Showing papers on "Cooperativity published in 1985"

Oxygen binding properties of human mutant hemoglobins synthesized in Escherichia coli

Abstract: Human beta-globin was synthesized in Escherichia coli as a cleavable fusion protein, using the expression vector pLcIIFX beta-globin [Nagai, K. & Thogersen, H. C. (1984) Nature (London) 309, 810-812]. The fusion protein cIIFX beta-globin was purified to homogeneity and cleaved at the junction by blood coagulation factor Xa; the authentic beta-globin was liberated. Beta-globin was folded in vitro and reconstituted with heme and alpha subunits to form alpha 2 beta 2 tetramers. The oxygen binding properties of reconstituted Hb are essentially the same as those of human native Hb. Two mutant Hbs (Hb Nympheas [Cys-93 beta----Ser] and Hb Daphne [Cys-93 beta----Ser, His-143 beta----Arg]) were constructed by site-directed mutagenesis using synthetic oligonucleotides. Hb Nympheas showed a slightly increased oxygen affinity and diminished cooperativity with normal 2,3-diphosphoglyceric acid and slightly reduced alkaline Bohr effects. Hb Daphne showed low cooperativity with high oxygen affinity. The alkaline Bohr effect was slightly reduced but the diphosphoglycerate effect was enhanced by 50% by the His-143 beta----Arg mutation. As arginine is fully charged at physiological pH and has a long flexible side chain, diphosphoglycerate binds more strongly to Hb Daphne.

Cooperativity theory in biochemistry

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Allosteric effects in organic chemistry: binding cooperativity in a model for subunit interactions

Abstract: Synthese, structure et dynamique d'un systeme faisant intervenir deux ethers crowns connectes par l'intermediaire d'un squelette biphenyle

Mechanism of intercalation: Ion effects on the equilibrium and kinetic constants for the interaction of propidium and ethidium with DNA

Abstract: The effects of sodium ion concentration on the binding isotherms and association and dissociation reaction rates for the interaction of the closely related intercalating dication, propidium, and the monocation, ethidium, with DNA have been determined by spectrophotometric binding and stopped-flow kinetics methods. The binding of propidium to DNA is best described by a neighbor-exclusion binding isotherm (two base pairs per binding site) with negative ligand cooperatively on binding. The cooperativity parameter is fairly independent of salt concentration, while the log of the observed equilibrium binding constant varies with −log [Na+], with a slope of two for the propidium–DNA interaction. These effects of the sodium ion on the equilibrium binding of propidium with DNA are similar to those previously described for the dication, quinacrine [Wilson, W.D. & Lopp, I.G. (1979) Biopolymers, 18, 3025–3041]. Ethidium behaves, as a function of salt, as a monocation binding to DNA with neighbor exclusion and without ligand cooperativity. Equations are derived for two limiting kinetics models for intercalation involving binding of the intercalator to a preequilibrium, open state of DNA (model I) or binding form a preequilibrium externally associated state of the intercalator with DNA (model II). Model II gives the best fit to all of the kinetic results if it is assumed that the initial external interaction of the intercalator with DNA is similar to the exchange of free and condensed simple counterions. Intercalation then occurs from this state following an opening transition of the base pairs of the double helix. This model predicts a larger effect of salt concentration on the association than on the dissociation reaction, and that is what is experimentally observed. The intercalation conformational change makes a significant contribution to the ionic effects for both the equilibrium binding and the kinetic constants. The dissociation results and the association reaction results under pseudo-first-order conditions could be fit with single exponential curves under the conditions of our experiments.

Structural junctions in DNA: the influence of flanking sequence on nuclease digestion specificities

Abstract: When a protein binds to DNA, the affinity of this protein for its primary site of interaction may be influenced by the nature of flanking sequences. This is thought to be a consequence of local cooperativity in the DNA molecule, where the conformation at one point along the helix can influence the conformation at another, and thereby modulate the free energy of protein-DNA recognition. In order to learn more about this process, we have carried out experiments of two sorts. First, we have constructed sequences of the type (dA)11 (dG)8, where the conformational preferences of the DNA molecule switch from one extreme to another over just a single base pair, and subjected them to digestion by DNAase I and DNAase II. This is to learn whether the structure changes abruptly at the junction point, or more gradually with an influence extending into residues on either side. Secondly, we have subjected long plasmid DNA to digestion by restriction enzymes Fnu DII, Hae III, Hha I and Msp I, to look for correlations between cutting rate and the identity of nucleotides on either side of the restriction site. The influence of flanking sequence on nuclease digestion specificities is clearly evident in both kinds of experiment, but the rules governing this seem complex and not easily formulated. The best that can be done at present is to divide the problem into two parts, "analogue" and "digital", representing sugar-phosphate and base components of recognition.

