approximately 2 and a cooperativity parameter omega approximately 4. The association constant for (Phen)2CuI binding decreased with increasing concentration of phenanthroline in excess over that required to stoichiometrically generate (Phen)2CuI, indicating that free phenanthroline was a weak competitive inhibitor of (Phen)2CuI binding. The maximal association constant for DNA binding of (Phen)2CuI in 0.2 M NaCl and 9.8% ethanol, extrapolated to zero concentration of excess phenanthroline, was 4.7 x 10(4) M-1 (DNA base pairs). The magnitude of the neighbor-exclusion parameter, the changes in spectral properties of (Phen)2CuI induced by DNA binding, and the increase in DNA solution viscosity upon (Phen)2CuI addition are consistent with a model for DNA binding by (Phen)2CuI involving partial intercalation of one phenanthroline ring of the complex between DNA base pairs in the minor groove as suggested previously [Veal & Rill (1989) Biochemistry 28, 3243-3250]. Viscosity measurements indicated that the mono(phenanthroline)copper(I) complex also binds to DNA by intercalation; however, no spectroscopic or viscometric evidence was found for DNA binding of free phenanthroline or the bis(2,9-dimethyl-1,10-phenanthroline)copper(I) complex. DNA binding of free phenanthroline may be cooperative and induced by prior binding of (Phen)2CuI.

Activation of skeletal alpha-actin gene transcription: the cooperative formation of serum response factor-binding complexes over positive cis-acting promoter serum response elements displaces a negative-acting nuclear factor enriched in replicating myoblasts and nonmyogenic cells.

Abstract: Three upstream CBAR cis-acting promoter elements, containing the inner core CC(A/T)6GG of the serum response element (SRE), are required for myogenic cell type-restricted expression of the avian skeletal alpha-actin gene (K.L. Chow and R.J. Schwartz, Mol. Cell. Biol. 10:528-538, 1990). These actin SRE elements display differential binding properties with two distinct nuclear proteins, serum response factor (SRF) and another factor described here as F-ACT1. SRF is able to bind to all actin SREs with various affinities. This multisite interaction is marked by cooperative binding events in that the two high-affinity proximal and distal SREs facilitate the weak central-site interaction with SRF, leading to the formation of a higher-order SRF-promoter complex. Functional analyses reveal that undisrupted multiple SRF-DNA interactions are absolutely essential for promoter activity in myogenic cells. F-ACT1, present at higher levels in nonmyogenic cells and replicating myoblasts than in myotubes, binds solely to the proximal SRE, and its binding is mutually exclusive with that of SRF owing to their overlapping base contacts. The cooperative promoter binding by SRF, however, can effectively displace prebound F-ACT1. In addition, an intact F-ACT1 binding site acts as a negative promoter element by restricting developmentally timed expression in myoblasts. F-ACT1 may therefore act as a repressor of skeletal alpha-actin gene transcription. This interplay between F-ACT1 and SRF may constitute a developmental as well as a physiologically regulated mechanism which modulates sarcomeric actin gene expression.

Characterization of multiple [3H]ryanodine binding sites on the Ca2+ release channel of sarcoplasmic reticulum from skeletal and cardiac muscle: evidence for a sequential mechanism in ryanodine action.

