Showing papers on "Cooperativity published in 1986"

Factor VII binding to tissue factor in reconstituted phospholipid vesicles: induction of cooperativity by phosphatidylserine

Abstract: The binding of factor VII and tissue factor produces a membrane-associated proteolytic complex which may be the primary biological initiator of coagulation Homogeneous tissue factor, a glycoprotein purified from bovine brain, was reconstituted into phospholipid vesicles ranging from neutral (100% phosphatidylcholine) to highly charged (40% phosphatidylserine) with octyl glucoside The vesicles were characterized with respect to size and tissue factor content and orientation Employing data from protease digestion, we deduced that tissue factor is randomly oriented; thus, its effective concentration in these vesicles was half its total concentration In all binding experiments, 1 mol of enzyme was bound per mole of available activator at saturation This stoichiometry was not affected by the form of the enzyme employed or the phospholipid composition of the vesicles With tissue factor incorporated into phosphatidylcholine vesicles, the Kd was 132 +/- 072 nM for factor VII and 454 +/- 137 nM for factor VIIa Thus, the one-chain zymogen binds to the activator with only slightly less affinity than the more active two-chain enzyme Active-site modification of factor VII and factor VIIa with diisopropyl fluorophosphate resulted in tighter binding of the derivatized molecules Inclusion of phosphatidylserine in the vesicles altered the binding both quantitatively and qualitatively With increasing acidic phospholipid, the concentration of enzyme required to occupy half the activator sites was decreased In addition, positive cooperativity was observed, the degree of which depended on the vesicle charge and the form of the enzyme An explicit two-site cooperative binding model is presented which fits these complex data In this model, tissue factor is at least a dimer with two interacting enzyme binding sites

Evidence for identity between the equilibrium unfolding intermediate and a transient folding intermediate: a comparative study of the folding reactions of alpha-lactalbumin and lysozyme.

Abstract: The refolding kinetics of alpha-lactalbumin at different concentrations of guanidine hydrochloride have been investigated by means of kinetic circular dichroism and stopped-flow absorption measurements. The refolding reaction consists of at least two stages, the instantaneous accumulation of the transient intermediate that has peptide secondary structure and the subsequent slow process associated with formation of tertiary structure. The transient intermediate is compared with the well-characterized equilibrium intermediate observed during the denaturant-induced unfolding. Stabilities of the secondary structures against the denaturant, affinities for Ca2+, and tryptophan absorption properties of the transient and equilibrium intermediates were investigated. In all of these respects, the transient intermediate is identical with the equilibrium one, demonstrating the validity of the use of the equilibrium intermediate as a model of the folding intermediate. Essentially the same transient intermediate was also detected in the folding of lysozyme, the protein known to be homologous to alpha-lactalbumin but whose equilibrium unfolding is represented as a two-state reaction. The stability and cooperativity of the secondary structure of the intermediate of lysozyme are compared with those of alpha-lactalbumin. The results show that the protein folding occurring via the intermediate is not limited to the proteins that show equilibrium intermediates. Although the unfolding equilibria of most proteins are well approximated as a two-state reaction, the two-state hypothesis may not be applicable to the folding reaction under the native condition. Two models of protein folding, intermediate-controlled folding model and multiple-pathway folding model, which are different in view of the role of the intermediate in determining the pathway of folding, are also discussed.

Cooperative and noncooperative binding of protein ligands to nucleic acid lattices: experimental approaches to the determination of thermodynamic parameters.

Abstract: Many biologically important proteins bind nonspecifically, and often cooperatively, to single-or double-stranded nucleic acid lattices in discharging their physiological functions. This binding can generally be described in thermodynamic terms by three parameters: n, the binding site size; K, the intrinsic binding constant; omega, the binding cooperativity parameter. The experimental determination of these parameters often appears to be straightforward but can be fraught with conceptual and methodological difficulties that may not be readily apparent. In this paper we describe and analyze a number of approaches that can be used to measure these protein-nucleic acid interaction parameters and illustrate these methods with experiments on the binding of T4-coded gene 32 (single-stranded DNA binding) protein to various nucleic acid lattices. We consider the following procedures: (i) the titration of a fixed amount of lattice (nucleic acid) with added ligand (protein); (ii) the titration of a fixed amount of ligand with added lattice; (iii) the determination of ligand binding affinities at very low levels of lattice saturation; (iv) the analysis of ligand cluster size distribution on the lattice; (v) the analysis of ligand binding to lattices of finite length. The applicability and limitations of each approach are considered and discussed, and potential pitfalls are explicitly pointed out.