Virus-receptor interaction in the adenovirus system: characterization of the positive cooperative binding of virions on HeLa cells.

Abstract: The established positive cooperativity of adenovirus 2 binding to HeLa cells revealed a strong temperature dependence. The degree of cooperativity, quantified by means of Hill coefficients, progressively increased from 10 degrees C to reach a maximum level, which was maintained between 20 and 37 degrees C. On the other hand, negative cooperativity of virion attachment was apparent at 3.0 degrees C and on glutaraldehyde-stabilized cells. The corresponding monovalent ligand of the system, the fiber antigen, demonstrated only weak-positive cooperativity of the binding at 37.0 degrees C, which was absent at 3.0 degrees C. Dithiothreitol and dansylcadaverine, reagents inhibiting clustering of ligand-receptor complexes in the plasma membrane, markedly reduced the degree of positive cooperative binding at 37.0 degrees C. Evidently, the positive cooperative binding of adenovirus to HeLa cells at 37.0 degrees C is a consequence of both the multivalency of virus attachment proteins, i.e., fibers, on the virion and of the capacity of the receptor sites to migrate in the plane of the plasma membrane, forming local aggregates of virus-receptor site complexes.

Protonic conductivity of hydrated lysozyme powders at megahertz frequencies.

Abstract: Dielectric losses were measured for lysozyme powders of varied hydration level by a dielectric-gravimetric technique in the frequency range of 10 kHz to 10 MHz. The relaxation showed an isotope effect and pH dependence, indicating that the inferred conductivity is protonic. The transport process is likely restricted to the surface of individual macromolecules and involves shifting of protons between ionizable side chain groups of the protein. The time constant of the relaxation shows cooperativity in its seventh-order dependence on bound protons. The process develops in the hydration region critical for the onset of the catalytic properties of the enzyme. The binding of a substrate increases the relaxation time by a factor of 2. These observations suggest that the megahertz dispersion reflects behavior at the protein surface, specifically the cooperative channeling of proton flow through the active site, that may be of particular significance for the enzymatic and other functional properties of proteins.

Specificity of the binding of bacteriophage M13 encoded gene-5 protein to DNA and RNA studied by means of fluorescence titrations.

Abstract: The fluorescence quenching of the bacteriophage M13 encoded gene-5 protein was used to study its binding characteristics to different polynucleotides. Experiments were performed at different salt concentrations and in some instances at different temperatures. The affinity of the protein depends on the base and sugar composition of the polynucleotides involved and may differ appreciably, i.e. by orders of magnitude. The salt dependence of binding is within experimental accuracy equal for all single stranded polynucleotides. A method is presented to estimate values of the cooperativity constant from salt titration curves. These values are systematically higher than those obtained from titration experiments in which protein is added to a polynucleotide solution. A comparison is made between the binding constants of the gene-5 protein and the gene-32 protein encoded by the T4 phage. Possible implications of the binding characteristics of the gene-5 protein for an understanding of its role in vivo are discussed.

Mechanism of allosteric regulation of the Ca,Mg-ATPase of sarcoplasmic reticulum: studies with 5'-adenylyl methylenediphosphate.

Abstract: Four mechanisms for the allosteric regulation of the calcium and magnesium ion activated adenosinetriphosphatase (Ca,Mg-ATPase) of sarcoplasmic reticulum were examined. Negative cooperativity in substrate binding was not supported by 3H-labeled 5'-adenylyl methylenediphosphate (AMPPCP) binding, which was best fit by a single class of sites. Although calcium had no effect on the absence of cooperativity, it did increase the affinity of the enzyme for AMPPCP. Allosteric regulation via an effector site for AMPPCP or ATP on the same ATPase chain was eliminated by the stoichiometry of ATP and AMPPCP binding, 1 mol of site per mole of enzyme. The possibility that AMPPCP acts at an effector site was eliminated by showing that it competitively inhibits the rate of phosphoenzyme formation. Allosteric regulation of kinetics via site-site interaction in an oligomer was eliminated by showing that the inhibition of ATPase activity by fluorescein isothiocyanate is linearly dependent upon its incorporation into the sarcoplasmic reticulum. The fourth mechanism considered was stimulation of ATPase activity by the binding of ATP or AMPPCP at the active site after departure of ADP but before the departure of inorganic phosphate. This hypothesis was supported by site stoichiometry and by the observation that AMPPCP or ATP stimulates v/EP, the rate of ATP hydrolysis for a given level of phosphoenzyme. Computer simulation of this branched monomeric model could duplicate all experimental observations made with AMPPCP and ATP as allosteric regulators. The condition that the affinity of ATP binding to the enzyme be reduced when it is phosphorylated, which is required by the computer model, was confirmed experimentally.