Abstract: Kinetic and equilibrium measurements of [3H]ryanodine binding to the Ca2+ release channel of rabbit skeletal and rat cardiac sarcoplasmic reticulum (SR) are examined to ascertain the nature of cooperative interactions among high and low affinity binding sites and to quantitate their distribution. Equilibrium studies reveal affinities of 1-4 nM for the highest affinity binding site and of 30-50 nM, 500-800 nM, and 2-4 microM for the lower affinity sites in both preparations, with Hill coefficients of significantly less than 1, and initial rates of association and dissociation increase with increasing concentrations of ryanodine. SR vesicles are actively loaded in the presence of pyrophosphate, and fluctuations in extravesicular Ca2+ are measured by the absorbance change of antipyrylazo III. The data demonstrate a biphasic, time- and concentration-dependent action of ryanodine on the release of Ca2+, with an initial activation and a subsequent inactivation phase. Kinetic analysis of the activation of Ca2+ release by ryanodine, in consonance with the binding data, demonstrates the existence of multiple binding sites for the alkaloid on the channel complex, with nanomolar to micromolar affinities. Based on the present findings obtained by receptor binding analysis and Ca2+ transport measurements, we suggest a model that describes four, most plausibly negatively cooperative, binding sites on the Ca2+ release channel. Occupation of ryanodine binding sites produces sequential activation followed by inactivation of the SR channel, revealing the strong possibility of an irreversible uncoupling of the native function of the receptor/channel complex by high concentrations of ryanodine. A model relating ryanodine receptor occupancy with SR Ca2+ release stresses two important new findings regarding the interaction of ryanodine with its receptor. First, ryanodine binds to four sites on the oligomeric channel complex with decreasing affinities, which can be best described by allosteric negative cooperativity. Second, binding of ryanodine to its receptor activates the Ca2+ release channel in a concentration-dependent and saturable manner in the range of 20 nM to 1 mM and produces a kinetically limited and sequential inactivation of the Ca2+ channel, with the concomitant attainment of full negative cooperativity. The results presented suggest that driving of the complex toward full negative cooperativity with high concentrations of ryanodine promotes a long-lived conformational state in which ryanodine is physically occluded and hindered from free diffusion from its binding site.

Electrostatic contributions to the binding of ca2+ in calbindin d9k

Abstract: A set of accurate experimental data is provided for Ca2+ ion binding to calbindin D9k, a protein in the calmodulin superfamily of intracellular regulatory proteins. The study comprises both the role of protein surface charges and the effects of added electrolyte. The two macroscopic Ca2(+)-binding constants K1 and K2 are determined for the wild-type and eight mutant calbindins in 0, 0.05, 0.10, and 0.15 M KCl from titrations in the presence of Quin 2 or 5,5'-Br2BAPTA. The mutations involve replacement of surface carboxylates (of Glu17, Asp19, Glu26, and Glu60) with the corresponding amides. It is found that K1K2 may decrease by a factor of up to 2.5 x 10(5) (triple mutant in 0.15 M KCl as compared to the wild-type protein in 0 M KCl). Ca2(+)-binding constants of the individual Ca2+ sites (microscopic binding constants) have also been determined. The positive cooperativity of Ca2+ binding, previously observed at low salt concentration [Linse et al. (1987) Biochemistry 26, 6723-6735], is also present at physiological ionic strength and amounts to 5 kJ.mol-1 at 0.15 M KCl. The electrolyte concentration and some of the mutations are found to affect the cooperativity. 39K NMR studies show that K+ binds weakly to calbindin. Two-dimensional 1H NMR studies show, however, that potassium binding does not change the protein conformation, and the large effect of KCl on the Ca2+ affinity is thus of unspecific nature. Two-dimensional 1H NMR has also been used to assess the structural consequences of the mutations through assignments of the backbone NH and C alpha H resonances of six mutants.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of iC3b with recombinant isotypic and chimeric forms of CR2.

Abstract: CR2 is a component of a signal transduction complex on B lymphocytes that augments B cell responses to Ag. We have quantitatively assessed binding by the two isotypic forms of CR2 for two of its ligands, the polymerized iC3b (p(iC3b)) fragment of C3, and gp350/220, the EBV membrane protein. The recombinant 15-SCR or 16-SCR forms of CR2 bound p(iC3b) with identical affinities. Full binding activity of CR2 for p(iC3b) was observed with a chimera comprised of SCR-1 and -2 of CR2 fused to SCR-17 through -30 of CR1. Therefore, the alternatively spliced SCR-10a has no role in binding p(iC3b), and the binding activity of wild type receptor for iC3b can be reconstituted with SCR-1 and -2 of CR2. The binding affinities of the two isoforms of CR2 for soluble gp350/220 were also similar. Additional sites in the C3c region of C3 have been postulated also to interact with CR2. However, monomeric iC3b and C3d were equally effective in inhibiting the binding of p(iC3b) to CR2, indicating that the C3c region of iC3b does not contribute to the interaction of iC3b with CR2. Finally, the relative abilities of C3b and iC3b to bind to CR1 and CR2 were compared. The conversion of C3b to iC3b generated a ligand with an approximate 100-fold decrease in affinity for CR1 and a 10-fold increased affinity for CR2, resulting in a 1000-fold greater likelihood for binding to the latter receptor that may then promote B cell activation.