Ca2+-induced alteration in the unfolding behavior of alpha-lactalbumin.

Abstract: Comparative studies of the unfolding equilibria of two homologous proteins, bovine alpha-lactalbumin and hen lysozyme, induced by treatment with guanidine hydrochloride have been made by analysis of the peptide and the aromatic circular dichroism spectra. The effect of the specific binding of Ca2+ ion by the former protein was taken into account in interpreting the unfolding equilibria of the protein. Proton nuclear magnetic resonance spectra of alpha-lactalbumin were also measured for the purpose of characterizing an intermediate structural state of the protein. In previous studies, alpha-lactalbumin was shown to be an exceptional protein whose equilibrium unfolding does not obey the two-state model of unfolding, although lysozyme is known to follow the two-state unfolding mechanism. The present results show that the apparent unfolding behavior of alpha-lactalbumin depends on Ca2+ concentration. At a low concentration of Ca2+, alpha-lactalbumin unfolds with a stable intermediate that has unfolded tertiary structure, as evidenced by the featureless nuclear magnetic resonance and aromatic circular dichroism spectra, but has folded secondary structure as evidenced by the peptide circular dichroism spectra. However, in the presence of a sufficiently high concentration of Ca2+, the unfolding transition of alpha-lactalbumin resembles that of lysozyme. The transition occurs between the two states, the native and the fully unfolded states, and the cooperativity of the unfolding is essentially the same as that of lysozyme. Such a change in the apparent unfolding behavior evidently results from an increase in the stability of the native state relative to that of the intermediate induced by the specific Ca2+ binding to native alpha-lactalbumin. The results are useful for understanding the relationship between the protein stability and the apparent unfolding behavior.

Energetics of cooperative protein-DNA interactions: comparison between quantitative deoxyribonuclease footprint titration and filter binding

Abstract: Using the binding of cI repressor protein to the lambda right and left operators as a model system, we have analyzed the two common experimental techniques for studying the interactions of genome regulatory proteins with multiple, specific sites on DNA. These are the quantitative DNase footprint titration technique [Brenowitz, M., Senear, D. F., Shea, M. A., & Ackers, G. K. (1986) Methods Enzymol. 130, 132-181] and the nitrocellulose filter binding assay [Riggs, A., Suzuki, H., & Bourgeois, S. (1970) J. Mol. Biol. 48, 67-83]. The footprint titration technique provides binding curves that separately represent the fractional saturation for each site. In principle, such data contain the information necessary to determine the thermodynamic constants for local site binding and cooperativity. We show that in practice, this is not possible for all values of the constants in multisite systems, such as the lambda operators. We show how these constants can nevertheless be uniquely determined by using additional binding data from a small number of mutant operators in which the number of binding sites has been reduced. The filter binding technique does not distinguish binding to the individual sites and yields only macroscopic binding parameters which are composite averages of the various local site and cooperativity constants. Moreover, the resolution of even macroscopic constants from filter binding data for multisite systems requires ad hoc assumptions as to a relationship between the number of ligands bound and the filter retention of the complex. Our results indicate that no such relationship exists. Hence, the technique does not permit determination of thermodynamically valid interaction constants (even macroscopic) in multisite systems.