Cooperativity among calmodulin's drug binding sites.

Abstract: The binding of felodipine, a dihydropyridine Ca2+ antagonist, to calmodulin has been studied by equilibrium dialysis and fluorescence techniques. Analysis using the Hill equation gives a Hill coefficient of 2. A plot of bound [felodipine] vs. free [felodipine]2 gives a Bmax of 1.9 mol/mol and a K0.5 of 22 microM. Two calmodulin antagonists, prenylamine and R24571, which have previously been shown to potentiate the fluorescent enhancement observed when felodipine binds to calmodulin [Johnson, J. D. (1983) Biochem. Biophys. Res. Commun. 112, 787], produce a reduction in Hill coefficient to 0.7 and 1.0, respectively, and account for the observed potentiation of felodipine binding. Titrations of felodipine with calmodulin in the absence and presence of prenylamine and R24571 suggest that these drugs decrease the K0.5 of calmodulin for felodipine by 25-fold. Thus, potentiating drugs (prenylamine and R24571) bind to either of the two felodipine binding sites and, through an allosteric mechanism, result in felodipine binding to the remaining site with greatly enhanced affinity. Two types of potentiating drugs are observed. Prenylamine exhibits a Hill coefficient of 0.8 whereas felodipine, R24571, and diltiazem exhibit Hill coefficients of 2 in their potentiation of felodipine binding. Titrations of felodipine and calmodulin with Ca2+ exhibit cooperativity with a Hill coefficient of 4. Half-maximal binding occurs near pCa 6.0. In the presence of R24571, the calcium dependence of felodipine binding is biphasic, now exhibiting a much higher affinity (pCa 7.6) component. A model is presented to explain the relationship of these various allosterically regulated conformers of calmodulin and their interactions and activation with its target proteins.

Oxygen equilibria of Octopus dofleini hemocyanin

Abstract: Oxygen binding by Octopus dofleini hemocyanin was examined under very nearly physiological conditions. The effects of pH, ionic composition, temperature, and aggregation were controlled so that the role each plays in modulating oxygen binding can be isolated. There is a very large effect of pH on affinity, the Bohr effect (delta log P50/delta pH = -1.7), which is the same at 10 and 20 degrees C. However, cooperativity is substantially altered over the same range of pHs at the two temperatures. The allosteric properties were examined by comparing the experimental data points to curves generated by use of the Monod-Wyman-Changeux model. A computer-fitting process was developed which allowed the individual allosteric parameters to be varied independently until the best fit could be determined. The relationship between kR and kT is responsible for the effect of pH on cooperativity. A change in the allosteric properties of the T form is primarily responsible for the differences due to temperature. Changing cation concentrations when the molecule is in the fully aggregated 51S form alters affinity without influencing cooperativity. The effect of Mg2+ is much greater than that of Na+. If the 51S decamer is dissociated to 11S monomers by removing divalent cations, oxygen binding is noncooperative. There is evidence for negative cooperativity, indicating heterogeneity of function within the subunit which contains seven oxygen binding domains. Association into decamers generates conformational change which results in a much wider range of allosteric function.

Mechanism of action of gentamicin components. Characteristics of their binding to Escherichia coli ribosomes.

Abstract: The binding of gentamicin (Gm) to Escherichia coli ribosomes and ribosomal subunits has been studied. By means of equilibrium dialysis and of statistical interpretation of the data it was found that [3H]gentamicin C2 and 6'-N-[3H]methylgentamicin C1a interact with three classes of sites on tight-coupled 70-S species: a first class concerning the tight and non-cooperative interaction with one drug molecule (Kd = 0.6 microM), a second class in which about five Gm molecules bind cooperatively (mean Kd = 10 microM), and a third class of very high capacity in which up to 70 drug molecules may interact. The extreme cooperativity of the third class of sites induces such an increase in the affinity for Gm that it may allow the shift of molecules already bound from high-affinity sites towards lower-affinity sites. The alteration of a ribosomal protein, L6, in a gentamicin-resistant mutant of E. coli abolished the multiclass and the cooperative aspects of ribosomes--gentamicin interaction. The large ribosomal subunits from E. coli MRE 600 strain interact cooperatively with Gm, whereas 50-S particles from the resistant mutant bind the drug in a diffuse way with high capacity and low affinity. The small subunits from both strains behave identically towards Gm. A good correlation is observed in comparing the gentamicin concentrations capable of saturating the different ribosomal classes of sites with concentrations inducing its multiphasic effects on protein synthesis.