H chain C domains influence the strength of binding of IgG for streptococcal group A carbohydrate.

Abstract: We have produced a panel of murine anti-streptococcal mAbs, expressing identical V domains and different H chain C domains, corresponding to the IgG3, IgG1, and IgG2b subclasses. We have used these mAb to evaluate the role of IgG subclass-specific C region determinants in modulating the interaction between antibody and the bacterial surface. We report, for the first time, that V region-identical murine IgG of different subclasses exhibit substantial differences in binding to specific Ag; IgG3 mAb binds more strongly to streptococci than the IgG1 and IgG2b mAb or IgG3-derived F(ab')2 fragments. Furthermore, the IgG3 mAB binds cooperatively to the bacteria, whereas the IgG1, IgG2b, and IgG3-derived F(ab')2 fragments do not exhibit significant cooperativity, which suggests that differences in Fc region structure can affect antibody binding to multivalent Ag by modulating the potential for cooperative binding. These results suggest a plausible mechanism by which murine IgG3 could be more effective, than other antibodies bearing identical V domains, but of different gamma-subclass, in mediating bacterial immunity.

DNA-binding specificity of the fushi tarazu homeodomain.

Abstract: The fushi tarazu (ftz) gene of Drosophila melanogaster encodes a homeodomain-containing transcription factor that functions in the formation of body segments. Here we report an analysis of the DNA-binding properties of the ftz homeodomain in vitro. We provide evidence that the homeodomain binds to DNA as a monomer, with an equilibrium dissociation constant of 2.5 x 10(-11) M for binding to a consensus binding site. A single ftz binding site occupies 10 to 12 bp, as judged by the ability of protein bound at one site to interfere with binding to an adjacent site. These experiments also demonstrated a lack of cooperative binding between ftz homeodomains. Analysis of single-nucleotide substitutions over an 11-bp sequence shows that a stretch of 6 bp is critical for binding, with an optimal sequence of 5'CTAATTA3'. These data correlate well with recent structural evidence for base-specific contact at these positions. In addition, we found that sequences flanking the region of direct contact have effects on DNA binding that could be of biological significance.

Highly sequential binding of protein kinase C and related proteins to membranes.

Abstract: Protein kinase C belongs to a class of proteins that displays simultaneous interaction with calcium and phospholipids. Other members of this class include two proteins (Mr 64K and 32K) isolated from bovine brain. The association of these proteins with membranes exhibited highly unusual properties that were not consistent with a simple equilibrium. Titration of protein-phospholipid binding as a function of calcium showed an apparently normal curve with a low degree of cooperativity. The binding was rapid and quickly adjusted to changes in the calcium concentration. Calcium was readily exchanged from the protein-phospholipid complex. However, at each calcium concentration, membrane-bound protein was not in rapid equilibrium with free protein in solution; the half-time for dissociation exceeded 24 h. Titration of phospholipid vesicles with proteins showed different saturation levels of bound protein at different calcium concentrations. The amount of protein bound was almost entirely determined by the concentration of calcium and was virtually unaffected by the free protein concentration. These properties suggested that protein-phospholipid binding involved a sequence of steps that were each irreversible upon completion. These binding properties were consistent with high-affinity interaction between protein and phospholipid, high cooperativity with respect to calcium (N greater than or equal to 10), clustering of acidic phospholipids, and negative cooperativity with respect to protein density on the membrane. A major apparent problem with the complete titration of PKC-membrane interaction was a requirement for calcium in excess of intracellular levels. However, a highly sequential binding process showed that a number of protein-binding sites on the membrane would be saturated with calcium at physiological levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Stability of a Lac repressor mediated "looped complex".