Distinct subtypes of the opioid receptor with allosteric interactions in brain membranes

Abstract: The binding isotherms of opioid receptors in rat brain membranes with [3H]D-Ala2-D-Leu5-enkephalin ([3H]DADLE), [3H]dihydromorphine ([3H]DHM), and [3H]etorphine were analysed to show the effects of Mg2+, Na+, and guanine nucleotides. Four opioid receptor subtypes of delta, kappa, mu 1, and mu 2 specificities were differentiated, where necessary with the aid of specific displacing ligands. Both a guanine nucleotide [guanosine-5'-(beta, gamma-imido)triphosphate] and the cations (Na+, Mg2+) affect the affinity state of all four subtypes of the receptor. The opioid binding behaviour is found on detailed inspection to be complex, with cases of "half-of-the-sites" reactivity and of cooperativity. By their behaviour under the various ionic conditions noted, it was concluded that these subtypes are distinct, without the need to assume interconvertibility by such agents. The evidence suggests that the formation of heterologous kappa-delta or mu 1-mu 2 receptor complexes is required for stabilization of the high-affinity conformational state of the receptor. Important effects of cations in increasing the binding and regulating the equilibria of receptor association-dissociation were observed when these studies were conducted, not in the Tris-HCl buffer commonly used in opioid binding assays, but in N-tris[hydroxymethyl]-methyl-2-aminoethanesulphonate (K+) buffer (TES-KOH; 10 mM, pH 7.5): it was found that ionic species of Tris can substitute for divalent cations. Dithiothreitol effects on agonist binding in the presence and absence of the cations suggested that those cation effects involve the exchange of -SH/-SS- bonds between receptor subunits. All of the behaviour is interpreted in terms of a model involving association-dissociation equilibria of homologous and/or heterologous receptor subunits of an oligomeric opioid receptor structure.

Calcium Binding to Calmodulin. Cooperativity of the Calcium-Binding Sites

Abstract: The effects of Mg2+ ion, pH, and KCl concentration on Ca2+ binding to calmodulin were studied by using a Ca2+ ion-sensitive electrode. The Ca2+ ion affinity of calmodulin increased with increasing pH or decreasing KCl concentration. Cooperativity between the Ca2+-binding sites was observed, and increased with decreasing pH or increasing KCl concentration. Free Ca2+ ion concentration was decreased by adding MgCl2 ion at low Mg2+ concentration and increased at higher concentrations in the presence of small amounts of Ca2+ ion. The decrease of free Ca2+ ion concentration by Mg2+ ion strongly suggests cooperativity between the Ca2+-binding sites, and it is difficult to explain the decrease in terms of the ordered binding models previously proposed. These results can be explained by a simple model which has four equivalent binding sites that bind Ca2+ and Mg2+ competitively, and showing cooperativity when either Ca2+ or Mg2+ is bound. Mg2+ ion binding to calmodulin was measured in the presence or absence of Ca2+ to confirm the validity of this model, and no Mg2+-specific site was observed.

THE CO2 SENSITIVITY OF THE HEMOCYANINS AND ITS RELATIONSHIP TO Cl- SENSITIVITY

Abstract: The effect of CO2 on hemocyanin-oxygen binding is not generally related to the effect of Cl-. Some hemocyanins respond to both and some to either one alone. The direction of the responses of O2 affinity of the various hemocyanins to CO2 is poorly correlated with the direction of responses to other effectors. The influence of CO2 on Busycon and Limulus hemocyanins reaches its maximum at high pH. Since the effect can be abolished by restoring divalent cation activities to the control levels prior to the addition of CO2, we suggest that the effect is not specific but rather indirect, by the pairing of the allosteric effectors Ca+2 and Mg+2 with the CO2 anions. In contrast the effect of CO2 on crustacean hemocyanins is greaterat low pH and it can be enhanced by maintaining HCO3- levels within narrow limits and permitting PCO2 to vary by a large factor. This finding suggests that the effective species is molecular CO2. While a influences only oxygen affinity, CO2 may influence cooperativity as well. Indifferen...

Crystallographic analysis of mutant human haemoglobins made in Escherichia coli.