Abstract: The quantitation of the stability of a protein-mediated "looped complex" of the Lac repressor and DNA containing two protein-binding sites whose centers of symmetry are separated by 11 helical turns (114 bp) was accomplished by footprint and gel mobility-shift titration techniques. Lac repressor binding to this DNA was only moderately cooperative; a cooperative free energy of -1.0 kcal/mol was calculated in a model-independent fashion from the individual-site loading energies obtained from the footprint titration studies. In order to partition the cooperative binding energy into components representing the dimer-tetramer association of Lac repressor and the cyclization probability of the intervening DNA, advantage was taken of the presence of experimental measures that were in proportion to the concentration of the looped complex present in solution. One measure was the DNase I hypersensitivity observed in footprint titrations in bands located between the two binding sites. The second measure resulted from the electrophoretic resolution in the gel mobility-shift titrations of the band representing the doubly liganded "tandem complex" from the band representing the singly liganded complexes, including the looped complex. Analysis of the footprint and mobility-shift titration data utilizing this additional information showed that approximately 65% of the molecules present in solution are looped complexes at pH 7.0, 100 mM KCl, and 20 degrees C when the binding sites on the DNA are saturated with protein. Reconciliation of the observed low binding cooperativity and the high proportion of looped complexes could only be obtained when the titration data were analyzed by a model in which Lac repressor tetramers dissociate into dimers in solution. The proportion of looped complexes present in solution is highly dependent on the dimer-tetramer association constant, delta Gtet. This result is consistent with the determination by high-pressure fluorescence techniques that Lac repressor tetramers dissociate with an association free energy comparable to their DNA-binding free energies [Royer, C. A., Chakerian, A. E., & Matthews, K. S. (1990) Biochemistry 29, 4959-4966]. However, when the value of delta Gtet of -10.6 kcal/mol (at 20 degrees C) reported by Royer et al. (1990) is assumed, the titration data demand that tetramers bind DNA with much greater affinity than dimers: a result inconsistent with the destabilization of tetramers by the operator observed in the dimer-tetramer dissociation studies.(ABSTRACT TRUNCATED AT 400 WORDS)

Cooperative binding of the E2 protein of bovine papillomavirus to adjacent E2-responsive sequences.

Abstract: The DNA-binding properties of purified full-length E2 protein from bovine papillomavirus type 1 have been investigated by utilizing a quantitative gel shift analysis. By using a recombinant baculovirus which express the E2 open reading frame from the polyhedrin promoter, the full-length E2 protein was synthesized in insect cells and purified to homogeneity by using an E2 binding site (ACCGN4CGGT)-specific oligonucleotide column. The Kd of E2 binding to a 41-bp oligonucleotide containing a single binding site was found to be 2 x 10(-11) M. When two binding sites were included on an oligonucleotide, cooperative binding to these sites by the E2 protein was observed. A cooperativity parameter of 8.5 was determined for E2 binding to two sites. An 86-amino-acid peptide encompassing the C terminus of the protein retains the ability to bind E2 binding sites with a Kd of 4 x 10(-10) M but exhibits slight cooperativity of binding to two adjacent sites. A major determinant for cooperative binding of the full-length E2 protein is thus encoded by the N-terminal amino acids outside the minimal DNA binding domain.

Cooperative Binding oftheE2Protein ofBovinePapillomavirus to Adjacent E2-Responsive Sequences

Abstract: The DNA-binding properties of purified full-length E2 protein from bovine papillomavirus type 1 have been investigated by utilizing a quantitative gel shift analysis. By using a recombinant baculovirus which express the E2 open reading frame from the polyhedrin promoter, the full-length E2 protein was synthesized in insect cells and purified to homogeneity by using an E2 binding site (ACCGN4CGGT)-specific oligonucleotide column. The Kd of E2 binding to a 41-bp oligonucleotide containing a single binding site was found to be 2 x 10(-11) M. When two binding sites were included on an oligonucleotide, cooperative binding to these sites by the E2 protein was observed. A cooperativity parameter of 8.5 was determined for E2 binding to two sites. An 86-amino-acid peptide encompassing the C terminus of the protein retains the ability to bind E2 binding sites with a Kd of 4 x 10(-10) M but exhibits slight cooperativity of binding to two adjacent sites. A major determinant for cooperative binding of the full-length E2 protein is thus encoded by the N-terminal amino acids outside the minimal DNA binding domain.

Computer simulations and experimental studies of gel mobility patterns for weak and strong non-cooperative protein binding to two targets on the same DNA: application to binding of tet repressor variants to multiple and single tet operator sites.

Abstract: A series of computer simulations of gel patterns assuming non-cooperative binding of a protein to two targets on the same DNA fragment was performed and applied to interprete gel mobility shift experiments of Tet repressor-tet operator binding. While a high binding affinity leads to the expected distribution of free DNA, DNA bound by one repressor dimer and DNA bound by two repressor dimers, a lower affinity or an increased electrophoresis time results in the loss of the band corresponding to the singly occupied complex. The doubly occupied complex remains stable under these conditions. This phenomenon is typical for protein binding to DNA fragments with two identical sites. It results from statistical disproportionation of the singly occupied complex in the gel. The lack of the singly occupied complex is commonly taken to indicate cooperative binding, however, our analysis shows clearly, that cooperativity is not needed to interprete these results. Tet repressor proteins and small DNA fragments with two tet operator sites have been prepared from four classes of tetracycline resistance determinants. The results of gel mobility shift analyses of various complexes of these compounds confirm the predictions. Furthermore, calculated gel patterns assuming different gel mobilities of the two singly occupied complexes show discrete bands only if the electrophoresis time is shorter than the inverse of the microscopic dissociation rate constant. Simulations assuming increasing dissociation rates predict that the two bands first merge into one, which then disappears. This behavior was verified by gel mobility analyses of Tet repressor-tet operator titrations at increased salt concentrations as well as by direct footprinting of the complexes in the gel. It is concluded that comparison of the intensities of the single and the double occupation bands allow a rough estimation of the dissociation rate constant. On this basis the sixteen possible Tet repressor-tet operator combinations can be ordered with decreasing binding affinities by a simple gel shift experiment. The implications of these results for gel mobility analyses of other protein-DNA complexes are discussed.

EPR and 1H-NMR spectroscopic studies on the paramagnetic iron at the active site of phenylalanine hydroxylase and its interaction with substrates and inhibitors.

Abstract: The paramagnetic iron at the active site of highly purified, catalytically active phenylalanine hydroxylase was studied by EPR at 3.6 K and one-dimensional 1H-NMR spectroscopy at 293 K. The EPR-detectable iron of the bovine enzyme was found to be present as a high-spin form (S= 5/2) in different ligand field symmetries depending on medium conditions (buffer ions) and the presence of ligands known to bind at the active site. At 3.6 K and in phosphate buffer, the paramagnetic iron is coordinated in an environment of rhombic symmetry (g= 4.3), whereas Tris buffer favours an environment of axial ligand field symmetry (g= 6.7, 5.3 and 2.0). The latter axial type of signals resembles those observed at g= 7.0, 5.2 and 1.9 for the enzyme in phosphate buffer when L-noradrenaline is added as an active-site ligand (inhibitor). The same proportion of iron that coordinates to L-noradrenaline seems to be reduced by the pterin cofactor and participate in catalysis. Experimental evidence is presented that Tris inhibits the enzyme by interacting with the enzyme-bound ferric iron and decreases its rate of reduction by the tetrahydropterin cofactor. Preincubation with dithiothreitol also inhibits the enzyme activity and prevents the reduction of its catalytically active ferric iron by pterin cofactors as well as binding of catecholamines to the enzyme. 1H-NMR spectroscopy revealed that the substrate (l-phenylalanine) and l-noradrenaline bind close to the paramagnetic iron, and that the catecholamine displaces the substrate from its binding at the active site. The results support our recently proposed model for the cooperative binding of inhibitor and substrate at the active site [Martinez, A. et al. (1990) Eur. J. Biochem. 193, 211–219].