Abstract: The expression of beta-globin in Escherichia coli has enabled us to study the functional role of individual amino-acid residues in haemoglobin (Hb) by site-directed mutagenesis. In contrast to mammalian Hbs, some teleost fish haemoglobins show a drastic lowering of oxygen affinity and cooperativity at low pH, a phenomenon known as the Root effect. We have produced the two mutant haemoglobins Hb Nympheas [Cys(F9)93 beta----Ser] and Hb Daphne [His(H21)143 beta----Arg, Cys(F9)93 beta----Ser] to investigate this allosteric property. Although these substitutions were thought to be responsible for the Root effect, Hb Nympheas and Hb Daphne show an increased oxygen affinity and a reduced effect of pH on oxygen affinity. Our X-ray crystallographic studies show that the hydroxyl group of Ser 93 beta forms a hydrogen bond with Asp 94 beta which is in equilibrium with the salt bridge between Asp 94 beta and His 146 beta. The oxygen-binding properties of Hbs Nympheas and Daphne are accounted for by the partial disruption of the salt bridge.

Modulation of Haemocyanin Oxygen Affinity by L-Lactate — A Role for Other Cofactors

Abstract: Evidence for the modulation of crustacean haemocyanin oxygen affinity by L-lactate is reviewed. The specificity of the lactate effect, its magnitude and correlation with pH, Bohr effect, cooperativity and calcium concentration is shown. The magnitude and the identity of dialysable non-lactate effectors of haemocyanin oxygen affinity are discussed.

Effect of amino acid substitutions on the catalytic and regulatory properties of aspartate transcarbamoylase.

Abstract: Although intensive investigations have been conducted on the allosteric enzyme, aspartate transcarbamoylase, which catalyzes the first committed reaction in the biosynthesis of pyrimidines in Escherichia coli, little is known about the role of individual amino acid residues in catalysis or regulation. Two inactive enzymes produced by random mutagenesis have been characterized previously but the loss of activity is probably attributable to changes in the folding of the chains stemming from the introduction of charged and bulky residues (Asp for Gly-128 and Phe for Ser-52). Site-directed mutagenesis of pyrB, which encodes the catalytic chains of the enzyme, was used to probe the functional roles of several amino acids by making more conservative substitutions. Replacement of Lys-84 by either Gln or Arg leads to virtually inactive enzymes, confirming chemical studies indicating that Lys-84 is essential for catalysis. In contrast, substitution of Gln for Lys-83 has only a slight effect on enzyme activity, whereas chemical modification causes considerable inactivation. Gln-133, which has been shown by x-ray crystallography to reside near the contact region between the catalytic and regulatory chains, was replaced by Ala. This substitution has little effect on catalytic activity but leads to a marked increase in cooperativity. The Gln-83 mutant, in contrast, exhibits much less cooperativity. Since a histidine residue may be involved in catalysis and His-134 has been shown by x-ray diffraction studies to be in close proximity to the site of binding of a bisubstrate analog, His-134 was replaced by Ala, yielding a mutant with only 5% wild-type activity, considerable cooperativity, and lower affinity for aspartate and carbamoylphosphate. All of the mutants, unlike those in which charged or bulky residues replaced small side chains, bind the bisubstrate analog, which promotes the characteristic "swelling" of the enzymes indicative of the allosteric transition.

Beta nerve growth factor binding to PC12 cells. Association kinetics and cooperative interactions.