Cooperative binding of oligonucleotides to DNA by triple helix formation: dimerization via Watson-Crick hydrogen bonds

Abstract: [Introduction] Cooperative binding by proteins to DNA results in higher sequence specificity as well as greater sensitivity to concentration changes. We recently reported cooperative binding of two oligonucleotides at abutting sites by triple helix formation on double helical DNA. However, the enhanced binding observed was modest (a factor of 3.5) and likely due to favorable basestacking interactions between adjacent oligonucleotides and/or induced conformational changes propagated to adjacent binding sites. Thus, the issue arises whether cooperativity in oligonucleotide-directed triple helix formation can be enhanced by the addition of discrete dimerization domains. We report here the binding properties of oligonucleotides that dimerize by Watson-Crick hydrogen bonds and bind neighboring sites on double helical DNA by triple helix formation.

The C-terminal 70 amino acids of the adenovirus E4-ORF6/7 protein are essential and sufficient for E2F complex formation

Abstract: E2F is a cellular transcription factor that binds to the adenovirus (Ad) E1A enhancer and E2aE promoter regions, to the cellular c-myc P2 and dihydrofolate reductase promoters, and to other viral and cellular regulatory regions. The binding activity of E2F to the Ad E2aE promoter is dramatically increased during an adenovirus infection (termed E2F induction). E2F induction is dependent on the expression of the 150 amino acid E4-ORF6/7 protein which forms a direct, physical complex with E2F to mediate the cooperative and stable binding of E2F to inverted sites in the E2aE promoter. Using in vitro DNA binding assays to measure the formation of the infection-specific complexes, we have defined the minimal domain of the E4-ORF6/7 protein, the C-terminal 70 amino acids, required to complex with E2F and stabilize its binding at the E2aE promoter. The ability of mutant E4-ORF6/7 proteins to form the stable E2F-E2aE promoter complex in vitro correlated well with their ability to trans-activate E2 transcription in vivo. These observations support a model in which the E4-ORF6/7 protein binds to E2F to induce the cooperative binding of two E2F molecules to the E2aE promoter thereby activating E2 transcription.

Cooperative Binding of Porphyrin by Anti-Porphyrin Antibodies

Abstract: One of the monoclonal antibodies specific for MESO-tetrakis(4-carboxyphenyl)porphine (TCPP) causes a large shift of the Soret band of TCPP to a longer wavelength and large induced Cotton effects (ICD) on TCPP. Quantitative analyses of the binding of TCPP to the antibody by ICD show that not only one-to-one binding but two-to-one (binding site: TCPP) binding occurs in excess of the antibody over TCPP.

Erythropoietin receptors induced by dimethyl sulfoxide exhibit positive cooperativity associated with an amplified biologic response.

Abstract: Erythropoietin triggers the differentiation of erythrocyte progenitors by binding to receptors on their plasma membrane. We report here that pretreatment of erythropoietin-responsive murine erythroleukemia cells with chemical inducers resulted in a striking increase in erythropoietin-specific hemoglobinization. This amplification of the erythropoietin biologic response was accompanied by the induction of a new population of high-density receptors (approximately 20,000 per cell) exhibiting marked positive cooperativity. Erythropoietin binding to new receptors displayed a convex upward Scatchard plot and a Hill coefficient (nH) of 6.75. Measurement of erythropoietin receptor mRNA demonstrated an initial decrease in receptor transcript followed by an approximately 2- to 3-fold increase after 24-48 hr. This increase in receptor message does not appear to account for the magnitude of the receptor up-regulation by dimethyl sulfoxide. We propose that this positive cooperativity reflects the interaction (clustering) of receptors, presumably through the formation of homooligomers or heterooligomers, and that this receptor interaction may amplify the erythropoietin signal transduction pathway.