Abstract: The association kinetics of 125I beta nerve growth factor (NGF) binding to the PC12 clonal cell line have been examined in detail at 0.5 and 37 degrees C. These data were examined by utilizing a reversible second-order integrated rate equation, and the results were not consistent with a simple bimolecular process. Two association rates were required to explain the results adequately. At 37 degrees C, the faster component was estimated to have a second-order association rate constant of 1.4 X 10(7) M-1 s-1, while the rate constant for the slower component (3.8 X 10(6) M-1 s-1) was about 4-fold lower. As shown by others, the temperature dependence of the dissociation kinetics indicated that while the rapidly dissociating component was only slightly slowed by lowering the chase temperature to 0.5 degrees C, the second component was slowed by about 270-fold, from 8 X 10(-4) s-1 to 3 X 10(-6) s-1. The binding data that describe the slowly dissociating component were obtained by utilizing this differential temperature dependence and revealed a concave downward Scatchard plot. The binding parameters determined from computer analysis using a nonlinear fitting program (LIGAND) suggest that this component consists of (a) an interacting class of about 4000 sites/cell that have a first stoichiometric steady-state dissociation constant of 65 pM and a second stoichiometric interaction constant of 16 pM, indicative of positively cooperative interactions, and (b) a class of sites consistent with a ratio of sites/Kd of about 11.1 sites/(cell X pM). The steady-state binding results at 37 degrees C indicated only one class of binding sites (155,000 +/- 18,000 sites/cell) that had an apparent Kd of 0.52 +/- 0.03 nM. One class of sites was also observed at 0.5 degrees C, and the receptor concentration was found to be reduced (99,000 +/- 7600 sites/cell) while the Kd was increased (1.7 +/- 0.14 nM). A significant level of positively cooperative interactions was observed frequently at 37 degrees C that was not due to a failure to reach steady-state conditions, internalization, or degradation. Since cooperativity of binding was never observed at 0.5 degrees C, a membrane event may be involved. Determination of the contribution of the different classes of NGF receptors found on PC12 cells to the biological actions of NGF requires a clear understanding of their kinetic properties and their relationship to each other. The studies presented here indicate that their interactions are more complex than previously described.

Cooperative interaction of histone H1 with DNA.

Abstract: labelled histone H1 . The titration data were analysed in terms of the large ligand model. The stoichiometric number, n = 65 ± 10 bases/Hi, was independent of NaCl concentration (0.02 - 0.35 M). The nucleation and the co- ~ operative binding constants, K' and K, and the cooperativity parameter q were # sensitive to salt concentration; K = 3.6 + 0.8 x 107 M~1 and q = 1.1 ± 0.4 x 103 at 0.2 M NaCl. The dependence of K' on NaCl concentration revealed that »* 6 Na+ ions were released from DNA upon complex formation. An extrapolation of K' to 1M NaCl yielded a small value, K1 = 5 + 2 M"1. Thus the binding of H1 i is essentially electrostatic, being compatible with its independence of tem- perature. A calculation of K' based on the counterion release reproduced the salt concentration dependence of K'. Therefore, the binding of H1 is of an electrostatic territorial type. Thus, H1 may move along the DNA chain to a ~X certain extent, when both salt concentration and the degree of saturation are sufficiently low. The condition is so restricted that the sliding would not •r play an important role ^ii vivo. It was concluded from the DNA concentration independent binding isotherm that H1 can cooperatively bind onto a single DNA ** molecule. A simple power law dependence of the cooperativity parameter q upon NaCl concentration was found; qoc(NaCl)h with h = 0.72, though the physical basis of this dependence remains unknown.

Dynamics of spin-state interconversion and cooperativity for ferric spin-crossover complexes in the solid state. 5. Variable-temperature spectroscopic, magnetic, and single-crystal x-ray structural characterizations of the spin-state and order-disorder transformations of a Schiff base complex.

Abstract: Two types of solid-state structural dynamics are exhibited by [ Fe( 3-OEt-SalAPA)2]C104C6H6, where 3-OEt-SalAPA is the monoanion of the Schiff base. condensate of 3-ethoxysalicylaldehyde and N-(3-aminopropyl)aziridine. Variable-temperature magnetic susceptibility and EPR data show that the compound undergoes a gradual, but complete, spin-crossover transformation for which there are equal populations of highand low-spin complexes at 205 K. Only a single quadrupole-split doublet is seen in the Mossbauer spectrum a t all temperatures from 11 1 to 306 K. Thus, this complex is interconverting between lowand high-spin states a t a rate faster than the inverse of the Miissbauer time scale. No hysteresis is seen in any of the spectroscopic or magnetic measurements. A multiple-temperature structural investigation has provided an independent characterization of the spin transformation. This characterization is in complete accord with the magnetic data but reveals a second structural transformation whose presence is not reflected in the spectroscopic or magnetic data. This second transformation, a t about 180 K, is an order-disorder transformation which results in a change of space group from C z l c at high temperature to P 2 , / c at low temperature. The abrupt nature of this transformation, the observed hysteresis (AT = 15 K), and the observation of two phases in the hysteresis region are characteristic of a first-order transition. The order-disorder transformation involves a dynamic interconversion of two crystallographically distinct sites. Preservation of the local environments a t the two iron sites accounts for the insensitivity of the spin-equilibrium thermodynamics to the transformation. Although the X-ray investigation provides no direct evidence of the rate of interconversion between the two environments, the possible influence of this dynamic process on the rate of spin-state interconversion is discussed. Solid-state spin-crossover transformations have been of recent interest because they provide a means for investigating just how a n intramolecular process ( the spin-state interconversion) is coupled to the intermolecular interactions intrinsic to a condensed phase. To date, research in this areas-* has centered to a greater (1) Part 4 in the series: Timken, M. D.; Wilson, S . R.; Hendrickson, D. (2) University of Illinois. (3) University of California, Los Angeles. (4) On leave from Faculty of Sciences at Sohag, Egypt. ( 5 ) KBnig, E.; Ritter, G.; Irler, W.; Goodwin, H. A. J . Am. Chem. SOC. N. Inorg. Chem. 1985,24, 3450. 1980. 102. 4681. . . . ., . . -, . . . . ( 6 ) Konig, E.; Ritter, G.; Kulshreshtha, S . K.; Nelson, S. M. J. Am. Chem. Soc. 1983, 105, 1924. 0002-786318611508-0395$01.50/0 extent on the solid-state factors that influence the thermodynamics a t the spin-crossover transformation and to a lesser extent on the factors t ha t influence spin-state interconversion rates. In fact, very little is known about the role of intermolecular interactions in inhibiting or enhancing these rates in the solid state. Within the past few years, several ferric spin-crossover solids have been discovered which m a y provide new insights into this problem. In two of t he preceding p a p e r ~ ~ ~ ' ~ in this series the magnetic and spectroscopic properties of the new ferric N4O2 spin-crossover (7) Purcell, K. F.; Edwards, M. P. Inorg. Chem. 1984, 23, 2620. (8) Giitlich, P. Sfrucf . Bonding (Berlin) 1981, 44, 8 3 . (9) Federer, W. D.; Hendrickson, D. N. Inorg. Chem. 1984, 23, 3861. (10) Federer, W. D.; Hendrickson, D. N. Inorg. Chem. 1984, 23, 3870.

Allosteric regulation of inducer and operator binding to the lactose repressor

Abstract: The effects of cysteine modification and variations in pH on the equilibrium parameters for inducer and operator binding to the lactose repressor protein were examined. Operator binding affinity was minimally affected by increasing the pH from 7.5 to 9.2, whereas inducer binding was decreased for both the unliganded protein and the repressor-operator complex over the same range. Inducer binding to the repressor became more cooperative at high pH. The midpoint for the change in inducer affinity and cooperativity was pH 8.3; this value correlates well with cysteine ionization. The differential between repressor-operator affinity in the presence and absence of inducer was significantly decreased by modification of the protein with methyl methanethiosulfonate (MMTS). In contrast to unreacted protein, the inducer binding parameters for MMTS-modified repressor were largely unaffected by pH variation. The free energy for formation of the completely liganded protein was calculated for two pathways; the delta G values for these two independent routes were equivalent only for stoichiometries of four inducers and two operators per repressor molecule. All of the binding data were analyzed quantitatively by using a Monod-Wyman-Changeux two-state model for allosteric regulation. The observed dependences of the isopropyl beta-D-thiogalactoside binding curves on pH, DNA concentration, and MMTS modification were fitted by varying only the equilibrium constant between the two conformational states of the protein. With this analysis, high pH favors the T (high operator/low inducer affinity) state, while modification of cysteine-281 with MMTS elicits a shift into the R (high inducer/low operator affinity) state.(ABSTRACT TRUNCATED AT 250 WORDS)